Clin. Biochem. 13 (i) 62-66 (1980)

Three Automated Quantitative Assays for Serum Proteins N E L L Y B L U M E N K R A N T Z '

U n i v e r s i t y o f C o p e n h a g e n , D e p a r t m e n t o f D e r m a t o l o g y , ( w i t h C o n n e c t i v e T i s s u e R e s e a r c h L a b o r a t o r i e s ) , R i g s - h o s p i t a l , C o p e n h a g e n , D e n m a r k

( A c c e p t e d N o v e m b e r 2, 1 9 7 9 )

Three quantitative assays for detection of proteins are reported. One is an adaptation to the Auto-Analyzer of the widely used method of Lowry et al "k In another procedure, the product of the reaction of proteins with a modified biuret ~z~ reacts with the phosphomolybdic - phosphotungstic reagent of Folin and Ciocalteu TM- The third method, performed on hydrolyzed sample, is based on the measurement of proline (Pro) and the conversion of the Pro content into protein ~4~. The universal presence of proline in serum proteins suggested this assay. The values for total serum proteins as assayed by the three procedures are similar. The second and the third assay for proteins, described in this paper, can also be per- formed manually. Studies on interfering mbstances and their elimination as well as on the sensitivity of the assays are reported.

MATERIALS AND METHODS

Reagents. All chemica ls were ana ly t i ca l grade . Au to -Ana lyze r . A Technicon A u t o - A n a l y z e r (Technicon I n s t r u m e n t s Corpora ton , N.Y.) was used fo r the procedures .

Assay I : A u t o m a t e d Lowry Procedure . The a lka l ine c a r b o n a t e - t a r t r a t e and cupr ic s u l f a t e solut ions as well as the a lka l ine c a r b o n a t e - t a r t r a t e - c u p r i c r e a g e n t were p r e p a r e d as descr ibed by Lowry et al. ~1~. The Fol in and Ciocal teu r e a g e n t ( B r i t i s h Drug Houses Ltd. ) was d i lu ted wi th 3 t imes i ts own volume of dis t i l led wate r .

The a s say was p e r f o r m e d as shown in the f low d i a g r a m (Fig . 1) . Standard. A se rum pro te in s t a n d a r d solut ion ( S i g m a Chem. Corp., St. Louis, Mo.) c o n t a i n i n g 8.0 g % to ta l s e r u m p ro t e in s was used. Di lu t ions c o n t a i n i n g 64 or 128 ~g p ro t e in pe r 0.1 ml sample were used.

Assay II. A u t o m a t e d B l u m e n k r a n t z Procedure . Biurct reagent. The b i u r e t r e a g e n t was p r e p a r e d by

d issolv ing 1.9 g of CuSO4-5H20, 3.85 g of E D T A (e thylene- d i a m i n e t e t r a a c e t a t e d isodium sa l t ) and 8.75 g of glycine (or ~.lanine) in abou t 750 ml of dis t i l led wa te r , a d d i n g 20 g N a O H and comple t ing to 1 l i t re wi th wate r . (All d r u g s were p u r c h a s e d f r o m E. Merck, A.G., D a r m s t a d t ) . The r eagen t , kep t in a p la s t i c bott le , is s t ab le for a t l eas t two m o n t h s a t room t e m p e r a t u r e .

Folin-Ciocalteu reagent ~ . The r e a g e n t ( B r i t i s h D r u g Houses Ltd.) was d i lu ted w i th 2 volumes of dis t i l led water .

Automated assay. The a s s a y was p e r f o r m e d as shown in the f low d i a g r a m (F ig . 2 ) .

Manual assay. 1 ml sample is mixed wi th 2 ml b i u r e t r e a g e n t and, a f t e r 5 minu tes , 0.35 ml of Fol in-Cioea l teu r e a g e n t was added. Absorbances were r ead immedia t e ly a t 620 nm on a Beckm an DU s p e c t r o p h o t o m e t e r a g a i n s t a s t a n d a r d c o n t a i n i n g 64 or 128 ~g p r o t e i n s / m l saline.

