Final Report

I

STUDIES ON THE HILL REACTION ACTIVITY OF SOLUBLE CHLOROPLAST EXTRACTS I

Prepared for:

OFFICE OF RESEARCH GRANTS AND CONTRACTS CODE SC NATIONAL AERONAUTICS AND SPACE ADMINISTRATION WASHINGTON, D.C. 20546 CONTRACT NASr 49-11

https://ntrs.nasa.gov/search.jsp?R=19650025979 2020-03-16T22:07:46+00:00Z

25 August 1965

Final Report

STUDIES ON THE HILL REACTION ACTIVITY OF SOLUBLE CHLOROPLAST EXTRACTS

Prepared for:

OFFICE OF RESEARCH GRANTS AND CONTRACTS CODE SC NATIONAL AERONAUTICS AND SPACE ADMINISTRATION WASHINGTON, D.C. 20546 CONTRACT NASr 49-11

By: L L O Y D K. MOSS JANICE E. COOMBER

S R I Project BU-44,335

Approved: G. W. NEWELL, DIRECTOR D I V I S I O N O F I N D U S T R I A L B I O L O G Y

W. A. SKINNER, EXECUTIVE DIRECTOR L I F E S C I E N C E S R E S E A R C H

2 '1 Copy No. ..............

CONTENTS

I

LIST OF ILLUSTRATIONS . . . . . . . . . . . . . . . . . . . . . . . iii

LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . i v

I INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . 1

I1 SUMMARY AND RECOMMENDATIONS . . . . . . . . . . . . . . . . . 2

I11 BACKGROUND . . . . . . . . . . . . . . . . . . . . . . . . . 3

I V EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . . . 7

A . Chloroplast Preparation. S t a b i l i t y . and Storage . . . . 7

1 . Typical Chloroplast Preparat ion . . . . . . . . . . 7 3 . Insonat ion . . . . . . . . . . . . . . . . . . . . . 8 4 . Immunochemistry . . . . . . . . . . . . . . . . . . 8

2 . Solut ions containing Digitonin. Dimethyl Sulfoxide. and Caffeine . . . . . . . . . . . . . 8

B . Centr i fuga l Frac t iona t ion . . . . . . . . . . . . . . . 9

C . Elec t rophore t ic Frac t iona t ion . . . . . . . . . . . . . 9

D . Measurement of H i l l Reaction Ac t iv i ty . . . . . . . . . 11 E . Spectrophotometric Analyses . . . . . . . . . . . . . . 13

V RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . 14 A . Preparat ion. Storage. and S t a b i l i t y . . . . . . . . . . . 14 B . Elec t rophore t ic Fract ionat ion . . . . . . . . . . . . . 26

ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . 38

LITERATURE CITED . . . . . . . . . . . . . . . . . . . . . . . . . 39

ii

I LLUSTRATI ONS

Fig. 1 Elphor FF: Electrophoretic Separation of a Chloroplast Preparation . . . . . . . . . . . . . . . . . . . . . . . . 47

Chloroplast Preparation Master Spectrum . . . . . . , . . . 48 Fig. 2a

aEle...en trz3sparmc, crerlaya fer chlr\roplast preparations and_ electrophoretic fract ions are contained i n the pocket of the rear cover.

iii

TABLES

I

Table I Ef fec t of Prepara t ive Methods and Storage on H i l l Reaction Ac t iv i ty . . . . . . . . . . . . . . . . . . . 15

Table I1 Ef fec t of Digi tonin Concentration on H i l l Reaction Act iv i ty . . . . . . . . . . . . . . . . . . . . . . . 16

Table I11 Effec t of D ia lys i s on H i l l Reaction Ac t iv i ty of Digi tonin Ex t rac t s . . . . . . . . . . . . . . . . . . 17

Table I V E f fec t of Period of Exposure t o Digi tonin on H i l l Reaction Ac t iv i ty . . . . . . . . . . . . . . . . . . . 18

Table V E f fec t of Lyophi l izat ion on H i l l Reaction Ac t iv i ty . . 19

Table V I Comparison on the Ef fec t of Freezing and Lyophi l izat ion on the H i l l Reaction Act iv i ty . . . . . . . . . . . . . 19

Table V I 1 E f fec t of Dimethyl Sulfoxide (DMSO) and Caffeine on H i l l Reaction Ac t iv i ty . . . . . . . . . . . . . . . . . 20

Table V I 1 1 E f fec t of Insonat ion and D i f f e r e n t i a l Cent r i fuga t ion on H i l l Reaction A c t i v i t y . . . . . . . . . . . . . . . 22

Table I X E f fec t of Sera and Colloids Added t o Ext rac t ion Medium on H i l l Reaction Act iv i ty . . . . . . . . . . . 25

Table X Operating Conditions f o r Prepara t ive Elec t rophores i s of Chloroplast Preparat ions . . . . . . . . . . . . . . 27

Table X I Chlorophyll Concentrations and H i l l Reaction A c t i v i t i e s of E lec t rophore t i ca l ly Separated Chloroplast Fragments. 30

Table X I 1 E f fec t of Chlorophyll Concentration on H i l l Reaction Act iv i ty Measured with the Polarographic Oxygen- Sensing Electrode . . . . . . . . . . . . . . . . . . . 34

iv

I INTRODUCTION

This study is concerned with the mechanisms and e s s e n t i a l r eac t an t s

of t h a t p a r t of t he photosynthet ic process i n which oxygen is produced

by photo lys i s of water. This is e s s e n t i a l l y the l i g h t absorbing

reac t ion , which can be s tud ied i n v i t r o separa te ly from the carbon

dioxide f i x i n g r eac t ions . The photolysis of water and evolut ion of

oxygen t h a t occurs when ch lo rop la s t s o r fragments of them a r e i l luminated

i n the presence of a s u i t a b l e e l ec t ron acceptor i s general ly r e fe r r ed t o

a s t h e H i l l r e a ~ t i o n . ~ ' ~

nonpar t icu la te s t a t e have general ly r e su l t ed i n lo s s of a c t i v i t y .

Progress toward t h i s goa l would be usefu l i n def in ing t h e minimum

components and condi t ions required f o r pho to ly t i c a c t i v i t y , a s w e l l a s

._

Attempts t o reduce the H i l l r eac t ion system t o a

i n determining t h e d e t a i l s of t h e mechanisms of energy t r a n s f e r . _-L

Recent experiments along these l i n e s i n ou r l abora to r i e s led t o

es tabl ishment of t h i s p ro j ec t t o study ch loroplas t e x t r a c t s .

The long range plan of study includes a n a l y t i c a l i nves t iga t ions t o

i d e n t i f y and cha rac t e r i ze t h e components of t h e photoactive complex, and

to determine t h e func t ion of each; examination of the r o l e of p ro te ins ,

expec ia l ly enzymes, i n t h e complex; s tud ie s on methods of preparing

e x t r a c t s of ch loroplas t s , and the s t a b i l i t y of t he prepara t ions ; de te r -

mination of t h e p a r t i c i p a t i o n and r o l e of var ious co fac to r s ; and

app l i ca t ion of spectroscopic and o ther phys ica l methods to e luc ida t ion

of t he c h a r a c t e r i s t i c s of t h e system.

1

I1 SUMMARY AND RECOMMENDATIONS

,

Car r i e r - f r ee continuous e lec t rophores i s has been appl ied t o chloro-

p l a s t fragments from common spinach, Spinacia o le racea . The e l e c t r o -

phore t i c f r a c t i o n s were t e s t e d a s biochemical c a t a l y s t s i n the H i l l

r eac t ion , t he l i b e r a t i o n of oxygen from water i n the presence of l i g h t

and f e r r i cyan ide .

Spec i f i c H i l l r eac t ion a c t i v i t y was sucessfu l ly demonstrated a f t e r

excursion through the Elphor FF, a commercial p repara t ive e lec t rophores i s

instrument. The a c t i v i t y was g r e a t e r f o r e l ec t rophore t i c e f f l u e n t s than

f o r unf rac t iona ted ma te r i a l . Maximum a c t i v i t y i n the e f f l u e n t s d id not

correspond t o t h e maximum chlorophyl l concentrat ions, i nd ica t ing e i t h e r

i n h i b i t i o n by c e r t a i n components or some optimum proport ion of c a t a l y t i c

co fac to r s . Differences i s absorp t ion spec t r a of f r a c t i o n s occurred i n the

u l t r a v i o l e t region but were not s i g n i f i c a n t i n the v i s i b l e region.

Novel methods t o preserve the biochemical i n t e g r i t y of components were

a l s o s tudied so t h a t extended experimentation could be performed wi th

minimum l o s s of a c t i v i t y . Although d ig i ton in f a c i l i t a t e d the so lub i l i za -

t i o n of t he pigment-protein complex, some H i l l r eac t ion a c t i v i t y was l o s t .

A t t he s a c r i f i c e of a c t i v i t y , however, f r a c t i o n a t i o n of d ig i ton in complexes

occurred during Sephadex chromatography. DMSO was a u s e f u l s o l u b i l i z i n g

agent with no de t r imenta l e f f e c t on H i l l a c t i v i t y . Preservat ion of

a c t i v i t y was obtained by adding co l lo ids , such a s normal and immune r abb i t

serum, g e l a t i n , and dextran, t o the solvent f o r ch loroplas t preparat ion.

I t is recommended t h a t f u t u r e research should involve a study of t h e

p ro te ins i n f r a c t i o n s separated e l ec t rophore t i ca l ly or by chromatography

on Sephadex or modified c e l l u l o s e ion exchangers. The l i p i d s and

pigments should be ex t r ac t ed with organic so lvents . The pro te ins should

then be separated chromatographically and charac te r ized by s t r u c t u r e and

amino a c i d composition.

2

I11 BACKGROUND

Chemical events i n photosynthesis can be descr ibed by two major

reac t ions . One is t h e l i g h t absorbing r e a c t i o n ; the o ther , the carbon

dioxide f i x i n g reac t ion . These two describe only the s t a r t i n g and

ending po in t s of a long chain of react ions, some of which a r e a s y e t

unknown.

For ins tance , t h e carbon dioxide f i x i n g r eac t ion has been followed

by using t r a c e r s and iden t i fy ing t h e progress of t h e tagged r eac t ions by

radioautography. The work of Calvin and h i s group has been la rge ly

successfu l i n following the main course of t h i s r eac t ion .

The l i g h t absorbing reac t ion can be s tudied separa te ly from carbon

dioxide f i x a t i o n . Under those conditions, i t is c a l l e d the H i l l r eac t ion .

I t proceeds i n the presence of an e l ec t ron acceptor , which is usua l ly a

complex inorganic ion or a compound of t h e quinone type. These e l ec t ron

acceptors a r e reduced when il luminated i n the presence of ch lo rop la s t s

o r ch lo rop la s t fragments, accompanied by photo lys i s of water and l i b e r -

a t i o n of oxygen.

