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A convenient modification of the method for hydroxyproline determination in proteins
ANALYTICAL
BIOCHEMISTRY
85,
550-555 (1978)
A Convenient Modification of the Method for Hydroxyproline Determination in Proteins W. GAEASI~KI,
ANNA GADEK, ANNA RATKIEWICZ,* AND W. RZECZYCKI*
Departments of General and Biochemistry,
Chemistry Medical
and *Biochemistry, Academy, 15-230
Institute Bialystok,
of Physiology Poland
Received March 8, 1977; accepted August 26, 1977 A modification of the method of hydroxyproline determination in proteins was devised. The modification consists of the hydrolysis of proteins in 72% perchloric acid of 100°C for 2 hr instead of 20 hr, autoclaving in 6 N HCl or 2 N Ba(OH)*. Determination of hydroxyproline by the modified method does not require any additional chromatographic purification, standardizes conditions of the assay, and increases the yield in a number of routine assays.
Most of the analytical methods for hydroxyproline determination are based on the method of Lang (l), Neumann and Logan (2), and Stegemann (3). Several modifications of this method have been developed (4-8), but they can be used only with relatively pure solutions. A quantitative method which can be applied for hydroxyproline determination in proteins was described by Prockop and Udenfriend (9). This method involves conversion of hydroxyproline to pyrrole, extraction into toluene, and reaction of pyrrole with Ehrlich’s reagent to yield red chromophore. The oxidation of hydroxyproline is performed in the presence of an excess of alanine to prevent the influence of the amino acids or similar substances on the yield of pyrrole. According to the method of Prockop and Udenfriend, the proteins placed in sealed tubes are hydrolyzed with a half volume of concentrated hydrochloric acid by autoclaving at 124°C for 1524 hr. In a method modified by Le Roy et al. (lo), the hydrolysis of proteins in a saturated solution of Ba(OH), by autoclaving at 124°C for 16 hr is also performed. An inconvenience of these two procedures lies in the length of time of the hydrolysis and the necessity of using the autoclave for that period. If the hydrolysis is carried out in a thermostat, the sealed tubes are very often broken. This is particularly unfortunate if, for example, the preparation of the sample for hydroxyproline determination is complicated by procedures that involve isolation, then separation with use chromatography or electrophoresis. Finding of a more reproducible and simple procedure for hydroxyproline determination in proteins was the objective of this work. Perchloric acid 0003-2697/78/0852-0550$02.00/O Copyright Q 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
550
HYDROXYPROLINE
DETERMINATION
IN PROTEINS
551
(PCA), 72% was satisfactorily used for DNA hydrolysis (11). DNA incubated with PCA is split into its four main bases with no distinct influence on the structures of the bases. We employed PCA reagent in the hydrolysis of hydroxyproline-containing proteins with good results. MATERIALS
AND METHODS
Materials
Plasma globulins were obtained from the defibrinated bovine plasma as described previously (12); p-dimethylaminebenzaldehyde (Sigma) for preparation of Ehrlich’s reagent was freshly recrystallized from ethanol. L-Hydroxyproline and L-alanine were from Calbiochem, Dowex 50-W was from Sigma, and other reagents were obtained from POCH Gliwice, Poland. The Hydrolysis
of Proteins
I. Prockop and Udenfriend’s method (9). About 8 mg of dried collagen was placed in a tube, and 4 ml of 6 N HCl were added; the tube was then sealed and autoclaved at 120°C for 16 hr. The hydrolysate was neutralized by the addition of 6 N NaOH with the use of phenolphthaleine as an indicator. The volume was made up to 200 ml with water in a volumetric flask. Two milliliters of this solution was made up to 8 ml with water, and the hydroxyproline contents were assayed by the method of Prockop and Udenfriend (9). 2. Le Roy et al.‘s method (IO). In this method 35-70 mg of plasma proteins were placed in a tube, then 2 ml of distilled water and 4 ml of a saturated solution of Ba(OH)* were added. The tube was sealed and autoclaved at 120°C for 16 hr. The hydrolysate was neutralized with 6 N H,SO, with the use phenolphthalein as an indicator. To the neutral solution, 0.2 ml of 6 N HCl were added, and the BaSO, precipitate was removed by centrifugation at 1OOOgfor 10 min. The supernatant solution was purified on a Dowex 50-W column (1 x 5 cm) equilibrated with 1 N HCl. The purified fraction of hydroxyproline eluted with 20 ml of 1 N HCl was neutralized with 1 N NaOH to pH 8.5 using phenolphthalein as an indicator. It was then concentrated to a volume of 8 ml, and hydroxyproline was assayed by the method of Prockop and Udenfriend (9). 3. The modified method. About 8 mg of dried collagen or 35 mg of dried plasma proteins (for instance, fibrinogen) were placed in a small tube (2-3 ml in volume), then 0.25 ml of 72% PCA was added. The tube was sealed with stopper and heated at 100°C for 2 hr. The hydrolysate was dark brown, but it did not influence the successive steps of hydroxyproline determination. To the hydrolysate, 2 ml of water were added, and this mixture was neutralized with 6 N NaOH using phenolphthalein as an indicator. Next, the hydrolysate was quantitatively transferred to a SO-ml centrifuge tube,
552
GAEASIfiSKI
ET
AL.