Standard. F o r the a u t o m a t e d as well as the m a n u a l a s s a y fo r s e r u m p ro t e in s a s e r u m p ro t e in s t a n d a r d

P r e s e n t a d d r e s s : Maon Olim P i n k u s Ka tzene lenson 63 R a m a t J o s e p h B a t Yam, I s rae l

so lu t ion ( S i g m a Chem. Corp.) was used. The s t a n d a r d solut ion was d i lu ted w i th sa l ine up to a f i na l concen- t r a t i o n of 64 or 128 ~g p ro t e in pe r ml solution. W h e n a lbumin was measured , a s t a n d a r d of h u m a n se rum al- bumin ( S i g m a ) in dist i l led w a t e r was p repa red . In case some o the r p ro t e in was q u a n t i t a t e d , a s t a n d a r d of tl/e same commerc ia l ly ava i l ab le pu r i f i ed p ro t e in was pre- f e r red , a l t h o u g h the above-ment ioned se rum pro te in s t a n d a r d solut ion can a lways be used. F i f t y ~ g / m l of g lycyl-glycyl-glycine (Gly -g ly -g ly ) , which gives a chrom- ogen s imi l a r to t h a t of 128 ~g of s e rum pro te ins , can also be used as s t a n d a r d .

Serum sample. ,Before the assay, s e rum m u s t be d i lu ted 1:1000 wi th sal ine.

Effect of the content of CuS04 in the biuret reagent. The con ten t of CuSO4 was decreased to 1) 1.5 g; 2) 1.33 g ; 3) 0.95 g; 4) 0.475 g, and 5) 0 g pe r l i t re of b i u r e t r e a g e n t . O the r b i u r e t r e agen t s ~'e~ were also t r i ed in a s s a y II.

Effect of glycine in the reagent. In o rder to de ter - mine the o p t i m u m con ten t of glycine, r e a g e n t s were pre- p a r e d w i t h o u t a n d w i th i n c r e a s i n g con ten t s of the men- t ioned amino acid. S u b s t i t u t i o n of glycine by o the r amino acids was also t r ied.

Assay I I I ( A u t o m a t e d Pro l ine P rocedure ) was pe r fo rmed accord ing to B l u m e n k r a n t z and Asboe-Hensen ' s modi f ica- t ion ~4~ of the p rocedure of Trol l and Linds ley ~'~ fo r the detec t ion of prol ine .

Reagent. 2.5 g of n i n h y d r i n was dissolved in a m i x t u r e of 80 ml of g lac ia l acet ic acid and 20 ml of fo rmaldehyde . (All r e a g e n t s were p u r c h a s e d f r o m E. Merck, A.G., D a r m s t a d t ) .

The a u t o m a t e d a s s a y was p e r f o r m e d accord ing to the f low d i a g r a m (Fig . 3). I t can also be p e r f o r m e d man- ua l ly by m i x i n g 1 ml of sample w i th 1 ml of r eagen t , boi l ing fo r 1 m i n u t e or h e a t i n g fo r 4 m inu t e s a t 75°C, cooling, and r e a d i n g the a b s o r b a n c e of the ch romogen which develops a t 515 nm in a Beckman DU spect ro- pho tomete r . A solut ion c o n t a i n i n g 2 E~g pro l ine pe r ml dis t i l led w a t e r was used as s t a n d a r d .

Preparation of samples . Fo r a s says I and II, p ro te in samples were dissolved in sal ine. S e r u m was d i lu ted in sa l ine , 1:100 fo r a s s a y I and 1:1000 for a s say II.