Most a t tempts t o r e f i n e t h e photoactive prepara t ion f u r t h e r , t h a t

is, t o a nonpar t icu la te s t a t e , have resu l ted i n l o s s of t h e H i l l

r eac t ion a c t i v i t y . Recent experiments i n our l abora to r i e s showed t h a t

i t is poss ib l e t o prepare a c t i v e e x t r a c t s of whole ch lo rop la s t s or

chloroplas t fragments, using d ig i ton in as a s o l u b i l i z i n g agent . Chloro-

p l a s t s were prepared according to the method of Arnon' and ex t rac ted

with d i g i t o n i n so lu t ion according t o t h a t of Nieman and Vennesland . 6 ,

W e have a l s o appl ied these procedures t o ch loroplas t fragments and a r e - - - - - - - - A X - - -&..A..J..-. the ....+rr"+4nn GLLI I Gll Ir .LY Y b U U Y &lrg A. iCGI .C, -vrr c? K i l l reaction activity during

s to rage of d i g i t o n i n e x t r a c t s under various condi t ions , The r e s u l t s

i n d i c a t e considerable s t a b i l i t y ,

"he H i l l r eac t ion is the photolysis of water and the evolut ion of

oxygen t h a t occurs when ch loroplas t s or fragments of them a r e i r r a d i a t e d

i n t h e presence of an e l ec t ron acceptor, e.g. , fe r r icyanide , quinone,

3

. I .

or 2,6-dichlorophenol indophenol. When d ig i ton in e x t r a c t s were subs t i -

t u t ed f o r ch loroplas t fragments, moderately high r a t e s of oxygen evolu-

t i o n were obtained only i f cofac tors , such a s adenosine diphosphate,

Mg++, inorganic phosphate, ascorb ic ac id , f l a v i n mononucleotide, and

menadione (vi tamin K 3 ) , were added.

was obtained unless condi t ions were favorable f o r photosynthet ic

phosphorylation. However, we obtained no evidence f o r conversion of

ADP t o ATP. These r e s u l t s may be compared w i t h some of those recent ly

reported by Wolken,8 who reported H i l l r eac t ion a c t i v i t y i n ch lo rop la s t in

prepared w i t h d ig i ton in .

I n f a c t , no H i l l r eac t ion a c t i v i t y

Chloroplas t in can be def ined a s a complex of pro te ins , l i p i d s ,

phospholipids, chlorophyl ls , and carotenoids obtained from green p lan t

t i s s u e s . The term "chloroplas t in t t was introduced i n 1938 by S t o l l and

Wiedemann.'

so lu t ions was summarized by Rabinowitch."

descr ibed by Lubimenkoll and Loring and co workers;12 these were

prepared without the use of so lub i l i z ing agents . Euler and Hahn prs-

pared ch lo rop la s t in by the S t o l l method and reported the presence of

r ibonucle ic ac id . l3

A desc r ip t ion of t h e ear ly work on suspensions and

Aqueous e x t r a c t s were

During the per iod 1938-1941, Smith made a r a t h e r thorough study

of the s o l u b i l i z a t i o n of ch loroplas t in w i t h de te rgents . 14-'' H e

was the f i r s t t o use d ig i ton in (a na tu ra l nonionic saponin gaining

favor a t t h a t t i m e with Tansley2' and Wald2'J2') a s an ex t r ac t an t of

photosens i t ive v i s u a l pigment-protein complexes, of sodium dodecyl

s u l f a t e , 1 3 and of t he b i l e s a l t s , sodium glycocholate and deoxy-

cho la t e .20 Smith's work has been repeated and extended by K e and

C l e n d e ~ m i n g ~ ~ and by Wolken and h i s coworkers. 5-28

Wolken is the only inves t iga to r iu i-apcrt 9111 r e ~ c t i n n a c t i v i t y

for ch lo rop la s t in preparat ions, which were prepared with d ig i ton in .

Numerous workers has been unsuccessful i n t h i s respect, e .g . , K e and

Clendenning,24 French,2s Chiba,30 Kok," and Vennesland and co-

~ u L ~ c ~ ~ . ~ ~ , ~ ~

t h e oxygen-evolving process33 and i n photosynthet ic phosphorylation. 32

-L--- I n f=cti d ig i ton in i s descr ibed a s an i n h i b i t o r i n

4

An explanat ion of t h i s lack of success may involve exclusion of one

of t he co fac to r s t h a t a r e necessary. Takashima described a ch loroply l l -

l i popro te in complex, but it was devoid of photochemical a c t i v i t y . 3 4

H e noted t h a t t h e carotenoids were excluded during the preparat ion of

h i s complex.

That c e r t a i n of the carotenoid pigments take p a r t i n the H i l l

r eac t ion under some condi t ions has been shown by experiments of French

and coworkers,35,36 Bishop,37-41 and Moss.42 Much needs t o be learned

about t h e i r place i n the sequence of s t eps making up the o v e r a l l

pho to ly t i c production of oxygen from water. Ident i fy ing the

carotenoids t h a t a r e e s s e n t i a l or a r e capable of taking pa r t is a l s o

not resolved. Some of the evidence on the a b i l i t y of 8-carotene t o

r e s t o r e H i l l r eac t ion a c t i v i t y t o ch loroplas t fragments a f t e r these were

ex t r ac t ed w i t h an organic solvent i s conf l i c t ing . A number of cofac tors

a r e apparently involved; they need to be i d e n t i f i e d and t h e i r funct ions

e luc ida ted .

During recent years , d ig i ton in and o the r so lub i l i z ing agents have

been widely used f o r t h e prepara t ion of var ious b io log ica l ly a c t i v e

ma te r i a l s . Friend and Mayer used sodium deoxycholate t o i s o l a t e a

carotenoid-destroying enzyme.43

of p r o t e i n s o l u b i l i z a t i o n w i t h Tr i ton X-100.44'45

Ji rgensons,46 the de tergents Aerosol 0" and HX form complexes with

p ro te ins and are much more e f f i c i e n t i n unfolding macromolecules than

a r e urea, guanidine s a l t s , a lcohol , and s i m i l a r denaturing agents .

Chiba used Duponol C and Span 80 f o r t h e s o l u b i l i z a t i o n of ch loroplas t

p ro t e ins . 30 K e and Clendenning extended t h e l ist of detergents ,

including Zephiran ch lor ide , Tween 20, and pur i f i ed saponin from

s e v e r a l sources .24 a t the ';ii:szrs:ty of Cbiz=go. venr?esl.rnd a n d her coworkers have

employed d ig i ton in t o fragment ch loroplas t s and have s tudied f r a c t i o n s

f o r s p e c i f i c enzyme aCtivity.6,7,32,3314l,4',4*,49,5l

Dowben and Koehler s tudied t h e e f f e c t

According t o

However, t he most p r o l i f i c group i s with Vennesland

Nieman and Vennesland character ized the enzyme cytochrome c

photooxidase. This enzyme was botained from chloroplas t in , wnicn was

5

I

f r ac t iona ted by e thanol p r e c i p i t a t i o n 6 or by d i f f e r e n t i a l c e n t r i -

fuga t ion .33

t h a t d ig i ton in inh ib i t ed oxygen l ibe ra t ion .

Nakamura,49 and Chow,48 t h e c a t a l y s t s involved i n photosynthet ic

phosphorylation a r e p a r t i c u l a t e i n nature . However, photophosphorylation

suf fered 50% i n h i b i t i o n i n 0.03% dig i ton in so lu t ion and was completely

inh ib i t ed by d ig i ton in concentrat ions g rea t e r than 0.1%.

Ardao and Vennesland observed chlorophyllase a c t i v i t y a s soc ia t ed with

ch loroplas t in . 4 7

was used by Kra l l and Purvis i n t h e i r study of nuc leo t ide acceptor

s p e c i f i c i t y of photophosphorylation. 5 0

No H i l l r eac t ion a c t i v i t y was evident , and i t was presumed

According t o Kouko1,32

Recently,

The Koukol method32 for preparing d ig i ton in fragments

We found t h a t the v i s i b l e absorption spectrum of ch lo rop la s t in

so lu t ions had a red peak with a maximum a t 675 xty,, i n agreement with

spec t ra reported i n the l i t e r a t u r e . 1 4 , 3 0 I n addi t ion , t he re was

s t rong absorpt ion i n the 450 t o 480 mp region c h a r a c t e r i s t i c of the

carotenoid components. The so lu t ions a l s o exhib i ted b r i l l i a n t red

f luorescence when i r r a d i a t e d with 365 mp l i g h t . One of the more

i n t e r e s t i n g experiments w e performed w i t h ch lo rop la s t in was column

chromatography on Sephadex. The migration of t he green band was e a s i l y

followed and i ts f luorescence was demonstrated during chromatography on

the column. We d id not, however, determine H i l l a c t i v i t y following

chromatography.

6

I V EXPER1IVIFN”TL

A . Chloroplas t Prepara t ion , S t a b i l i t y , and Storage

1. Typica 1 Chloroplast Preparation

Chloroplas t s and fragments were prepared from f r e s h spinach32

The leaves were c h i l l e d , washed i n d i s t i l l e d water, and deveined. They

were then cu t i n t o small pieces and ground with e i t h e r a mortar and

p e s t l e or minced i n a Waring blendor in a co ld s o l u t i o n (4OC) of 0 .1 M

KC1, 0.5 M sucrose, and 0.5 M phosphate b u f f e r (pH 6.6).

were e i t h e r i l lumina ted or kept i n the dark before gr inding . The

sucrose-phosphate b u f f e r was used a t the r a t e of 1 ml/g of leaf t i s s u e .

The leaves were ground only enough t o l i q u i f y t h e prepara t ion and then

s t r a i n e d through fou r layers of cheesecloth and one l a y e r of g l a s s wool.

The leaves

The f i l t e r e d suspension was cent r i fuged f o r 5 min a t 2000 rpm (480 x g )

t o remove l a rge c e l l deb r i s , then for 15 min a t 3000 rpm (1085 x g ) t o

c o l l e c t whole ch lo rop la s t s , and f i n a l l y f o r 15 min a t 15,000 rpm

(27,000 x g ) i n a Serva l1 cen t r i fuge t o c o l l e c t ch lo rop la s t fragments.

The p e l l e t s of ch lo rop la s t s or fragments were resuspended i n sucrose-

phosphate bu f fe r , using a g l a s s homogenizer. The sample prepara t ion

was a l l done under green l i g h t . 9 9 Samples not used immediately f o r H i l l

r e a c t i o n assays were f rozen i n 3 - m l a l i q u o t s and s to red i n t h e dark i n

a f r e e z e r (-40°C).

I n recent experiments, a T r i s buf fer (0.025 M T r i s [hydraxymethyl]

amino methane, ad jus t ed t o pH 7.0 with HC1) was used because it was

d i r e c t l y app l i cab le t o p repa ra t ive e l ec t rophore t i c s epa ra t ions .

