PHYSICOCHEMIICAL STEM1 - Plant physiology · the different solutions with such salts as K4Fe(CN)6 and AgNO3 also are recorded. The method adopted for determining the reactivity of

CERTAIN PHYSICOCHEMIICAL PROPERTIES OF PINEAPPLESTEM1 COLLOIDS*

C. P. SIDERIS

(WITH EIGHT FIGURES)

The investigations reported in this paper were conducted with the pur-pose of obtaining certain information on the behavior of pineapple planttissue colloids in health and disease. Some of this information is reportedin another publication by the writer (7).

It was found (6) about two years ago that pineapple plants grown inwater and soil cultures of different H-ion concentrations thrive best at pHvalues between 4.5 and 6.5. These findinge suggested that the inhibitoryeffect of H-ions above or below the values mentioned may have had someinfluence on the physicocheinical properties of the pineapple tissue colloids.As such studies could not have been conducted very satisfactorily with theliving tissues, it was thought best to obtain the fluid colloids and studythem in ritro.

Methods of experimentationPineapple stems, from which the leaves, the extreme basal portion, and

part of the exterior tissues of the base had been removed, were ground in ameat grinder. The pulpy mass was then placed in a fruit press and thefluids extracted by pressure. The extract was filtered through filter-paperfor the removal of particles of plant tissue. The product thus obtained wascentrifuged for further purification by means of a Sharples centrifuge, andthen subjected to the treatments, as outlined later, for the separation of thedifferent colloidal fractions. The reagents used for these treatments werein certain cases 0.1 normal NaOH and HCl, in others 0.1 normal NaOHand HNO3, and 0.1 normal ammonium hydroxide and acetic acid. The pHof the resulting solution never went above 10.0 nor below 2.0.

The procedure followed for the treatment of the different colloid frae-tions may be illustrated by the following outline and diagram.

OUTLINE1. Extracted pineapple fluids.2. Treatment of pineapple fluids with NH4OH. Formation of a pre-

cipitate (P-2) and filtrate (F-2).* Technical paper no. 4 of the Experiment Station of the Association of Hawaiian

Pineapple Canners, University of Hawaii.

309

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310 PL.ANT PHYSIOLOGY

3. Treatment of precipitate (P-2) with HNO3. Formation of a pre-cipitate (P-3) and filtrate (F-3).

4. Treatment of filtrate (F-2) with acetic acid. Formation of pre-cipitate (P-4) and filtrate (F-4).

5. Treatment of filtrate (F-3) with NaOH. Formation of precipitate(P-5) and filtrate (F-5).

6. Treatment of filtrate (F-4) with NaOH. Formation of a precipi-tate (P-6) and filtrate (F-6).

7. Treatment of filtrate (F-5) with HNO3. Formation of a precipitate(P-7) and a filtrate (F-7).

8. Treatment of filtrate (F-6) with HN03. Formation of a precipitate(P-8) and filtrate (F-8).

DIAGRAMPineapple fluids~1Fluids + NH4OH

Precipitate (P-2)+HNO3 Filtrate (F-2 )+acetic acid

Precipitate (P-3) Filtrate (F-3)+NaOH Precipitate (P-4) Filtrate (F-4)+NaOH

Precipitate (P-5) Filtrate (F-5)+HNO3 Precipitate (P-6) Filtrate (F-6) +HNO3

Precipitate (P-7) Filtrate (F-7) Precipitate (P-8) Filtrate (F-8)

Qualitative chemical tests of different fractionsThe different fractions were analyzed qualitatively with the following

results:TABLE I

QUALITATIVE TESTS ON THIE STEM COLLOIDS

DIFFERENT COLLOID FRACTIONSTESTS

P-3 P-4 P-5 P-6 P-7 P-8

Biuret.±.... + ++ +- 1- +

Sulphur.- - --.+ +

Xanthoproteic +.. + - + +

Molisch.- - + + - -



SIDERIS PINEAPPLE STEM COLLOIDS l1

The six different fractions may be placed in three groups as the resultof their chemical behavior. P-3 and P-A represent one and the same pro-tein; P-7 and P-8 likewise, represent another protein, and P-5 and P-6represent a carbohydrate.