F o r a s s a y I I I , 0.2 ml s e r u m was t r e a t e d wi th 2.5 ml of 0.75 M HC104. The sample was c e n t r i f u g e d a t 3000 r p m fo r 20 m i n u t e s a f t e r s t a n d i n g for 15 m i n u t e s a t room t e m p e r a t u r e . The s u p e r n a t a n t was d i scarded and the pe l le t hydrolyzed in 6 N HC1 a t 118°C fo r 18 hours . The HC1 was e v a p o r a t e d a t 65°C u n d e r vacuum. The e v a p o r a t e d sample was dissolved in dist i l led wate r . S t a n d a r d s of p ro l ine were s u b m i t t e d to the t r e a t m e n t wi th HC1, e v a p o r a t i o n and di lut ion. The d i lu t ion of s e r u m a f t e r hydro lys i s was 1:1000.

Interfering substances. In o rde r to i nves t i ga t e the in- f luence of va r i ous subs t ances on as says I and II, they were p rac t i s ed wi th the r e a g e n t of Lowry et al. '~' or w i th our b i u r e t r e a g e n t wi th and w i t h o u t the phenol r e a g e n t "~' or w i th the phenol r e a g e n t alone.

E l im ina t i on of the i n t e r f e r ence due to subs t ances which can reduce d i rec t ly the phenol r e a g e n t was t r i ed by s h a k i n g the s amples w i th ac t i va t ed charcoa l for 10 min- u te s c e n t r i f u g i n g a t 2000 r p m fo r 5 m inu t e s and u s i n g the s u p e r n a t a n t fo r a s s a y II.

SERUM PROTEIN ASSAYS 63

37"C Dehvery ml lm,n i

~. (0 60 A,r

~ • 2 ~ ) Alkahrm CODD4r Re~

° ' ° " Jo:,!2::::" % COLOR~M E TER

fdle, 735nm

Fig. 1 - - Flow diagram of assay I. Automated assay ]or proteins. Modified Lowry procedure.

OFt-iV(FlY ml Imln

_ IO.6 AIFI

A¢ ~ 0.~4 Pha~Ol

• 2.O WATFR . . . ~ . J

2.39 VII& ~TI~ oouet-( Leuest-['wAs~[ ~ - I [ COl L ~ PROPORTIONATING PUMP

A¢ A(;, Acsdflet QII lubes transpOrt*nO ~ef~DI fllO~llnt

143. Co¢lu| connecto¢ DOUBt.( . . . . I "',--.' l u l I . . . . . . . . . . . . .

CO~.OFIlMETER hirer ~4~ nm

Fig. 2 - - Flow diagram of assay II. Automated assay for proteins with a new biuret reagent and the reagent of Folin and Ciocalteau.

Other proteins. Besides human serum and various al- bumin samples (Tables 1 and 2), the following proteins were assayed: collagenase and urease (Worthington Biochem. Corp., Freehold, N.J . ) ; papain, protamine sul- fa te (Sigma Chem. Corp., St. Louis, Mo) ) and pronase (E. Merck, A.G., Darmstadt) .

Activated charcoal. 200 g of norite (Charcoal active g ranu la r 11/2 ram, E. Merck, A.G., Darmstad t ) were placed in a large flask and mixed with 1 l i tre of 10 per cent HC1. The mixture was heated to boiling and fi l tered with suction. The cake of norite was moved to a large beaker and washed with distilled water until the washing gave negative or fa in t reaction for fer r ic ions. The washed carbon was then dried at 115°C. Af te r cooling, it was ready for use (8)

Interfering substances. D-penic i l lamine , N-ace ty l -D -penic i l lamine , d i t h i o t h r e i t o l ( D T T ) , homocys t ine , p ipeco l i c ac id , h o m o s e r i n e and a s c o r b i c ac id gave fa lse pos i t ive p ro t e in reac t ion . The ment ioned sub- s tances r eac ted d i rec t ly wi th the phenol r eagen t . The i n t e r f e r e nc e was not iceable a t 0.6 ~M concent ra - t ion of the ment ioned subs tances . T r i s ( h y d r o x y - m e t h y l ) a m i n o e t h a n e in concen t r a t ions above 0.005 M gave fa lse p ro t e in reac t ion .