++Purchased a t a l o c a l market.

7

2. Solu t ions containing Digitonin, Dimethyl Sulfoxide, and

Caffeine . Preparat ions were made a s described above, through the f i r s t c e n t r i -

fuga t ion a t 2,000 rpm. The supernatant was cent r i fuged a t 15,000 rpm,

and t h e p e l l e t was resuspended i n 30 m l of sucrose-phosphate bu f fe r w i t h

var ious amounts of d i g i t o n i n added. The homogenized mixture was allowed

t o s tand i n t h e cold f o r 15 min and was then cent r i fuged a t 3,000 rpm

f o r 10 min. The supernatant (SI) was separated from the p e l l e t ( P I ) ,

and a por t ion was cent r i fuged a t 15,000 rpm for 15 min. The S, super-

na tan t was separa ted from t h e P, p e l l e t , and a po r t ion was again c e n t r i -

fuged a t 15,000 rpm f o r 60 min t o prepare p e l l e t P, and supernatant S,.

The p e l l e t s were resuspended i n sucrose-phosphate b u f f e r f o r H i l l

r eac t ion measurement.

Dimethyl su l foxide and ca f f e ine (both a s reagent grade chemicals )

were s u b s t i t u t e d f o r sucrose i n the typ ica l b u f f e r s o l u t i o n used through-

out t h e e x t r a c t i o n and resuspension of ch lo rop la s t p repara t ions . The

DMSO was present e i ther as a 0.4 M or 10% so lu t ion .

ca f f e ine , a 1% so lu t ion was employed.

I n t h e case of

3 . Insonat ion

Whole ch lo rop la s t s were prepared from 100 g of spinach leaves.

P e l l e t s obtained by low speed cent r i fuga t ion were resuspended i n 50 m l

of T r i s - H C 1 bu f fe r (pH 6 . 9 ) and soni f ied i n a Raytheon 10-kc s o n i f i e r

modified t o achieve a 250-watt output . The ch lo rop la s t s were son i f i ed

f o r b r i e f i n t e r v a l s a t maximum power for 4 min.

4. Immunochemistry

The crude supernatant so lu t ion from a t y p i c a l ch lo rop la s t fragment

prepara t ion was d ia lyzed overnight against 4 liters of d i s t i l l e d water.

The experiment was c a r r i e d out i n a cold room a t 4OC but , because of

hea t generated by a magnetic st irrer, t h e temperature of t he s o l u t i o n

rose t o 3OoC during t h e d i a l y s i s .

8

t

The dialyzed ma te r i a l was concentrated by lyoph i l i za t ion ; 1.1 g

was recovered from 150 g of spinach leaf t i s s u e . A 250-mg por t ion of

the lyophi l ized ma te r i a l w a s resuspended i n 20 m l of T r i s buf fer , and

the suspension was centr i fuged f o r 15 min a t 15,000 rpm.

c l e a r yellow s o l u t i o n was used i n H i l l r eac t ion experiments, i t had no

e f f e c t on the photo ly t ic a c t i v i t y of t y p i c a l ch loroplas t fragments.

When the

The dialyzed, lyophi l ized m a t e r i a l was suspended i n a n e u t r a l bu f fe r

( 2 mg/ml Bacto-hemagglutination bu f fe r ) and s t e r i l i z e d by Mil l ipore

f i l t r a t i o n . One pa r t of t h i s so lu t ion was mixed w i t h an equal volume

of Freund's complete adjuvant. The ant igen so lu t ion (1 m l , containing

1 mg of lyophi l ized m a t e r i a l ) was in jec ted intramuscularly i n t o each

of three New Zealand white r a b b i t s every o the r day f o r the f irst week

of immunization and then, a s an ant igen so lu t ion without t he adjuvant,

for t h e next t h ree t o four weeks on t h e same schedule.

Rabbits were bled one week a f t e r t h e f i n a l i n j e c t i o n . The serum

was separated from the c l o t t e d blood and preserved frozen i n s ter i le

serum b o t t l e s .

B. Cen t r i fuga l Frac t iona t ion

A Beckman/Spinco Preparat ive Ul t racent r i fuge (Model L-2) was used

t o f r a c t i o n a t e the ch loroplas t preparat ion a f t e r sonica t ion . The super-

na t an t suspension was centr i fuged a t 20,000 x g f o r 15 min and a p e l l e t ,

Po-zo, co l lec ted . The r e s u l t i n g supernatant suspension was then cen t r i -

fuged an add i t iona l 15 min a t 50,000 x g. The p e l l e t w a s resuspended a s

f r a c t i o n Pzo-so. The supernatant f r a c t i o n S s 0 was s t i l l green. Various

spectrophotometric, H i l l r eac t ion , and chlorophyl l determinat ions were

made on these f r a c t i o n s .

C . E lec t rophore t ic Frac t iona t ion

Prepara t ive e lec t rophores i s was performed i n an Elphor FF, a

carrier-free continuous e l ec t rophore t i c separa tor . The instrument was

designed by HannigS2 and is d i s t r ibu ted i n the United S t a t e s by

Brinkmann Instruments, Inc. , Westbury, New York 11590.

9

In con t r a s t t o c u r t a i n e lec t rophores i s a t 3 0 wat ts , a ch loroplas t

fragment prepara t ion can be s tud ied eas i ly on a prepara t ive s c a l e and

under high power condi t ions a t about 300 wa t t s i n t h e Elphor FF. The

Elphor FF has no c a r r i e r ; i t is e s sen t i a l ly two air-cooled g l a s s p l a t e s

about 50-cm square, separated by a 0.5-mm space through which the e l ec t ro -

l y t e passes a s a f i lm. The instrument was run a t a constant cur ren t and

observed under dim green l i g h t . Forty-eight f r a c t i o n s d iv ide the

e f f l u e n t a t cent imeter i n t e r v a l s , t h e f r a c t i o n numbers increasing from

l e f t (anode) t o r i g h t (cathode) .

were masked t o decrease t h e i r i n t ens i ty .

The red p i l o t l i g h t s on t h e instrument

Concent ra tedTr is bu f fe r 0.075 M, pH 7.0, was used i n the e l ec t rode

chambers. The e l e c t r o l y t e passing through the chamber was T r i s 0.025 M,

pH 7.05. The buf fe r flow r a t e through t h e separa t ing chamber was 363 m l

e f f l u e n t per h r ( e l e c t r o l y t e p e r i s t a l t i c pump).

10.5 m l per h r (sample p e r i s t a l t i c pump s e t t i n g ) .

34-35. With these flow r a t e s and e l e c t r o l y t e concentrat ions, t h e power

s e t t i n g s were 160 ma and 1900 v o l t s (approximately 304 w a t t s ) .

temperature con t ro l was s e t a t 4OC.

ice was placed over t he p l a s t i c tubing leading from the dosing pump i n t o

the chamber.

The dosing r a t e was

The i n l e t port was

The

A p l a s t i c bag containing crushed

The chloroplas t fragment sample was prepared a s follows f o r dosing

i n t o t he Elphor FF. The supernatant l i g u i d containing ch loroplas t

fragments (P,) was cent r i fuged f o r 15 min a t 15,000 rpm (27,000 x g ) i n

a Servall cen t r i fuge . The high-speed p e l l e t s were resuspended i n 20.0

m l of T r i s bu f fe r and homogenized w i t h a hand homogenizer. The so lu t ion

was then centr i fuged f o r 10 min a t f u l l speed i n a c l i n i c a l cen t r i fuge .

The p e l l e t was discarded and a portion of t he suspension of P, was used

i n t h e high vol tage e l ec t rophores i s u n i t . Figure 1, appended t o the

report , is a photograph of the Elphor FF, showing a ch loroplas t e l ec t ro -

phoret i c separa t ion .

The ch loroplas t fragment sample (P, ) was subjec ted t o approximately

38 v/cm during about 18 min i n the separa t ion chamber,

90 min w a s required f o r t h e e lec t rophore t ic separa t ion .

Approximately

10

In experiments involving d ig i ton in ex t r ac t ion , t h e p e l l e t s (PI) from

s l o w speed cen t r i fuga t ion were resuspended i n 20 m l T r i s bu f fe r containing

l$ d ig i ton in , allowed t o s tand f o r 15 min,and centr i fuged f o r 10 min a t

1000 X g The p e l l e t s were discarded but supernatant s o l u t i o n was then

cent r i fuged i n a Serva l1 cen t r i fuge f o r 15 min a t 15,000 rpm (27,000 x g )

t o recover l a rge r d ig i ton in /ch loroplas t fragments (PD,).

na t an t so lu t ion , containing very small d ig i ton in /ch loroplas t fragments,

was recent r i fuged f o r 1 h r a t 27,000 x g, and the p e l l e t s (m,) were

resuspended i n 5 . 0 m l of T r i s bu f fe r .

t he 15-min cen t r i fuga t ion were a l s o resuspended i n T r i s bu f fe r and

prepared f o r the Elphor FF a s described f o r t he P, fragments.

The super-

The l a r g e r fragments (PD,) from

The lyophi l ized ma te r i a l used a s the an t igen mixture for r abb i t

immunization was a l s o analyzed by e lec t rophores i s i n the Elphor FF,

using a T r i s buf fer (pH 6 .9) .

s e t t i n g , 3.0; buf fe r d i a l s e t t i n g , 2.5; res idence t i m e i n the chamber,

41.7 min; 130 ma; 1500 v o l t s . The e f f luen t f r a c t i o n s from the Elphor

were examined i n a spectrophotometer a t 260 q and 280 9.

came o f f i n f r a c t i o n s 22 through 36, showing t h a t most of i t was a c i d i c

a t pH 6.9. A f a i n t yellow co lo r w a s v i s i b l e i n f r a c t i o n s 25 through 30.

I t is probable t h a t a f r a c t i o n of t h i s so lub le ma te r i a l is r e l a t e d t o

the Frac t ion I of Singer, 53,7s4 Wildman, 55- -67

The i n l e t por t was 34-35, dosing d i a l

The mater ia l

and o the r s .5 8-63

D . Measurement of H i l l Reaction Act iv i ty

H i l l r eac t ion a c t i v i t y of t he f r ac t ions was determined by the

usua l manometric methods f o r oxygen evolution64 i n an Aminco Warburg

apparatus or a constant volume re f r ige ra t ed respirometer. A l l manipula-

t i ons , before the exposure t o red l i gh t during the photo lys i s reac t ion ,

were performed under dim green l i g h t , The temperature was mairiiaiiieC a t

14.4 f 0.1' C during runs. The conical Warburg vesse l s were i l luminated

with red l i g h t from below by a c i r c u l a r , f ive- tube neon bulb. The l i g h t

i n t e n s i t y was approximately 9,000 lux. The 3 - m l

Warburg f l a s k cons is ted of i . 0 mi T,, c s z t a i r ? i ~ ~

chlorophyl l , 0.8 m l T r i s bu f fe r (pH 7.05), and 1

reac t ion volume i n each

about 0.25-1.6 mg

m l of H i l l r eac t ion

11

oxidant (0.01 mg potassium fer r icyanide) . There was 0 . 2 m l of 10% KOH

i n the cen te r wel l s . Vessels and l i n e s were f lushed w i t h prepur i f ied

ni t rogen. Evolution of 0, i n each Warburg manometer was cor rec ted

aga ins t a thermobarometer.