Physicochemical properties of the different fractionsThe different fractions were purified as thoroughly as possible by

dialysis through parchment paper. Folded parchment paper cones con-taining the different colloidal fractions were immersed in four-liter beakersof distilled water. The water was changed three times a day. A piece ofthymol, about the size of a pea, was kept in the water to prevent biologicalaction. The electrical resistance of the dialysate was determined every

TABLE IIPHYSICOCHEIICAL BEIIAVIOR, OF FRACTION P-3 IN ACIDS AND ALKALIES

REACTIONREAGENT

SAM1- 24 HOURS TuR-PLE -- A AFTER BID- AgNO3 KFe(CN). REMARKS

BER 0.1 N. 0.1 N. STERI- ITYHNO,, NaOH TRA- LIZA-

MENT TION

CC. CC. pH pH1 5.0 2.25 2.30 _ _ - Soluble2 4.4 2.40 2.60 - _ _3 3.8 2.50 2.65 _ _ _4 3.2 2.70 2.85 + _ -H Insoluble5 2.6 2.80 3.20 ++ _ ++6 2.0 3.15 4.10 HI-I ++ i-7 1.4 4.10 4.65 +11 - +8 0.8 5.00 4.85 H-- - - Insoluble

(isoelectricpoint)

9 0.45 5.80 4.85 -11H-A - - Insoluble10 0.22 6.20 4.85 -H-H- + _11 0 0 6.47 5.10 1+1 + -

12 0.3 6.65 5.50 -I--H- ++13 0.7 6.70 5.60 A-A--I- ++-H-14 1.3 6.90 6.05 ++ -HI-115 1.9 7.15 6.20 ++ l-H- -16 2.5 7.30 6.60 + +H-If17 3.1 7.50 7.00 - - - Soluble18 3.7 7.80 7.50 - - -19 4.3 8.10 7.80 - - -20 4.9 8.60 8.00 - - -



312 PLANT PHYSIOLOGY

time the solution was changed. After this value had risen from 200 to15,000 ohms, the nitrate content from the added HN03 of the dialysate,was also determined. It was found that when an electrical resistance ofabout 20,000 ohms had been reached, the nitrate content of the dialysatewas considerably less than one part per million. At this stage the differentfractions were removed from the bags and studied for their physicochemicalbehavior.

Each fraction was made up to a certain volume with distilled water.This was acted upon, then, by a mechanical stirrer, in order to disperseevenly through the solution, the different sizes of the colloidal particles,

TABLE IIIPHYSICOCHEMICAL BEHAVIOR OF FRACTION P-4 IN- ACIDS AND ALKALIES

SAM- REAGENTPLE -NUM- 0.1 N. 0.1 N.BER HNO, NaOH

CC. CC.

1 5.02 4.53 4.04 3.55 3.06 2.57 2.08 1.59 1.010 0.511 0.2512 0.125

Check 0 014 0.125

1516171819202122232425

0.250.51.01.52.02.53.03.54.04.55.0

REACTION

p112.102.152.202.252.352.402.452.803.203.804.404.604.654.90

4.955.105.305.405.756.006.156.356.506.656.75

TURBID-ITY

++

+1

++

+

AgN3,

++

++

++

-H-Fl

K4Fe (CN\)6 REMARKS

Soluble

_±]- InsolubleI++

+4+ft

+ Insoluble(isoelectric

point)_ Insoluble

l- I11Soluble



SIDERIS-PINEAPPLE STEM COLLOIDS 313

before the solution was distributed into a number of Erlenmeyer flasks.Each flask received 100 cc. of the colloidal suspension drawn off by meansof a pipette. The different flasks received, in addition to the colloidalsuspension, a definite volume of 0.1 normal HNO3 or NaOH. The numberof cubic centimeters of the acid or alkali solution together with the pHvalue and turbidity that were produced are reported in tables II, III, IV,V and VI, and in fig. 7. In tables II and III and fig. 8, the reactivity ofthe different solutions with such salts as K4Fe(CN)6 and AgNO3 also arerecorded. The method adopted for determining the reactivity of thesesalts with protein P-3 and P-4 was as follows: Protein solutions, about25 cc. of each, were taken into a dark room and there treated with 5 cc. of1 per cent. concentration of either salt. The mixture was left to standover night and was then filtered through hardened filter-paper. Theresidue was collected and placed in a solution having the same pH as thatof the original solution. The intensity of the color of the precipitate servedas an index of its reactivity. It may be added that before removing theprecipitate from the filter-paper it is necessary to wash it with distilledwater repeatedly until all traces of the unaffected salt have been removed.Equally good results were obtained when protein suspensions of different

TABLE IVPHYSICOCHEMIICAL BEHAVIOR OF FRACTION P-7 IN ACIDS AND ALKALIES

--

REAGENTSAM1PLENUMBER 0.1 N. -REACTION TURBIDITY REMARKS

HNO3 NaOH

C.- CeC. pH4.03.53.02.52.01.51.00.50.250.1250

2.63 - Soluble2.672.702.883.00 + Insoluble3.13 +3.33 XH-4.16 ++i-5.32 -H-1-6.45 +-H Insoluble (isoelectric point)

0 7.30 +++--0.125 8.65 ++0.25 9.45 +0.5 9.90 - Soluble1.0 10.42 -1.5 10.58 -

2.0 11.00 -

1

34

6789

1011121314151617

I I .