F i f t y ~g per ml of Gly-Gly-Gly gave a chromogen s imi l a r to t h a t c o r r e spond ing to 128 ~g se rum pro- te ins pe r ml.

RESULTS

A s s a y I : A u t o m a t e d L o w r y P r o c e d u r e .

The a s s a y us ing a lbumin or s t a n d a r d se rum pro- t e ins fol lowed L a m b e r t and Bee r ' s law for p ro t e in concen t r a t ions be tween 33 and 128 ~g/0.1 ml ( F i g . 4) .

Effect of temperature. No s i g n i f i c a n t d i f f e r ences in the chromogen ob ta ined w i th a lbumin was not iced w h e t h e r the r eac t ion was al lowed to develop a t 37°C or a t 50°C. A t room t e m p e r a t u r e (23°C) a lower s e n s i t i v i t y was found (not shown) .

TABLE I.

COMPARISON OF THE RESULTS OBTAINED WITH THE THREE AUTOMATED ASSAYS

Solutions in saline of various commercially available albumin samples were prepared. Aliquots of each albumin solution were quantitated with the three automated assays. Results are ex-

pressed as g of protein per litre of solution.

Protein g / l

Assay I Assay II Assay II I

Human serum albumin (Sigma) 51.0 (essentially fatty acid free) Human serum albumin 48.6 Fraction V (Sigma) Human serum albumin (Sigma) 49.7 (crystallized and lyophilized) Bovine serum albumin (Sigma) 56.0 (crystallized and lyophilized) Bovine serum albumin 62.0 Fraction V (Sigma) Bovine serum albumin 53.6 (Armour)

51.4 51.8

49.9 48.9

48.9 48.0

55.2 53.0

61.0 62.8

54.6 54.2

A s s a y I I : A u t o m a t e d B l u m e n k r a n t z P r o c e d u r e .

Assays p e r f o r m e d wi th a lbumin or s t a n d a r d s e r u m - p r o t e i n so lu t i on fo l lowed L a m b e r t a n d B e e r ' s l aw be tween p ro te in concen t ra t ions of 4 to 128 ~ l /ml ( F i g . 5) .

Effec t of glycine in the composition of the biuret reagent. Lower s e n s i t i v i t y of a s say I I was not iced by dec reas ing the concen t ra t ion of g lyc ine in the r eagen t . In the absence of g lyc ine the s ens i t i v i t y of the a s say was 5 7 ± 4 % of t h a t of the a s say w i th a complete r eagen t . When b i u r e t con t a in ing the double amoun t of all cons t i t uen t s was used, the s e n s i t i v i t y was reduced to 5 5 ± 7 % . In the absence of glycine, bu t wi th the t h r e e o the r components in double con- cen t ra t ion , the s e n s i t i v i t y o f a s s a y I I was 1 9 ± 1 % .

Effec t of substitution of glycine by other amino acids in the biuret reagent. No d i f f e r ence in a s say I I was o b s e r v e d when g l y c i n e was s u b s t i t u t e d by a l an ine or leucine. S l igh t ly lower s e n s i t i v i t y was no- t iced when lysine, a rg in ine , pheny la l an ine or g lu t amic acid were used as s u b s t i t u e n t s fo r g lyc ine in the b i u r e t r eagen t .

H y d r o x y - a m i n o a c i d s as h y d r o x y l y s i n e and s e r i n e gave a blue chromogen w i th k max a t 560 nm w i t h biure~ only (wi thou~ Fo l in -Cioca l t eu ' s r e a g e n t ) . I t fol lowed L a m b e r t and Bee r ' s law be tween 0.5 and 8 .8 -mg/ml .

Homocys t ine gave a yel low r e a g e n t which t u r n s blue a f t e r the add i t i on of t he phenol r eagen t .

64 B L U M E N K R A N T Z

TABLE 2.