The oxygen evolu t ion r a t e was ca lcu la ted

Chl - pl O,/min x 60 QO, - mg chlorophyl l

a s :

To measure oxygen by t h e polarographic e l ec t rode method, a Beckman

oxygen sensor (39065) and adapter box (96260) were used i n conjunction

with a Model 76 Beckman expanded sca le pH meter.

set up, s e v e r a l minor adjustments had t o be made t o adapt i t t o use with

a potent iometr ic recorder . The short ing s t r a p across the recorder output

on the pH meter was replaced with a r e s i s t o r . Using a 1-mv recorder

(Servo/Riter Model PSR, Texas Instruments, Inc. ) with an a t t enua t ion

path on t h e input c i r c u i t , a 49.5-ohm r e s i s t o r , f l%, $ watt, gave a

10-mv recorder s e n s i t i v i t y .

75 p l of oxygen evolved per char t d iv is ion .

a s s i s t a n c e of Frankl in M. Church during t h e i n s t a l l a t i o n of the new

instrumentat ion is g r a t e f u l l y acknowledged. )

Afte r the equipment was

The recorder span was ad jus ted t o read

(Note: t he t echn ica l

Experiments were conducted t o compare the accuracy of the r e s u l t s

obtained using t h e polarographic e lec t rode with t h e Warburg respirometer .

The oxygen-sensing polarographic e lec t rode was ca l ib ra t ed i n a i r using

the X10 a t t enua t ion and the 100% sca le on the oxygen adapter box.

gave a reading of 21$ p a r t i a l pressure of oxygen i n a i r on the pH meter;

the recorder was ad jus ted t o read accordingly.

This

The e lec t rode was in se r t ed i n a modified Warburg f l a s k . An addi-

t i o n a l s i d e tube was a t tached t o the f l a s k t o f a c i l i t a t e f lush ing with

p repur i f i ed n i t rogen t o obta in an anaerobic base l i n e . The f l a s k con-

ta ined 1 . 0 m l of ch loroplas t fragment suspension i n the s i d e arm, 1 . 0

m l of 0.01 M potassium fe r r i cyan ide so lu t ion and 0.8 m l of 0.025 M T r i s

12

I .

buffer (pH 7. 0 )

potassium hydroxide so lu t ion i n the center we l l .

i n the main port ion of t he f l a s k , and 0.2 m l of 10%

The f l a s k was immersed i n the constant temperature water bath

(14.4 f 0.1OC) of t h e Warburg apparatus (Model 5-134, American Instrument,

I n c . ) and shaken while being f lushed with p repur i f i ed n i t rogen .

approximately 20 min, a steady basel ine was obtained on the recorder .

The so lu t ions were then mixed and allowed t o s t a b i l i z e i n t h e dark f o r

a few more minutes before the l i g h t was turned on t o d r i v e the photo-

chemical r eac t ion . Comparable f l a sks , including a thermobarometer, were

run using s tandard manometric methods. The resu l t s by both methods

compared favorably. In one run, t h e m i c r o l i t e r s of oxygen evolved per

minute were 4.43 (manometric) and 4.5 (polarographic) . I n another

experiment, t h e y were 5.94 and 6.3, r e spec t ive ly ,

Af t e r

E . Spectrophotometric Analyses

A Cary spectrophotometer (Model 14 ) was used t o examine the spec t ra

of t h e Elphor F'F f r a c t i o n s i n an aqueous so lu t ion .

samples were a l s o analyzed. For t he more opaque so lu t ions , a 1 : l O O

d i l u t i o n was made t o g e t t he complete spectrum on t h e c h a r t ; the

spec t ra were scanned from 700 nw. t o 200 mp.

The P,,-,, and S,,

Chlorophyll determinat ions were made using t h e method of Arnon.lOs

Absorbancy A 0.5-ml sample was d i l u t e d t o 25 m l i n 80% aqueous acetone.

was measured a t 645, 652, and 663 v. The chlorophyl l concentrat ions

were ca l cu la t ed from t h e formula:

= mg/ml 'OD* 6 5 2 x 1000 25 34.5 1000 x 0.5

13

V RESULTS AND DISCUSSION

A . Preparat ion, Storage, and S t a b i l i t y

One of t h e major problems i n the study of t he H i l l r eac t ion i s the

preserva t ion of photosynthet ic a c t i v i t y ; t h i s a c t i v i t y of p l an t s i s

contained i n the ch lo rop la s t s . During t h e i s o l a t i o n of these s t ruc tu res ,

t he leave t i s s u e s a r e fragmented and b a r r i e r s a r e destroyed t h a t normally

preserve the biochemical i n t e g r i t y of a l l t h e components. The H i l l

r e ac t ion enzyme sys t em becomes rapidly and i r r e v e r s i b l y inac t iva ted i n

t h i s crude mil ieux unless i t is kept cold and protected from atmospheric

oxygen.

In a t tempting research on t h i s l a b i l e biochemical sys t em, an i n i t i a l

ob jec t ive was t h e prepara t ion of ch loroplas t s and fragments under

condi t ions t h a t would g ive t h e highest i n i t i a l a c t i v i t y . A coro l la ry of

t h i s ob jec t ive was t h e search f o r methods of preserving the H i l l

r eac t ion a c t i v i t y so t h a t extended experimentation could be performed

with minimum loss of a c t i v i t y .

I l lumina t ion and a l t e r n a t i v e methods of gr inding appeared t o have

r e l a t i v e l y l i t t l e e f f e c t on H i l l r eac t ion a c t i v i t y . The chloroplas t s

kept b e t t e r than t h e fragments while s tored frozen. Washing with

l a rge volumes of bu f fe r d id not appreciably a f f e c t t he a c t i v i t y . I n

genera l , t h e preserva t ion of a c t i v i t y i n s to red frozen samples was

q u i t e good. Storage r e s u l t s a r e summarized i n Table I .

I n the Background sec t ion , there i s a d iscuss ion on t h e use of

d i g i t o n i n as a s o l u b i l i z i n g agent f o r t he prepara t ion of what has been

c a l l e d ch lo rop la s t in . Preliminary s t u d i e s before t h i s p ro jec t was

e s t ab l i shed ind ica ted t h a t d i g i t o n i n so lu t ions might w e l l f a c i l i t a t e

t h e s o l u t i o n of protein-pigment complexes and, hopeful ly , those

demonstrating H i l l r eac t ion ac t iv i ty .

I t I 1

?he experiments summarized i n Table I1 suggested t h a t the presence

of d i g i t o n i n might diminish the H i l l r eac t ion a c t i v i t y , espec ia l ly i n t he

l a t e r supernatant f r a c t i o n s .

14

I n

0) rl 3

F

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c 0 .rl c, cd k cd a 0, k pc

E: 0 .I4 . . U

t4 rl H

L)

b P) E -

Q) a x !4

-: 2

0 m W

0 0 F3 rl

0 0 0

m u , w * b u ,

0 0 0 0 w c n

% Qcn 0 0 N w o h ] m * t - w

0 rl d( d

w r l o m * o r l r l (9 w r l w h ] CDa 8 m w m O W ( o m l-l 1-I

in cd d a 0 k 0 rl c u

m c, E: 0,

cd k k

El

in a rl a 0 k 0 rl c u

m c, E: Q)

cd k

Frc

El

in cd d a 0 k 0 d d u

in (d rl a 0 k 0 rl G u

in cd rl a 0 k 0 rl c u

m (d rl a 0 k 0 rl c u

m c, c

B cd k R

m cd rl a 0 k 0 rl c u

m Q) $I i?

k cd c, k

2

Fc 0 a E: Q? z

rd k cd c, k! iz

a k cd c, 2 52

k cd c, k

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15

Table I1

EFFECT OF D I G I T O N I N CONCENTRATION ON H I L L REACTION ACTIVITY

Attempts were made t o improve t h e a c t i v i t y by Sephadex chromato-

graphy and by d i a l y s i s . The Sephadex columns were prepared with d i f f e r e n t

grades suspended i n 0.1 M K C 1 and 0.5 M sucrose. The column was developed

with 0.5 M phosphate bu f fe r and 0.5 M sucrose, pH 6.6.

A t room temperature on Sephadex G-25, P3S3 formed two bands, one

of which was e lu t ed . P1P3S3 toge ther formed th ree bands, the f i r s t two

of which were e lu t ed . On Sephadex G-50, PlP3S3 toge ther formed fou r

bands, of which the f i r s t t w o were eas i ly e lu t ed , the o t h e r two not a t

a l l . On Sephadex G-100, flow rates were very f a s t , and no separa t ions

were achieved. Unfortunately, a l l of t he f r a c t i o n s e lu t ed from Sephadex

columns contained d ig i ton in . A t t h e low temperature of a co ld room

(4OC), t h e so lvents were too viscous t o provide u s e f u l flow rates.

Two chloroplas t p repara t ions were prepared with I$ d ig i ton in and

used t o inves t iga t e the e f f e c t of d i a l y s i s , I n each case, a f t e r

s o l u b i l i z a t i o n with d ig i ton in solut ion, ha l f of t he sample was dialyzed

i n t h e co ld aga ins t 3 liters of 0.5 M phosphate buf fer , pH 6.6, and was

s t i r r e d mechanically during the d i a l y s i s , The o t h e r ha l f of t h e sample

16

was kept a s a con t ro l a t t h e same temperature, Both d ia lyzed and

con t ro l samples were f r ac t iona ted i n t o p e l l e t and supernatant f r a c t i o n s .

The r e s u l t s of H i l l r eac t ion measurements on these f r a c t i o n s a r e shown

i n Table 111.

Dia lys i s f o r 4 h r d id not e f f ec t ive ly remove enough of t he

d ig i ton in , while d i a l y s i s f o r 19 h r removed most but not a l l . D ia lys i s

had a b e n e f i c i a l e f f e c t i n some cases , although not cons i s t en t ly . The

loss of a c t i v i t y i n con t ro l s during s torage a t 4OC discouraged the use

of extended d i a l y s i s t i m e .