. 11



314 PLANT PHYSIOLOGY

hydrogen-ion concentrations were treated with the above salts and left indarkness over night without subjeeting the resulting solution to filtration.The intensity of the color that develops in the solutions after they have beenexposed to the light may serve, in this case also, as an index of the reactivityof the salts and protein.

TABLE VPHYSICOCHEMICAL BEHAVIOR OF FRACTION P-8 IN ACIDS AND ALKALIES

REAGENT

NUMBER 0.1 N. 0.1 N. pH TURBIDITY REMARKSHN03 NaOH

_ _ ----

CC. C.1 5.0 2.00 SSoluble2 4.5 2.10 -3 4.0 2.35 _4 3.5 [ 2.455 3.0 2.55 + Insoluble6 2.5 2.75 +7 2.0 3.10 --+8 1.5 3.50 ++9 1.0 3.90 +4410 0.5 5.35++v11 0.25 6.65 ++++ Insoluble (isoelectric point)12 0.125 7.20 +13 0. 0 7.50 l,14 0.125 7.9515 0.25 8.75 -16 0.5 9.82 - Soluble17 1.0 10.3718 1.5 10.8519 2.0 11.00 _

Discussion of resultsThe results presented in tables II, III, IV, V and VI indicate that the

difference in the physicochemical behavior of protein colloids and carbo-hydrate colloids is quite pronounced. The former react amphotericallyand have a definite isoelectric point, whereas the latter do not. Differentproteins may have different isoelectric points as is the case with fractionsP-3 and P-4, on the one hand, and P-7 and P-8 on the other.

The amphoteric behavior of the two proteins is due, according to LOEB(3) and others, to their component reactive groups, namely, the amino(NH,) and carboxyl (COOH) radicals, the former behaving as a cation,and the latter as an anion. In the case of the carbohydrate colloids, where



SIDERIS PINEAPPLE STEM COLLOIDS

usually only one reactive group occurs, that is, the carboxyl radical, thebehavior can not be amphoteric but points only in one direction. Theexperimental data are in complete agreement, therefore, with the theoreticalexpectations.

TABLE VIBEHAVIOR OF SAMPLES P-5 AND P-6 IN ACIDS AND ALKALIES

REAGENTSAMPLE _REACTION ITURBIDITYNUMBER O.1 N. 0.1 N.

HNO3 NaOH

CC. CC.pH1 12.0 10.0 2.302 8.0 2.50 +3 6.0 2.90 +4 4.0 5.30 -t5 2.0 6.70 ++6 1.0 7.27 0.5 7.3 1H1 -8 0.0 0.0 7.4 -H1-H-9 0.5 7.6 -H1H-

10 1.0 7.711 2.0 7.912 4.0 8.4 +13 6.0 8.514 8.0 8.6 by15 10.0 8.8 1±1-

During the different studies both the protein and carbohydrate colloidswere subjected to various treatments. One hundred cc. of suspensions ofprotein P-3 and P-3 of pH values above and below the isoelectric point weretreated with 10 cc. of 0.1 molal NaOH and HCL. The water of the suspen-sion was evaporated at room temperature and the residue containing NaClcrystals and protein was examined under the microscope. The examinationproved that at pH values above the isoelectric point of the protein theprotein occupied part of the central portion of the crystal, wrhereas thearrangement was reversed at pH values below the isoelectric point, as shownby figs. 1, 9 and 3. At the isoelectric point the protein forms transparentthread-like structures, which, under the microscoj)e, are transpareut flakeswithout anv definite arrangement.