COMPARISON OF RESULTS OBTAINED WITH THE THREE ASSAYS FOR PROTEINS.

Assay I Assay II Assay II I

mg protein/lO mg ~g Pro/lO mg mg protein/lO mg*

Human serum albumin (Sigma) (essentially fatty acid free) Human serum albumin Fraction V (Sigma) Human serum albumin (Sigma) (crystallized and lyophilized) Bovine serum albumin (Sigma) (crystallized and lyophilized) Bovine serum albumin Fraction V (Sigma) Bovine serum albumin (Armour) Urease Worthington Biochem. Corp.) apain (Sigma)

Pronase (E. Merck, A.G.; Darmstadt). Protamine sulfate (Sigma) (essentially histone-free) Collagenase (Worthington Biochem. Corp.)

7650 7800 382 7640

8400 8200 418 8260

8900 9000 452 9040

10000 9800 489 9780

9900 10000 495 9900

9800 9900 479 9580

4700 4600 228 4560

9200 8800 389 7780 8900 10000 460 9200

7600 7800 584 11680

9600 10000 248 4960

*Converted by multiplying ~g Pro by the factor 20.

Ef fec t of decreasing content of CuSO, in the biuret reagent. When the conten t of CuSO, was decreased or omi t ted , the s e n s i t i v i t y of a s s a y I I was accord ing ly decreased.

Ef fec t of d i f ferent concentrations of NaOH used instead of the biuret reagent. When 0.5; 0.25 or 0.125 N N a O H was used in s t ead of the b iu r e t r eagen t , the s e n s i t i v i t y of the N a O H phenol ( c o r r e s p o n d i n g to b iu re t -pheno l ) was 19---0.8%; 18+-0.8% and 14+-0.1% of t h a t g iven by a s s a y I I . The reac t ion s t i l l fol lowed the L a m b e r t and Bee r ' s law. The peak of m a x i m u m abso rp t ion of the chromogen ob ta ined was h igh ly de- penden t on the a lkal i concen t ra t ion in the b i u r e t r eagen t . A t h i g h e r a lka l in i ty , the peak of h -max was 530 nm a t lower a lka l i n i t y h - m a x was 735 nm.

Ef fec t of the alkalinity of the biuret reagent on the sensi t ivi ty of assay II. When the n o r m a l i t y of N a O H in our b i u r e t r e a g e n t was reduced to 0.25 or 0.125 N, the s e n s i t i v i t y of a s say I I d ropped to 50+-0.5% and 17+-0.7%.

When, in a s s a y I I , our b i u r e t r e a g e n t was sub-

DELIVERY ml Im,n

HEC=TING BATH (7 TURNS) 75"C ~ • 0 4 2 A I R

- 1 19 R E A G E N T [

_ 1 SINGLE ~ 2.0 WATER COIL •

COLORI M J TIeR h l t t r 5 2 5 nm

Fig. 3 ~ Flow diagram of assay tl i . Automated assay ]or protein by determination of the proline content of a hydrolyzed sample.

s t i t u t e d by t h a t r e p o r t e d by Ya tz id i s ~' c o n t a i n i n g the double a m o u n t of the same componen t s plus NaC1, the s e n s i t i v i t y was 55+--7% lower t han t h a t of a s s a y I I .

The b i u r e t used by Pe l ley et al (s) c on t a in ing CuS04, E D T A and N a O H was also t r i ed in a s s a y II . The s e n s i t i v i t y ob ta ined was 17+-0.7% of t h a t of our a s s a y I I .

Interfer ing substances. The same as i nd i ca t ed fo r a s say I.

0.7

0.6

0.5

0.4

k,. 0.3

.,J 0.2

q.

0.1

A S S A Y |

P R O T E I N S T A N D A R D CURVE

,,.c AA 2 3 °C

• ° . ° .

6 4 192 3 2 0 4 4 8 6 4 0 pg P R O T E I N

Fig. 4 - - Assay I. S tandard curve for proteins.