Table I11

EFFECT OF DIALYSIS ON H I L L REACTION ACTIVITY OF DIGITONIN EXTRACTS

Preparat ion

Number 1

Before d i a l y s i s

19-hr d i a l y s i s

19-hr con t ro l

Number 2

Before d i a l y s i s

4-hr d i a l y s i s

4-hr con t ro l

Whole Ext rac t

18 0 ---*

90

23 1

240

90

P l

* --- Ind ica t e s neg l ig ib l e a c t i v i t y

Ch 1 0 2

---

180 I p, .- p3

-50 - 156

S l s2

3 90

109

A new prepara t ion of ch loroplas t s and fragments was made with 1%

d i g i t o n i n . H i l l r eac t ion a c t i v i t y was measured a s soon a s p r a c t i c a l

a f t e r preparat ion, again a f t e r one and two periods of incubation i n the

17

cold, and f i n a l l y a f t e r four days frozen s torage . A s seen by the

r e s u l t s i n Table I V , the main e f f e c t of d i g i t o n i n is prompt, w i t h very

l i t t l e f u r t h e r change during incubation or s torage .

46 9

1 1216

Table I V

EFFECT OF PERIOD OF EXPOSURE TO DIGITONIN ON HILL REACTION ACTIVITY

3.5 h r Prepa r a t ion

Chloroplasts

Fragments

4 days s torage 7 h r

Digi tonin

3 h r 10 min

I 444

8 00 i

Y e s

No

Yes

No

6 h r 40 min 4 days s torage

48 0 23 0

816 1043

Ch 1 0 2

45 min

4 95

1207

45 min

1013

A number of preparat ions of both whole and fragmented ch loroplas t s

were quick-frozen and lyophi l ized. Results of H i l l r eac t ion s t u d i e s on

these a r e given i n Table V.

The e f f e c t of lyophi l iza t ion seemed t o be q u i t e e r r a t i c and

e spec ia l ly de t r imenta l t o the preparat ions of fragments. A s seen w i t h

p repara t ion F, i t was poss ib le t o obtain a highly a c t i v e lyophi l ized

prepara t ion . A l imi ted s torage study was performed on a lyophi l ized

ch loroplas t p repara t ion . H i l l r eac t ion a c t i v i t y was measured on a

po r t ion of t h e sample before the s torage per iod, A f t e r one month, i t

was again measured immediately and a t l a t e r i n t e r v a l s a f t e r thawing

and resuspending the products. The r e s u l t s a r e snown lii Tabla VI.

Obviously, much less a c t i v i t y was l o s t by f reez ing than by lyophi l iz ing .

Apparently,considerable care is required i n the f reez ing and dehydrating

Steps t o preserve the H i l l r eac t ion a c t i v i t y .

18

Table V

EFFECT OF LYOPHILIZATION ON H I L L REACTION ACTIVITY

Chloroplast Prepara t ion and

Storage Time

A . Frozen: 2.5 months

B. Lyophilized: 1 month

Prepara t ion

Ch 1 0 2

After s to rage

I n i t i a 1 0 3 h r 5.5 h r

8 63 811 48 6 4 05

833 28 1 290 18 0

A .

B.

C.

D.

E .

F.

Chloroplasts

Chloroplasts

Fragments

Chloroplasts

Ch loroplas t s

Fragments

Chloroplasts

Fragments

Chloroplasts

Fragment s

Ch 1 0 2

1143

1014

882

Afte r Lyophilizing

600

618

2 90

5 70

470 --- 45 1

3 45

1105

Table V I

COMPARISON OF THE EFFECT OF FREEZING AND LYOPHILIZATION

ON THE HILL REACTION ACTIVITY

19

The d i f f i c u l t i e s encountered i n the Sephadex chromatography of

d ig i ton in - so lub i l i zed ch lo rop la s t p repara t ions prompted u s t o search f o r

o t h e r substances t h a t might have a p ro tec t ive e f f e c t and serve a s an

adequate s u b s t i t u t e f o r the t y p i c a l sucrose-phosphate bu f fe r . D i m e t h y l

su l fox ide (DMSO), which has been described a s a u s e f u l nonaqueous so lvent

f o r many p r o t e i n s 6 5 , 6 6 y i e l d s so lu t ions without marked changes i n t h e

b i o l o g i c a l p rope r t i e s of t h e p ro te ins . DMSO has unique s o l u b i l i t y

p r o p e r t i e s and is misc ib le i n both polar and nonpolar so lven t s . When a

0.4 M s o l u t i o n showed no i n h i b i t o r y e f f e c t on t h e H i l l r e a c t i o n a c t i v i t y

of a ch lo rop la s t p repara t ion , t h e concentration of DMSO was increased

t o 10% wt/vol. The r e s u l t s of t h e s e s t u d i e s a r e included i n Table V I I .

Solvent Systems

DMSO may become a u s e f u l s o l u b i l i z i n g agent . Within experimental

e r r o r , t h e H i l l r eac t ion a c t i v i t y of s o l u t i o n s containing it was

unchanged or s l i g h t l y enhanced. Future s t u d i e s should include i t s

e f f e c t on t h e s to rage s t a b i l i t y of ch loroplas t so lu t ions kept a t 4OC

and a t -4OoC, i t s e f f ec t iveness i n increas ing d ig i ton in s o l u b i l i t y i n

aqueous s o l u t i o n s , and i ts po ten t i a t ion or a t t enua t ion of energy

t r a n s f e r i n the H i l l r eac t ion system.

Table VI1

EFFECT OF DIMETHYL SULFOXIDE (DMSO) AND CAFFEINE ON HILL REACTION ACTIVITY

I Chloroplas t s I 0.4 M DMSO

Cont ro 1

l O $ DMSO

Cont ro l

1% Caffe ine

Cont ro l

460

55 1

61 1

551

387

48 7

- Fragment s

3 83

43 Y

520

611

352

851 1 20

The recent paper by Fredericks and Jagendorfs7 descr ibed both the

preserva t ion of H i l l r eac t ion a c t i v i t y and t h e marked s t imula t ion of some

photosynthet ic systems by high molecular weight polymers, such a s

Carbowax and dextran. The e a r l i e r work of Clendenning and Brown was

c i t e d .68

f o r t he inac t iva t ion of ch lo rop la s t s by leaf tannins re leased i n t o

in t imate mixture with the H i l l react ion components. The H i l l r eac t ion

r a t e s were inversely r e l a t e d t o the tannin con ten t , Furthermore, it

was impossible t o rega in H i l l r eac t ion a c t i v i t y once it was l o s t a s a

consequence of tannin-protein p rec ip i t a t ion .

Clendenning advanced the thes i s t h a t t h e Carbowax compensated

On another p ro jec t , we have been using tannic a c i d a s a q u a n t i t a t i v e

p ro te in p r e c i p i t a n t , The pro te ins were recovered unchanged by applying

the novelcaffeinemethod of Mejbaum-Katzenellenbogen and coworkers. 69-71

Although spinach has a neg l ig ib l e tannin content , w e t e s t e d the e f f e c t

of 18 ca f fe ine content on the H i l l r eac t ion a c t i v i t y of a t y p i c a l spinach

ch loroplas t p repara t ion w i t h the r e s u l t s shown i n Table V I I .

Although t h e r e was a decrease i n the H i l l r eac t ion a c t i v i t y of both

whole ch lo rop la s t s and fragments, w e do not consider 1% ca f fe ine a s a

photosynthet ic i n h i b i t o r . Stimulation of H i l l r eac t ion a c t i v i t y was

reported by MacDo~a11'~ f o r another c l a s s of a lka lo ids , e.g. , brucine and

s t rychnine. Caffeine should be tes ted a s a H i l l r eac t ion s t imula tor ,

using a photosynthet ic s y s t e m high i n t annin content . In addi t ion , the

enhancement of H i l l r eac t ion a c t i v i t y by s t rychnine should be confirmed.

Information of t h i s na ture would be he lpfu l i n extending the b io log ica l

h a l f - l i f e of the H i l l r eac t ion system.

In search f o r highly a c t i v e chloroplast preparat ions, w e turned t o

a i f f e r e n i i a l cei i t r i fagat isn sc) effectively demonstrated by Gross, Becker,

and Shefner73'76 as an i d e a l method for separa t ing ch loroplas t fragments.

In addi t ion , w e used t h e i r method of d i s rup t ing t h e ch lo rop la s t s by

insonat ion. The r e su l t s of two experiments a r e summarized i n

Table V I I I .

21

Table V I 1 1

EFI.XCT OF INSONATION AND D1FFEREXL"TAL CENTRIFUGATION ON

H I L L REACTION ACTIVITY

F rac t ion

Nonsonified

Sonif i e d

CF1-2 0

CF2 o s

CF2 0- 5 0

CF5 Os

Expt. 1

&02

Ch 1

m g h l

0.430 --- 1.18

0.193 --- ---

Expt. 2

C h l

&02

--- 25 0

5 60 --- 43 7

299

mg /m 1

--- 0.613

0.948 --- 0.906

0.121

The da ta suggest t h a t the smaller p a r t i c l e s i n the supernatant

s o l u t i o n have higher H i l l r eac t ion a c t i v i t y than the l a r g e r p a r t i c l e s

sedimented a t 20,000 x g. We have previously reported t h a t

t h e Q e' v a r i e s a s an inverse funot ion.of the ch lorophyl l concent ra t ion du r ing the H i l l r eac t ion run. Thus, t h e low a c t i v i t y of

CF1-,, may r e su l t from the use of two- t o f ive- fo ld amounts of chlorophyl l .

The a c t i v i t y of CFl,20 was almost equal t o t h a t of CF20-50, according t o

the resu l t s of Experiment 2. The crude mixture of son i f i ed ch loroplas t

fragments, as w e l l as the supernatant s o l u t i o n of CF20-50, showed less

a c t i v i t y . Under condi t ions previously used t o prepare ch loroplas t

fragments, t he re would be low concentrat ions of ch lorophyl l and

i n s i g n i f i c a n t H i l l r eac t ion a c t i v i t y i n supernatant so lu t ions . In

genera l , t hese r e s u l t s confirm t h e repor t of Becker, Shefner, and

Gross t h a t sonic d i s rup t ion of ch loroplas t s f a c i l i t a t e s f r ac t iona t ion

i n t o p a r t i c l e s whose a c t i v i t y v a r i e s w i t h sedimentation r a t e .

Gross and coworkers had observed t h e g r e a t e s t H i l l r e ac t ion

a c t i v i t y i n the CFzo-so f r a c t i o n , I n subsequent experiments, w e have

22

c a l l e d it the P20-50 f r a c t i o n .

w i l l be descr ibed below.

I ts use i n e l ec t rophore t i c s t u d i e s

During t h e prepara t ion of chloroplast fragments, the so luble

f r a c t i o n i n the supernatant so lu t ion i s discarded a s t h e components a r e

not required f o r t he pho to ly t i c processes of photosynthesis. An

experiment was performed t o i s o l a t e a l l t h e nondialyzable components i n

the so lub le f r a c t i o n f o r the production of a n t i s e r a .