When the carbohydrate colloids, of fractions P-5 and P-6, dissolved ina beaker or other container, were treated at intervals with drops of 0.1normal NaOH, it was found that certain membranous spherical or ringbodies are formed, as shown in fig.. 4. These bodies are hollow inside; that

31.5



PLANT PHYSIOLOGY

R.~~ ~~~~~om

~~~~~~~at..........a~~~~~

FIG. 3. Sodium chloride crystals in pineapple protein (P-7) suspension at pH values

sa. e. bu like alrubberballoon fomsa membranous shspelli. at thicness

of the membrane of such bodies may vary from 0.5 to 0.05 mm. They mayremain in the solution from a few seconds to many minutes depending on

the acidity or alkalinity of the solution. If the solution is too acid theirduration is short; if, however, it is but slightly acid or close to neutral theymay last for many minutes and possibly hours. Their formation may beexplained as follows: The COOH radical of this colloid fraction beingchemically highly reactive is able to enter into a chemical combination withsubstances having an opposite electric charge and to form salts which may

or may not be soluble. In this particular case the COGH reacting withNaGH according to the equation,-RC'OOH ± NaOH -*> RCOONNa ±~-11,0,-forms a certain insoluble salt. Whether the reaction, as stated, representsthe actual conditions is not known. Qualitative tests of the carbohydratecolloid gave a positive test for calcium. The presence of calcium suggeststhat the substance may be a calcium pectate or some other similar salt.Such a salt when acted upon by an acid or a base may behave as follows:

(1) (RCOO) 2Ca + 211N ica>2RCOOH +oCa (NOi ) 2.(2) 2RCOOri + Ca (uNiO n3 2+ 2NaOH --* (RCOO) ACa 2NaNO, + 2120,

316



SIDERIS-PINEAPPLE STEM COLLOIDS

These reactions represent the conditions that might possibly develop withthe different treatments. The equations are merely illustrative, and thebalancing should not be interpreted as indicative of precise knowledge ofthe reactions.

The spherical or doughnut shaped bodies that are formed, when thiscarbohydrate colloid is treated with NaOH, may be the insoluble salt ofcalcium pectate or some other similar substance. The physicochemical con-ditions that enter into their formation may be explained as follows: TheCOOH radical upon coming in contact with the NaOH of the solution formswater while at the same time the H-ion is replaced by Na or Ca. With thereplacement of the H-ion by Ca the membrane of the body or calciumpectate salt is formed. The water that is being formed is apparently sur-

FIG. 4. Carbohydrate colloid bodies of spherical and ring shapes formed by adding dropsof 0.1 N. or 1.0 N. NaOH in an acid suspension of the colloids. The bodies are

membranous, containing in their cavities w ater and some salts.

rounded by the membrane waith the Na or Ca ions projecting into it. Theinert part of the colloid substance or R occupying the exterior surface ofthe membrane projects into the exterior acid solution. With conditionslike these it is possible to have the membrane lasting in the acid solutionfrom a few minutes to a few hours. The decomposition or dissolution ofthe membrane may be brought about by the interplay of osmotic forces.The H-ions that are present in the outside solution penetrate the membraneeither slowly or rapidly depending on their concentration. Their penetra-tion into the interior of the spherical body may reverse the reaction, that is,the H-ion may replace the Ca or Na ions and reestablish the original condi-tion as follows: (COO)2Ca + 2HNO3-> 2COOH1 Ca (NOQ).2

317



PLANT PHYSIOLOGY

According to these observations, then, the structure of a carbohydratecolloid body, such as the one under discussion, may be imagined as a hollowsphere the shell of which is formed by aggregated micellae held in closep-'oximity by electrostatic forces as represented by the sketch in fig. 5.Membranous bodies of this type have been observed to form in differentsizes, varying between 10 and 0.5 mm.

(S)4 ADSORPTION Z1 0 N E

,,'MICELLAE ')X

(DIOS&IONS RI O

S OLUTION1

COOH X

v-<EXTERIOR SOLUTION

FIG. 5. Pattern of a carbohydrate colloidal particle.