. * =

SERUM PROTEIN ASSAYS 65

TABLE 3.

SERUM PROTEINS COMPARISON OF THE THREE AUTOMATED ASSAYS

Assay

Sample No. I II mg Pro/1 III

Serum proteins Serum proteins* g/1 g/1 g/1

Human serum 42 66 4- 4 67 =t= 3 3340 ± 258 66.8 ± 5

*Converted from Pro values by the factor 20.

TABLE 4.

CONTENT OF PROLINE IN THREE DIFFERENT SAMPLES OF COLLAGEN

RESULTS ARE EXPRESSED As ttG/10 MG COLLAGEN

Proline % Pro per sample Factor

Calf skin collagen (Sigma) 2223 22.23 4.618 Calf skin collagen (Sigma) 2085 20.85 Calf tendon collagen (Sigma) 2187 21.87 Average 2165 21.65 ± 0.58

I f the samples are shaken for 10 minutes with activated charcoal and the supernatant af ter centri- fugation used for assay II, the interference of as- corbic acid, DTT, glutathion, cysteine, NADH, D-peni- cillamine and N-acetyl-D-penicillamine is eliminated (Not shown). After this treatment, there is a con- sistent decrease of 14% in the chromogen obtained with assay II.

Assay I I I : Automated Proline Procedure.

This reaction follows Lambert and Beer's law at concentrations of Pro between 1 and 10 ~g/ml (Fig. 6). The content of Pro in the hydrolyzed pellet (HCI04 precipitate of serum) expressed as mg Pro per 1000 ml serum multiplied by the fac tor 20 gives the protein content as g/1 of serum. Content of proline in three different samples of collagen is shown in Table 4.

I n t e r f e r i n g s u b s t a n c e s . High content of salts de- creased the intensity of the chromogen developed. By using a s tandard of Pro in a solution containing the same salt concentration as the test solution, this

problem can be overcome. Under these conditions, although the sensitivity of the reaction will be de- creased, it will still follow Lambert and Beer's law.

Comparison of the results obtained with the three assays for different proteins are presented in Tables 1-3.

DISCUSSION

The automation of the most widely used method for quantitative determination of proteins, i.e. the Lowry method (') , gives the same sensitivity and is liable to the same as that of the heated biuret-Folin-Ciocalteu assay. Under the conditions reported herein the procedure follows Lambert and Beer 's law and has the general advantage of automation, viz. rapidity and higher reproducibility. The sensitivity o f this auto- mation of the Lowry procedure is comparable to that of the continuous flow versions of the same procedure as reported by Gaunce and D ' Io r i#" , Huemer and Lee "°' and Klosse et al. (m, although the present flow diagram is simpler.

Assay I I combines the specificity of a biuret re- action with the sensitivity of the phenol reaction of Folin and Ciocalteu '3). The principle of assay II is the same as that of the heated biuret-Folin-Ciocalteau protein assay of Dorsey et alY'. The difference is that with a different composition of the biuret reagent the latter requires heating of the sample and biuret reagent at 100°C for 100 minutes, s t irr ing for 15 minutes at 10°C after addition of the phenol reagent and standing for one hour before reading the ab- sorbances. As indicated by Dorsey et al. 's' one of the inconveniences of their assay (reported as a manual procedure) is that instant results cannot be obtained. By using a different biuret reagent, which reacts almost instantaneously with proteins we have ob- tained a very quick and easy procedure for quantita- tive detection of proteins. The interference of thiols and ascorbic acid can easily be avoided by previous shaking with activated charcoal. The assay can be used as manual or automated with identical qualities. Pelley et al. '5), in a recent paper, presented a b i u r e t method for the detection of proteins in the presence of thiols. However, the sensitivity of their assay is quite low.

07

O(

0."

0,4

i °' ~.o2

ASSAY 2

PROTEIN STANOARD CURVE

12.8 256 34~.4 51.2 6 4 0 78.8 89.6 102.4 115.2 1280 PROTEIN

Fig. 5 - - A s s a y I1. S t a n d a r d curve /or prote ins .