The r a t i o n a l e f o r studying soluble components is based on conclusions

of Arnon and W h a t l e ~ ~ ~ and numerous repor t s descr ib ing t h e successfu l

i n h i b i t i o n of enzymes by s p e c i f i c a n t i s e r a , i . e . , u s e of anti enzyme^.^^^'*-*^

Af ter confirming the importance of ch lor ide ion a s a so luble co fac to r

of the H i l l r eac t ion , Arnon and W h a t l e ~ ~ ~ sought o the r

f a c t o r s . Their r e s u l t s suggested an unavoidable l a rge degree of de t e r io r -

a t i o n during t h e r e l a t i v e l y long period of high speed cen t r i fuga t ion

when the cytoplasmic f l u i d and ch loroplas t fragments were combined.

I t soluble" co-

Without exception, a l l ava i l ab le information on the nature of t h e

components a c t i v e i n the H i l l r eac t ion emphasizes the i r p a r t i c u l a t e

s t r u c t u r e . The s p e c i f i c H i l l r eac t ion enzymes a r e a t tached t o p a r t i c l e s

and a r e not unbound, so luble substances. I n con t r a s t , many enzymes

involved i n t h e

so lub le p ro te ins .

hypothesis t h a t numerous degradat ive enzymes can be found among t h e

so luble components, e .g . , phosphatases,s8 proteases ,82 and c h l ~ r o p h y l a s e . ~ ~

When t h e i n t a c t p l an t leaf i s f i r s t fragmented i n the Waring blender,

a l l t h e so lub le components a r e brought i n t o in t imate contact with the

p a r t i c u l a t e ch loroplas t fragments, exposing them t o undesirable e f f e c t s ,

11 dark" r eac t ions have been charac te r ized a s t y p i c a l ,

(See d iscuss ions by Losada and Arnon.8') I t is our

The e f f e c t of small amounts of rabbit S8i -Z wzs studied. Noy?nal

r a b b i t serum (NRS) and immune r a b b i t serum (IRS) d i l u t e d 1 : l O O i n t h e

0.025 M T r i s b u f f e r (pH 6.97) had a neg l ig ib l e e f f e c t on the H i l l

r eac t ion a c t i v i t y . However, when both NRS and I R S were d i l u t e d 5:100,

~ G L I V I L Y -*'--: &-- ---- w a s d=rAaAA,,,J - f i n * + ~ l ~ ~ o---. r r r e n t e r . The ch loroplas t fragment preparat ions

were r e f r i g e r a t e d overnight a t 4'C and t e s t e d for a c t i v i t y approximately

23

24 and sometimes 48 h r a f t e r p repa ra t ion , The a c t i v i t y was s l i g h t l y

g r e a t e r i n preparat ions containing sera . Immune se ra showed a s l i g h t

advantage over normal s e r a . In each experiment, a con t ro l preparat ion

was made using T r i s bu f fe r a lone. Surpris ingly, s to red con t ro l pre-

para t ions were a l s o a c t i v e .

These r e su l t s suggest t h a t enhancement might occur when any moder-

a t e l y high molecular weight s o l u t e is present when t h e H i l l r eac t ion

components a r e re leased from the leaf s t r u c t u r e . Some s t u d i e s were

performed i n 0.4% ge la t in -Tr i s bu f fe r and 1% dextran-Tris bu f fe r .

Although the a c t i v i t y i n the g e l a t i n so lu t ion showed enhancement a t 4

h r , t he re was a considerable decrease i n a c t i v i t y a t 24 h r . The dextran

so lu t ion was higher i n i n i t i a l a c t i v i t y than the con t ro l and a l s o showed

improved a c t i v i t y a f t e r one day of s torage.

Normal horse s e r u m (NHS), when present i n the bu f fe r a t a d i l u t i o n

of 5:100, enhanced t h e a c t i v i t y more than d id NRS. Immunodiffusion

ana lys i s of IRS aga ins t the immunizing an t igen from t h e ch loroplas t

fragment supernatant s o l u t i o n indicated a t l e a s t two p r e c i p i t i n a r c s on

micro-Ouchterlony p l a t e s . This was confirmed by immunoelectrophoresis

of t h e supernatant s o l u t i o n , I t is possible t h a t t h e NHS used i n

these s t u d i e s conta ins an t ibodies against p l an t an t igens and t h a t i t s

enhancing a c t i v i t y is s i m i l a r t o tha t of IRS.

I n an experiment using 5:lOO IRS, the i n i t i a l a c t i v i t y of QEhl 450 u 2

increased t o QChl 1215 one day a f t e r s torage a t 4OC. One of the 0, -

problems of t h e immunochemical approach is t h e l a rge quant i ty of IRS

requi red f o r ch loroplas t fragment preparat ion. The minimum volume of

so lvent i n the Warning blendor is approximately 50 m l .

3 m l of serum is use6 io prepare e c o l l o i d a l bu f fe r with enhancing

a c t i v i t y . The da ta for s e v e r a l experiments a r e given i n Table I X .

Hence, a t l e a s t

The r e su l t s of sus ta ined H i l l r eac t ion a c t i v i t y a r e encouraging.

Since i t appears t h a t c o l l o i d a l so lu t ions of ch loroplas t fragments may

withstand considerable manipulation, prepara t ive eiectrophcresis will

be app l i ed t o the more i n t e r e s t i n g , highly a c t i v e c e n t r i f u g a l f r a c t i o n s

24

rl ' E e-. " 1 v

3 1 2 1 8 1 1 3 N * I N I N I I N

I . I . I I .

d

2160"

h c d N -

N I I I i m ~ m ( 0 1 I I I m r l m N I I I i m v v )

d

80" cp

N I I I I N O ~ ( 0 1 I I i r l r n r l N I I I I d N v

m i P - i t - i i i v 1 0 1 r n 1 1 1 N l d l d l l l

I - I - I N I I I I N N I N I I I I m m i m i i i i

ala,- o r l 1 0 1 1 1 1 v r l i m i i i ~ m m l N l I I I

P

3 0 2 rl

m

B ? 0

rl

3 0 0

In TI

; Y

0 u

such a s CF20-50.

w i t h c o l l o i d a l enhancing agents i n the e l e c t r o l y t e .

I t should be possible t o perform t h e e lec t rophores i s

The s tud ie s of var ious methods and substances f o r s o l u b i l i z a t i o n

and p ro tec t ion of t h e H i l l r e ac t ion s y s t e m were not g rea t ly rewarding.

In general , the following conclusions were drawn:

1. Freezing proved a much more s a t i s f a c t o r y method of s to r ing

a c t i v e ch loroplas t fragments than lyoph i l i za t ion .

2. When used for so lub i l i za t ion , d i g i t o n i n lowered Hill r eac t ion

a c t i v i t y immediately, and the fragments appeared t o r e t a i n t h i s lower

l e v e l of a c t i v i t y when s to red frozen.

3. A 10% s o l u t i o n of DMSO w a s found t o have no inh ib i tory a c t i o n

on ch loroplas t a c t i v i t y and might be use fu l i n f u t u r e s t u d i e s .

4. When 1% c a f f e i n e was included i n the ex t r ac t ing medium, H i l l

r eac t ion a c t i v i t y diminished.

5. With t he exception of dextran, c o l l o i d s such a s r abb i t and

horse serum and g e l a t i n produced a pro tec t ive ac t ion , e spec ia l ly when

the ch loroplas t p repara t ion was s tored a t 4OC f o r a few days.

B. E lec t rophore t i c Frac t iona t ion

One of t h e simplest methods of separat ing ch loroplas t preparat ions

i n t o f r a c t i o n s is by sedimentation, which can be performed rap id ly

enough t o permit t he study of H i l l r eac t ion a c t i v i t y i n the f r a c t i o n s .

Other b iophys ica l procedures, however, are so t i m e consuming t h a t most

of t h e a c t i v i t y i s l o s t during f r ac t iona t ion .

W e have gained considerable experience i n using high vol tage

prepara t ive e l ec t rophores i s on t h e c a r r i e r - f r e e instrument, the

Elphor FF, designed by Hannig. I n cont ras t t o c u r t a i n e lec t rophores i s

a t 30 w a t t s , ch loroplas t preparat ions can be s tud ied easily on a

prepara t ive s c a l e and under high power condi t ions of about 300 wat t s ,

Table X is a summary of operat ing conditions and t h e r e s u l t i n g spread

of pigmented ch loroplas t ma te r i a l s i n t o viirifi i is fracticzs cf the

r ece ive r assembly.

26

:EL4 E 0 k

n- € 0 2 O w

m c, rl

c , . n o x z w

I n I n I n I n I n I n I n m I n m m I n I n I n I n N N N N N N N N N N N 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 . . . . . . . . . . . . . . . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

I n I n I n I n I n I n m I n I n I n I n I n I n I n I n

N N N N N b N N N N N N b b N . . . . . . . . . . . . . . .

0 0 0 0 0 0 0 0 0 0 0 0 0

l - l r l N N * m m m I n * * * I n I n I n . . . . . . . . . . . . .

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

a m o a m I n I n o m I n g g o I n m I n o * m

27

Whole ch lo rop la s t s ( P I ) agglu t ina ted spontaneously i n the separa t ion

chamber during e l ec t rophores i s . Since t h i s phenomenon was r a r e l y

observed w i t h ch loroplas t fragments (Pz ), preparat ions of them were s tudied

i n d e t a i l .

I n genera l , most of the p ro te ins i n the mixtures migrated toward

t h e anode i n the pH 7.5 buf fer . The suspensions gave green p a t t e r n s

t h a t broadened a s much a s 23 c m from t h e i r i n l e t po in ts (Experiments 4

and 5 ) .

formed c h a r a c t e r i s t i c s t reaked pa t te rns . Ef f luent was uniformly

d ispersed green suspension t h a t gave t y p i c a l f luorescence when i r r a d i a t e d

with 365 ny, l i g h t .

During the residence per iod i n the chamber, t h e suspensions

With t h e exception of Experiments 2, 3, and 4, a l l of t h e experiments

were performed with ch loroplas t fragments (Pz ). was s tudied i n Experiment 2 , and t h e smaller d ig i ton in fragments PD,

were used i n Experiment 3. The corresponding supernatant so lu t ion SD,

was a l s o s tud ied ; it gave the very broad pa t t e rn under the condi t ions

of Experiment 4.

Digi tonin f r a c t i o n PD,

Experiments 6 and 7 show t h e e f f e c t of var ied bu f fe r flow rate. A t

t h e high flow r a t e , the residence time i n the e l e c t r i c f i e l d i s shor t e r .

Consequently , there is less reso lu t ion (spreading ), but t h e e f f l u e n t

con ta ins a higher concentrat ion of chloroplast fragments, Experiments

7 and 8 can be compared a s a measure of t h e e f f ec t iveness of e lec t rophores i s ,

Experiment 8 (no power) showed a maximum spread of f i v e f r a c t i o n s

c o l l e c t e d i n a d i r e c t l i n e below t h e i n l e t por t , whereas Experiment 7

(38 volts/cm f o r 42 minutes) produced a broad pa t t e rn of 15 f r a c t i o n s

skewed f a r enough toward t h e anode t o be c l e a r of any components w i t h

negligible ~chi1itx.r .r > had ---- they exis ted . In Experiment 9, t he increased

rate of sample dosing broadened t h e pa t t e rn s l i g h t l y .