The question now arises: Is it possible to have similar bodies formed withproteins at pH values above or below their isoelectric points? Assumingthat isoelectric protein is like a collapsed rubber balloon or a flat membrane,is it not possible for such a membrane to assume a spherical or other similarform when suspended in solutions of a higher or lower pH from that of theisoelectric point of the protein? By analogy, one would expect in the caseof the formation of spherical bodies, the COOH group to project inward atpH values above the isoelectric point and outward at pH values below thispoint. The position of the NH, group must be then, exactly the reverseof that of the COOH group, as in fig. 6. Although the work of previousinvestigators favors this view considerably, no one has ever been able todemonstrate that such a structure occurs in proteins. MEYER (4) has ob-served with starch and water the formation of a net structure made updistinctly of globules. HARDY (2) believes that concentrated gelatin jelliesconsist of drops of water suspended in a gelatin-rich phase. LOEB'S (3)work on the swelling of proteins, suggests that proteins must undergo acertain modification in their isoelectric structure to make them hold watervery tenaciously. From a theoretical consideration the formation of suchmembranous structures is possible only at p11 values slightly above or belowthe isoelectric point of the particular protein. At considerably higher con-centrations of H+ or 011- ions the electrostatic forces that keep the indi-

318




NllH .vigICELLAE

CO ~~~~~~~~~~~~~~~OH10NS (INTE R IOP) )1tCoos~~~~~~~~~SOLUTI00'

a., XTERIOR SOLUTION

FIG. 6. Pattern of a protein particle.

vidual micellae together in a membrane-like structure are destroyed. Thedevelopment of this condition is due to the increase of the electrical poten-tial of the micellae. These, acted upon by high concentrations of H+ or OH-

'4- 7. b~ &64 b O 5. C iS..'.:

__... ...

FIG. 7. Pineapple proteiii (P-7) in solution of different pH values. At 6.4, theisoelectric point, the protein precipitated completely; slightly at pH 6.8, 6.0, and 7.9;remained tuihid at a.6, 5.1, 4.6, 4.0, and 7.4, but dissolved completely at 7.8, 8.4,8 .4and 3.0.

ions, become highly electrified, which condition makes them repel each otherand disperse themselves thoroughly in the solution.

The phenomenon of dispersion and precipitation has been studied quiteextensively by LOEB (3), NORTHROP (5), FAURE-FREMIET and 'NICHITA (1)

319



PLANT PHYSIOLOGY

FIG. 8. Pineapple protein (P-7) treated with K4Fe(CN)6 and AgNOa. It re-

acted well, and developed a yellowish green color at pH 5.6 and 6.2, which did not pho-tograph well; at 3.1 it was green. With AgNO, it reacted well, as showa at pH6.7-8.5.

and others. These investigators found that the stability of colloidal lar-ticles or bacteria in a suspension depends on the electrical potential of thesolution. When this potential reaches a very low figure the particles or

bacteria, as the case may be, become precipitated.Further work on the structure of protein as well as carbohydrate colloids

is necessary in order to obtain a clearer picture of their physical andchemical behavior.

SummaryThe results obtained by the investigations reported in this paper indicate

that it is possible to separate mixtures of colloids by making use of theirisoelectric or critical precipitation point. Both carbohydrate and proteincolloids may be separated in this way.

The writer expresses an opinion about the structure of colloidal particleswhich may be represented by a membranous body of spherical or othershape, the interior space of which contains water, with the reacting radicalof the colloid and other ions in solution.

Pineapple stem protein has been obtained in an imperfectly crystallineform. The crystalline structure is flake-like, transparent, and macroscopi-cally fibrous. The isoelectric point of this protein is at pH 6.4.

The writer wishes to express his appreciation to Dr. A. L. DEAN for

reading the manuscript, for his constructive criticisms, and many helpfulsuggestions.

EXPERIMENT STrATION, Assoc. OF HAWAIIAN PINEAPPLE CANNERS,

UNIVERSITY OF HAWAII, HONOLULU.

:3 20




LITERATURE CITED1. FAURE-FREMIET, E., and NICHITA, G. Charge electrique et agglutination

chez les ambiocytes d'invertebres marins. Ann. Physiol. et dePhysicochimie Biol. 3: 247-306. 1927.

2. HARDY, W. B. Uber den Mechanismus der Erstarrung in umkehrbarenKolloidsystemen. Zeitschr. physikal. Chemie. 33: 326-343. 1900.

3. LOEB, JACQUES. Proteins and the theory of colloidal behavior. Mc-Graw-Hill. 2d ed., 1924.

4. _MEYER, A. Beitrage zur Kenntnis der Gallerten, besonder der Starke-gallerten. Kolloidehemische Beih. 5: 1-48. 1913.

5. NORTHROP, JOHN H., and DE KRUIF, PAUL H. The stability of bacterialsuspensions. II. The agglutination of the Bacillus of rabbit septi-cemia and of Bacillus typhosus by electrolytes. Jour. Gen. Physiol.4: 639-654. 1922.

6. SIDERIS, CHRISTOS P. The effect of H-ion concentration on the growthof pineapple plants. Exp. Sta. Assoc. Hawaiian Pineapple Can-ners, University of Hawaii, Bull. no. 4. 1925.

7. . Similarity between physicochemical and biological reac-tions. Plant Physiol. 3: 79-83. 1928.

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Documents

PHYSICOCHEMIICAL STEM1 - Plant physiology · the different solutions with such salts as K4Fe(CN)6 and AgNO3 also are recorded. The method adopted for determining the reactivity of