0 7

06

05,

O4

0.3

~ 0~

OI

0

ASSAY 3

l/l/lit VII/I V V V V V V / VVvVvv""

PROLINE STANOARO CURVE

~g PROLINE

Fig. 6 - - A s s a y II1. S t a n d a r d curve f o r proline.

66 BLUMENKRANTZ

As the interference of thiols with the method of Lowry et al. c~ as well as with our assay I I is mainly due l:o their capability to reduce the phenol reagent of Folin and Ciocalteu, the use of only biuret (no phenol) by Pelley et al ~5~ does not really solve the men- tioned problem, besides the low sensitivity for proteins. Under the present conditions, assay I follows Lambert and Beer's law, while in the original procedure of Lowry et al '" the quantity of protein is read from a graph constructed from the optical density readings for known amounts of the protein assayed. The sensitivity of assay II is higher than that of assay I wiCh the advantage for the former of having the Cu s+ containing reagent (biuret) ready for use and stable for two months, while for the latter, the alkaline re- agent has to be combined with CuS04 at the moment of the reaction.

The serum-protein standard has the advantage over albumin standard of containing albumin as well as globulins. This standard is preferable, specially when determining total proteins in serum. Bovine serum albumin is widely used as standard in quantitative assays of proteins, i.e. those of Lowry et al (1) and Yatzidis '2), and Pelley et al (5). Using albumin as standard when determining serum proteins we noticed lower values than those of a standard of serum proteins of the same concentration. As a consequence, and considering the presence of albumin, globulins etc. in serum, we adopted serum proteins as standard. Klosse et al (") reported that the chromogen produced by albumin with the Lowry reaction was 80% of that of globulin.

Butcher and Lowry "~) reported on an assay for quantitation of proteins based on the determination of the glutamate content in their acid or alkali hydro- lysates. The assay is based on the relatively high content of glutamate in most proteins. The same consideration led us to use the proline content for the quantitation of serum proteins. The content of Pro is quite constant, and we found a factor to convert the value of Pro into that of serum protein. The assay is simple and gives similar values for serum proteins as those obtained with the other two assays presented. In case of purified collagen, a factor of 4.6 should be used to convert Pro into collagen. Due to the different content of Pro in the proteins analyzed, a common factor for the conversion of Pro into protein obviously cannot be found. This is valid also for the procedure of Butcher and Lowry "2~, which is more complicated than ours.

I f so desired, assays II and I I I presented in this paper can be used as manual procedures.

The three procedures can be used for quantitation of proteins in milk. Procedures I and II are success- fully used to determine proteins in serum, in cerebro- spinal fluid and in urine, in the latter after intro- duction of counter current analyses, as suggested by Klosse et al "~) to eliminate low molecular weight interfering substances. Procedure I shares with the procedures of Gaunce and D'Iorio c°), Huemer and

Lee '°,, and Klosse et al. (m the disadvantage of the original Lowry procedure (~), i.e. the intensity of the developed color varies with different proteins. This disadvantage is not overcome by procedure II.

SUMMARY

Three automated procedures for quantitative deter- ruination of proteins are presented. Assay I is a modified automation of the procedure of Lowry et al '" which, in the conditions presented herein, has the linearity which is lacking in the original procedure. Assay II combines a new quick biuret reagent and the phenol reagent of Folin and Ciocalteu. I t gives practically instantaneous quantitation of proteins. By assay I I I the proline content can be determined and converted into protein by the use of the factor 20. The three assays give comparable results for total serum and solutions of purified albumin of different sources. As for some enzymes and protamine sulfate, assays I and II gave similar results while, due to their different content of proline, the results with assay I I I differ in some. Assays I and II can be used to determine proteins in cerebrospinal fluid and in urine, in the latter af ter introduction of counter current dialysis.

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