An o l d sample of ch lo rop la s t s ex t rac ted with T r i s and s to red a t

4OC fo r 11 days w a s s tud ied i n Experiment 10-12.

s a t i s f a c t o r y separa t ion was obtaineci w i i i i chloroplast f r z c t i n m prepared w i t h

I n con t r a s t , no

28

t he sucrose-phosphate bu f fe r and s tored a t 4OC f o r 14 days before e l ec t ro -

phoresis i n the Elphor FF.

A t a bu f fe r flow r a t e of 153 ml/hr/48 tubes, the e f f l u e n t i s

co l l ec t ed a t 3.2 ml/hr/tube. A t yp ica l sample dosing r a t e of 6.7 ml/hr

produces f r a c t i o n s containing chloroplast ma te r i a l t h a t is fou r t o e igh t

t i m e s a s d i l u t e a s t he dosing so lu t ion . Consequently, some method of

concentrat ing t h e e f f l u e n t was comtemplated. However, i t w i l l be

shown t h a t concent ra t ion was not necessary.

Based on t h e preliminary s t u d i e s of f r e s h and aged ch loroplas t

preparat ions i n the Elphor FF, the u l t imate experiment was performed.

I t combined the t y p i c a l prepara t ion of ch loroplas t fragments, followed

by prepara t ive e lec t rophores i s i n the Elphor FF, and terminat ing i n

the manometric determination of H i l l r e ac t ion a c t i v i t y i n the

e l ec t rophore t i ca l ly separated e f f luen t f r a c t i o n s . We would l i k e t o

emphasize t h a t i t was c r u c i a l t o ca re fu l ly place a p l a s t i c bag containing

ice over t h e p l a s t i c tubing from t h e dosing pump t o t h e separa t ing

chamber. The pigment was spread over a range of 7 cm i n t h e 48 f r a c t i o n s ,

from f r a c t i o n 27 through 34. The green e f f l u e n t fmm the Elphor FF

subsequently f luoresced c h a r a c t e r i s t i c a l l y red when i r r a d i a t e d with

l i g h t of wave length 365 9.

of chlorophyl l a and b, and t h e H i l l r eac t ion a c t i v i t y i n se l ec t ed

Elphor f r a c t i o n s a r e summarized i n Table X I .

The concentrat ion of t o t a l chlorophyl l ,

A second experiment involving an Elphor separa t ion and H i l l

r eac t ion determination was performed on an independent preparat ion of

ch loroplas t fragments. A s l i g h t l y higher e l e c t r o l y t e , pH 7.49, was

used. The condi t ions i n the Elphor FF separa t ion were e s s e n t i a l l y the

same, with the exception t h a t t h e v o l t s w e r e 21W; zorssqdsntly, t he

ch loroplas t fragments were subjected t o 42 v/cm f o r about 18 min a t 336

wat t s . Data f o r t he second experiment a r e shown i n Table X I .

The concent ra t ion of chlorophyll ( a + b ) i n each of t h e e f f l u e n t

f r a c t i o n s from the Elphor was approximately four t o f i v e t i m e s a s

d i l u t e a s t h e concentrat ion i n the dosing s o l u t i o n (usua l ly 1 .5 mg

29

T a b l e XI

Ch loro phy 11 ( mg /ml ) Fraction Total '

C h l a C h l b A p p r o x . Precise

CHLOROPHYLL CONCENTRATIONS AND H I L L REACTION A C T I V I T I E S OF

ELECTROPHORETICALLY SEPARATED CHLOROPLAST FRAGMENTS

Q?l

I pH 7.05

C o n t r o l

~

0.280

0.343 1 0.278

0.287 1-40 r 0.211

0*334 I 0*252 0.270 0.202 0.068 712

C o n t r o l

27

28

29

30

31

1.60

0.330

0.440

0.410

0.294

0.267

0.076

0.082

pH 7.49

1.39

0.291

0.389

0.368

0.263

0.238

0.910

0.183

0.245

0.233

0.153

0.148

0.480

0.108

0.144

0.135

0.110

0.090

327

42 4

5 67

382

43 3

472

30

chlorophyll/ml).

use the e f f l u e n t d i r e c t l y f o r t he manometric measurement of H i l l

r e a c t i o n a c t i v i t y without a concentrat ion s t e p . A l l of t h e f r a c t i o n s

showed H i l l r eac t ion a c t i v i t y . The middle f r a c t i o n of pigmented e f f l u e n t

had a lower H i l l r eac t ion a c t i v i t y than f r a c t i o n s ad jacent t o i t . T h i s

e f f e c t was not iceable i n both experiments.

Even a t t h i s d i l u t e concentrat ion, i t was possible t o

Some separa t ion of crude components occurred i n our experiments.

For example, e l ec t rophore t i ca l ly d iver ted ch loroplas t fragments exhib i ted

h igher s p e c i f i c a c t i v i t y than d id unseparated fragments. The reproduci-

b i l i t y of lower a c t i v i t y of f r a c t i o n s from the middle region i n the

e l ec t rophore t i c p a t t e r n 1s in t e rp re t ed t o ind ica t e a chemically f r a g i l e

f r a c t i o n or one lacking components i n optimal proport ions. On the

o the r hand, it should be noted t h a t chloroplast fragments bear ing

ch lorophyl l a were not resolved from those containing chlorophyl l b.

There a r e seve ra l u s e f u l f ea tu re s about t he Elphor separa t ion .

Whole ch loroplas t s , ch loroplas t fragments, and even so lub le components

can be s tudied conveniently, a t l o w temperatures, under n e u t r a l pH,

and on a prepara t ive s c a l e . Moderately concentrated suspensions may

be s tudied , i .e. , mixtures of chloroplast fragments were appl ied

d i r e c t l y i n t o the Elphor. Some d i l u t i o n was incurred by the separat ion,

bu t it was not necessary t o apply a concentrat ion s t ep , such a s u l t r a -

f i l t r a t i o n o r concent ra t ion-d ia lys i s before t h e e f f l u e n t could be exam-

ined f o r H i l l r eac t ion a c t i v i t y . The experiment can be performed

r e l a t i v e l y rap id ly . Although the chloroplast fragments were i n t h e

e l ec t rophore t i c f i e l d f o r only 18 min, longer res idence per iods a r e

poss ib le . A t the upper l i m i t (about 3 h r ) , the extreme condi t ions may

prove useless aga ins t such f r a g i l e , b io log ica l ly a c t i v e ma te r i a l s w i t h

re la t ive ly s h o r t ha l f - l i ves . I n the Elphor, i t is poss ib ie tu use a

v a r i e t y of buf fer s y s t e m s , s e l ec t ing those t h a t provide a minimum of

an ion ic uncoupling of the type described by Good.84

t a i n i n g o the r p o t e n t i a l s t a b i l i z i n g agents, such a s carbowaxes, dextrans,

and poiyviny ipyrrol laoua -'-.--- U A --- c r w w y u ----n++hln ..-- -- w i t h .. - --- t h e Elphor. Fredericks

and Jagendorf6' r ecen t ly descr ibed an e x t r a c t a b l e p ro te in required i n

Buffers con-

31

t he H i l l r eac t ion i n Anacystis nidulans. Carbowax 4000 or dextran and

calcium ions not only preserved the H i l l r e ac t ion a c t i v i t y but gave

marked s t imula t ion . From our experience, these f a c t o r s do not appear

t o play a r o l e i n the H i l l r eac t ion system of spinach ch loroplas t

fragments because of the notable absence of tannins . However, t he

presence of t he cofac tors and p ro tec t ive agents could be included

during Elphor separa t ions .

U n t i l recent ly , t h e only preparat ive e l ec t rophore t i c method

involved a s o l i d support , such a s a paper cur ta in .$’ Unfortunately,

t he ch lo rop la s t pigment-protein complex has a marked a f f i n i t y f o r

c e l l u l o s e . A l l of our previous attempts t o study the mobil i ty of

ch lo rop la s t fragments by paper s t r i p o r paper c u r t a i n e lec t rophores i s

have been unsuccessful because t h e sample remains a t the poin t of

app l i ca t ion .

Using t h e Elphor FF f o r t h e separa t ion of ch loroplas t fragments

is not without problems, The c r i t i c a l s e n s i t i v i t y of H i l l r eac t ion

c a t a l y s t s t o l i g h t demands t h a t experiments be performed i n dim green

l i g h t . Another problem is encountered i n the method of c o l l e c t i n g

e f f l u e n t from t h e Elphor FF: From 48 small c o l l e c t i n g w e l l s adjacent

t o the chamber, e f f l u e n t is per iodica l ly a sp i r a t ed i n t o l a r g e r

c o l l e c t o r tubes i n an air-cooled receiver . A n i t rogen , r a t h e r than

oxygen, atmosphere would favor f r a c t i o n s with higher H i l l r eac t ion

a c t i v i t y .

The l i t e r a t u r e conta ins some references t o e l ec t rophores i s of

pigment-protein complexes.

descr ibed t h e continuous e l ec t rophore t i c separa t ion of the a l g a l b i l l i -

chromoproteins phycoerythrin and phycocyanin. Two chromoglobulins

conta in ing phosphate groups were included among six or seven components

Svensson and Bra t t s t en6” and l a t e r Brattsten’7

32

e l e c t rophoret i ca l l y resolved. from chloroplast p ro t e ins by S isakyan and

Mslik-Sarkisyan.88

d ig i ton in-ext rac ted ch loroplas t fragment preparat ion, a s e l ec t rophore t i -

c a l l y homogeneous; however, no d e t a i l s were given. A desc r ip t ion of

experimental condi t ions and r e s u l t s was omitted by Heber,” who claimed

t o have achieved H i l l r eac t ion a c t i v i t y of small ch loroplas t fragments

separated by c u r t a i n e lec t rophores i s .

Eversole and WolkenS9 descr ibed ch loroplas t in , a

Gross, Becker, and Shefnersl reasoned t h a t i t might be poss ib le t o

i s o l a t e , from a c e n t r i f u g a l f r a c t i o n of ch lo rop la s t fragments prepared

by osmotic shock, a chemically and s t r u c t u r a l l y pure f r a c t i o n t h a t

might be responsible f o r the high H i l l r eac t ion a c t i v i t y of t he parent

mixture and t h a t would e x h i b i t an even h igher r eac t ion r a t e . The

e l ec t rophore t i c method of was used i n a t tempts a t f u r t h e r

f r a c t i o n a t i o n . However, the volume of t h e f r a c t i o n s withdrawn from the

Kolin ce l l was so small t h a t t he re was i n s u f f i c i e n t ma te r i a l f o r assay

of both t h e chlorophyl l content and H i l l r eac t ion a c t i v i t y . While

the Kolin method may u l t ima te ly prove rap id , convenient, and s e n s i t i v e

f o r q u a l i t a t i v e a n a l y s i s of any biophysical separa t ion , it i s incomparable

with t h e prepara t ive s c a l e a f forded by t h e Elphor FF.

Recently, t h e prepara t ive e l ec t rophore t i c separa t ions of p l an t

p a r t i c u l a t e s by Hannig, Klofat , and Endresg5 came t o our a t t e n t i o n .

Their spinach homogenate gave a much narrower separa t ion p a t t e r n i n the

Elphor than w e have reported even though the i r vol tage and power were

higher . We t e n t a t i v e l y conclude t h a t t h e pH was l o w enough t o

restrict t h e mobil i ty of t he chloroplast fragments. I t was unfortunate

t h a t an attempt was not made t o demonstrate H i l l r e ac t ion a c t i v i t y .

A genera l problem of determining H i l l r eac t ion a c t i v i t y is the

s e n s i t i v i t y of t h e de t ec t ing system. Standard Warburg manometry is a11

extravagant method t o study the c a t a l y t i c a c t i v i t y of photosynthetic

sys tems ; l a rge amounts of t he b io-ca ta lys i s (ch loroplas t fragments

containing chlorophyl ls , e t c . ) a r e required t o demonstrate a c t i v i t y by

oxygen evolut ion. Among the aitei-ijjtiv- ~ i c m r n o t h n d ~ ~ the platinum

33

e lec t rode oxygen d e t e c t o r of Blinks and coworkers, 9 6 y 9 7 as employed

by ForkYs8 o f f e r s many advantages t o improve s e n s i t i v i t y and oxygen

s p e c i f i c t y while conserving the bio-catalyst . Consequently, w e

changed our H i l l r eac t ion ana lys i s t o t h i s method of de tec t ion .

According t o Spikes ,99 H i l l reaction a c t i v i t y is inversely r e l a t e d t o

the i n i t i a l concent ra t ion of oxidant . Resul t s reported by Arnon and

Whatley" s t rongly support t he suggestion t h a t smaller q u a n t i t i e s of

r eac t an t s and c a t a l y s t s should f a c i l i t a t e higher H i l l r eac t ion r a t e s .

Experiment Number

This method of sensing the evolution of oxygen was used i n a

series of experiments i n which the H i l l r eac t ion a c t i v i t y was determined

a s a func t ion of t he volume of chloroplast fragment suspension added a s

the b ioca ta lys t . The r e s u l t s f o r a s e r i e s of preparat ions a r e sumnarized

i n Table XII.

Q c1 Volume of Chloroplast Suspension ( m l )

b

Table XI1

33 9

---

EFFECT OF CHIOROPHYLL CONCENTRATION ON H I L L REACTION ACTIVITY MEASURED

WITH THE POLAROGRAPHIC OXYGEN-SENSING EIECTRODE~

528

3 23

--- 53 6

378

268

t 1.00 I 0.50

15 0

5 62

510

--- I I

0.25

--- 45 6

28 6

715

641

5 24

a Influenced by the quant i ty of b ioca ta lys t added t o the r eac t ion mixture.

b A l l experiments were conducted with a t o t a l volume of so lu t ion of 3.00 m l i n the 15-ml Warburg f l a s k s .

34

In genera l , t h e s lope of t h e curve of H i l l r eac t ion a c t i v i t y i s a

func t ion of ch lorophyl l concentrat ion in the s y s t e m and ind ica t e s

increas ing a c t i v i t y a s t he concentration of b ioca ta lys t approaches

i n f i n i t e d i l u t i o n . Unfortunately, the data a r e too incomplete t o permit

useful eva lua t ion . I n f u t u r e experiments, we plan t o study t h e var ia-

t i o n of a c t i v i t y a t a f ixed l e v e l of chlorophyll concentrat ion, e.p;.,

0.50 or 0.25 mg chlorophyl l per ml and t o study t h e shape of t h e curve

i n g r e a t e r d e t a i l a t lower concentrations of chlorophyl l .

One explanat ion frequent ly offered is t h a t some chloroplas t f rag-

ments sc reen o the r s from receiving adequate i l lumina t ion . Consequently,

the H i l l r eac t ion a c t i v i t y is proport ional t o the e f f i c i ency of exposure

of t he b i o c a t a l y s t . This would be improved a t h igher d i l u t i o n s a t a

s a c r i f i c e of prec is ion . However, there should be a concentrat ion below

which the a c t i v i t y is a l i n e a r funct ion of t h e b ioca ta lys t concentrat ion,

i .e., f r e e of screening in te r fe rence .

Another poss ib l i t y is suggested by the SchGtz ru l e - - i t is necessary

t o quadruple the quan t i ty .o f the enzyme i n order t o double the r a t e of

react ion.100 Assuming t h a t the concerted enzymatic a c t i v i t y i n the H i l l

r eac t ion is d i r e c t l y r e l a t e d t o t h e quant i ty of chlorophyl l , t he r e s u l t s

very c lose ly resemble those t y p i c a l of the Schctz phenomenon. F u r t h e r

s t u d i e s of t h e r ep roduc ib i l i t y of r e s u l t s w i l l h e lp i n the adoption of

t h i s i n t e r p r e t a t i o n .

The s tud ie s with t h e polarographic electrode were performed with

a s i n g l e f l a s k and i t s e lec t rode . It should be poss ib le t o monitor

several f l a s k s simultaneously through a mul t ipo in t recorder.

Since Gross and c o ~ o r k e r s ~ ~ ' ' ~ had charac te r ized a s p e c i f i c cent-

r i f u g a l f r a c t i o n of sonica l ly disrupted ch loroplas t a s having the

g r e a t e s t H i l l r eac t ion a c t i v i t y , w e repeated t h e i r methods of sonica t ion

and cen t r i fuga t ion , The son ica l ly disrupted ch loroplas t fragments were

r e a d i l y separated i n t o th ree f rac t ions : P20--50, and S s 0 . The

s p e c t r a of t h e l a t t e r two f r a c t i o n s iveze obtsinec! in 2quenu.s Tris

buf fe r . There is a d i s t i n c t absorption d i f f e rence between them only

35

i n t h e u l t r a v i o l e t region but not i n t h e v i s i b l e region. These

s p e c t r a l d i f f e rences a r e bes t seen by comparison of t h e overlays i n

the envelope a t t h e back of t h e r epor t , The o r i g i n a l spec t ra were

ad jus ted proport ionately t o a r e l a t i v e absorbance of 1,000 a t 678 mp,

the maximum of t h e chlorophyl l spectrum i n t h i s aqueous medium.

In seve ra l experiments, the P,,-,, f r a c t i o n was separated

e l ec t rophore t i ca l ly i n t h e Elphor FF. The separa t ion was s i m i l a r

t o t h a t of t h e heterogeneous f r a c t i o n c a l l e d ch loroplas t preparat ion.

An important d i f f e rence was t h e spread of maximum chlorophyl l concentra-

t i o n over a 3 cm d i s t ance i n comparison with only 2 c m i n the

heterogeneous pa t t e rn . Preliminary r e s u l t s ind ica ted t h a t t he P,,-,,

f r a c t i o n gave an Elphor separa t ion without the minimum of H i l l r eac t ion

a c t i v i t y so not iceable i n the heterogeneous pa t t e rn . Addit ional s t u d i e s

are required t o c l a r i f y t h i s r e s u l t . I n accordance with the conclusions

of Park and Biggins,lol w e t en t a t ive ly conclude t h a t t h i s re f ined

c e n t r i f u g a l f r a c t i o n may be devoid of some permeabili ty membrane t h a t

restricts H i l l r eac t ion a c t i v i t y of t h e components i n t h e middle of

t he e l ec t rophore t i c p a t t e r n of t he heterogeneous ch loroplas t preparat ion.

Another e f f e c t was observed and is a t t r i b u t e d t o competit ion of

p ro te ins f o r t h e energy i n the e lec t rophore t ic f i e l d , I f previously

separa ted f r a c t i o n s , e.g., Frac t ion 21 and 22, a r e re run through the

Elphor FF under t h e same experimental condi t ions a s they were obtained,

t h e p a t t e r n is e f f e c t i v e l y broadened toward t h e anode, although the

mobi l i ty of components on the cathodic edge of t he pa t t e rn remained

t h e same a s i n the previous separat ion.

An add i t iona l set of overlays fo r t he spectra of t h e e l ec t rophore t i c

frartians of a P,,,,, f r a c t i o n a r e included i n the envelope a t the

back of t he r epor t . They may be compared with each o the r and aga ins t

t he master spectrum of Fig. 2, appended t o the r epor t .

I n conclusion, w e have r ead i ly e s t ab l i shed t h a t ch loroplas t

fragments, produced by niilechanieal and s m i c dis rupt ion , r e t a i n H i l l

r e a c t i o n a c t i v i t y a f t e r prepara t ive e l ec t rophores i s , A s with heterogeneous

36

chloroplas t fragments, a minimum of H i l l a c t i v i t y is assoc ia ted w i t h

chlorophyl l - r ich components i n t h e middle of the e l ec t rophore t i c separa t ion

p a t t e r n ; t h i s i s probably not t h e case i n the highly a c t i v e P,,,,,

f r a c t i o n . The size of the ch loroplas t p a r t i c l e has l i t t l e , i f any,

e f f e c t on e l ec t rophore t i c mobil i ty . Spec t ra l c h a r a c t e r i s t i c s are

almost cons tan t and a r e independent of e l ec t rophore t i c mobil i ty .

A f r u i t f u l a rea f o r research, however, is t h e nature of the

p ro te ins assoc ia ted with the pigments and l i p i d s of t h e photoactive

p a r t i c l e s . The pro te ins can be s tudied i n terms of s t r u c t u r e and

composition a f t e r the pigments and l i p i d s have been removed by extrac-

t i o n with organic so lvents .

quantasome w i t h a molecular weight of 2 x l o6 . l i p i d , including a very complex inventory of components, The o the r

ha l f of t h e quantasome was pro te in . I t seems highly l i k e l y t h a t t he

p ro te in w i l l be an equally complex array of pro te in components.

E lec t rophores i s should a s s i s t i n f r ac t iona t ion s tud ie s .

Park and Biggins , lol have described the

One ha l f of i t was

37

ACKNOWUDGEMENTS

W e thank Luman F. Ney f o r h i s he lp fu l d i scuss ions and B. Berridge,

11Irs. J. G, Cummings, and W . Johnson fo r t h e i r t echn ica l a s s i s t ance .

Biochemist

/ J ,' (2

Janice E. Coomber Biochemist

LKM:JEC :eek

38

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