and the volume was made up to 8 ml with water. The hydroxyproline contents were assayed by the method of Prockop and Udenfriend (9) with some modifications which standardized the determination procedure. Complete determination can be carried out in a single centrifuge tube without transfer. It was also established that suitable saturations can be made if 3.5 g of KC1 are added before the oxidation step and 2 g of KC1 are added at the moment when the oxidation is stopped by the addition of 3.6 M sodium thiosulfate. To the mixture, 10 ml of toluene were added, then the tube was stoppered and shaken vigorously for 2 min in an electricshaker, instead of shaking by hand 100 times. The mixture was centrifuged at 3000 r-pm for 5 min, and the toluene was discarded. The tube was then capped and heated in a boiling-water bath for 30 min, after which the mixture was cooled and 10 ml of toluene were added, and the tube was stoppered, shaken in electric shaker, and centrifuged as above. Two parallel 2.5-ml portions of the toluene phase were placed in separate tubes, 1 ml of Ehrlich’s reagent was added with stirring, and after 15 min, E&&, was read on a Beckman-Du spectrophotometer using toluene as a blank. The contents of hydroxyproline were calculated from the standard curve or from the parallel assay of the hydroxyproline standard sample. RESULTS
In the preliminary experiments, the effect of hydrolysis conditions with PCA on the yielding capacity of hydroxyproline was studied. The results presented in Table 1 show no losses of added hydroxyproline and no increases in hydroxyproline content after the addition of proline to the sample. The following experiments examined the influence of interfering material in the method of Le Roy ef al. (10) and in our modified method. From Fig. 1 we can see that after hydrolysis of fibrinogen in a Ba(OH), solution (IO), absorbance at 450 nm is much higher in the sample assayed without ion exchange purification, then in that purified on the column. This is in agreement with Le Roy et al.‘8 results (10). After the hydrolysis of fibrinogen in PCA, no differences in absorbances at 450 nm were observed. Contrary to Le Roy ef al.‘s results (lo), the spectra of total plasma globulin PCA hydrolysates passed through the cation exchanger, then eluted with 1 and 3 N HCl showed very low levels of interfering material, and a second peak of absorbance at 560 nm was not observed. The results of hydroxyproline determinations in collagen and gelatin performed after hydrolysis in 6 N HCl or in PCA were quite similar when obtained either by Prockop’s and Udenfiiend’s method (collagen, 7.64 + 0.7%; gelatin, 9.00 ? 0.25%) or by the modified method (collagen, 7.60 + 0.3%; gelatin, 8.76 -C 0.7%). Our results, obtained by the modified method with or without use of the column, are similar to those obtained by Le Roy et af.‘s method with column purification. The hydroxyproline contents in fibrinogen hydro-
HYDROXYPROLINE
DETERMINATION TABLE
THE EFFECT
Expt
OF ADDING HYDROXYPROLINE OF HYDROXYPROLINE
Weight of sample bg)
Protein
1
OR PROLINE TO THE SAMPLE DETERMINED IN PROTEINS
Total hydroxyproline in sample (/a)
Added to examined protein
2 3 4 5
Collagen Collagen Collagen Collagen Collagen
8 8 8 8 8
Hydroxyproline Hydroxyproline Hydroxyproline Hydroxyproline
(500 (500 (500 (500
1 2 3 4
Fibrinogen Fibrinogen Fibrinogen Fibrinogen
35 35 35 35
Hydroxyproline Hydroxyproline Hydroxyproline
(5 kg) (5 pg) (5 pg)
1 2
Fibrinogen Fibrinogen
35 35
Proline (1 mg)
1
553
IN PROTEINS
Yield of added hydroxyproline 043)
(%)
570 530 480 510
110 105 96 102
8.9 13.8 13.6 13.8
-
98 94 98
9.9 9.6
-
670 1240 1200 1150 1180
pg) pg) pg) pg)
ON THE YIELD
4.9 4.7 4.9
-
lyzed in PCA were 22.80 -+ 1.85% (without column) and 21.90 k 1.83% (with column) and in Ba(OH), were 26.20% + 1.3% (without column) and 22.00 + 1.3% (with column). Similar results were also obtained by Le Roy et al.‘s method (without column, 31.20 + 1.2%; with column, 28.80 OD
-
BOG
wth
-
Ea(OH)2
without
cdumn column
-
PCA
with
o-+
PCA
wlthout
column column
FIG. 1. The spectra of fibrinogen hydrolysates in Ba(OH), and in PCA, with and without purification on the cation exchanger.
554
GAILASIfiSKI
ET AL.
+ 3.0%) and the modified method (without column, 28.8 + 1.4%) for plasma globulins.
column,
29.2 + 1.75%; with
DISCUSSION
The findings presented in this paper show the modified method described here can be used for hydroxyproline determination in proteins with quite good results. The advantages of this modification are the much shorter protein hydrolysis time and the fact that the autoclave is not required. Heating sealed tubes in a thermostat at 124°C instead the use of an autoclave, causes tube breakage. Moreover, the transfer of hydrolysate from tube to tube creates the possibility of losses. The purification of the hydrolysate on the ion exchange column chromatography is not necessary in our modified method. The higher standardization of the hydroxyproline assays makes this method quite reproducible. The performance of the total assay procedure in one centrifuge tube and shaking in an electric shaker for 2 min instead by hand 100 times makes this method much more efficient. Many samples can be assayed in parallel; thus, using the modified method, 10 samples can be assayed in 6 hr, whereas only one sample could be assayed in 4 hr before. As can be seen from the results presented, the reproducibilities of the modified method and other methods (9,lO) are similar. No losses of hydroxyproline during the hydrolysis with PCA and no excess hydroxyproline content after addition of proline to the sample in the assay conditions were noted. The results presented in Fig. 1 show that absorption spectra of chromophores from hydroxyproline determined in proteins by the PCA method are similar with and without purification on cation exchange resin. But in the Ba(OH), method (IO), OD,,, ,,,,, of the sample measured without purification is much higher in unpurified material. This means the interfering material is separated during hydrolysis with PCA. In Prockop’s and Udenfriend’s method the hydroxyproline is oxidized in the presence of an excess of L-alanine in order to prevent the influence of varying amounts of amino acids or similar substances on the pyrrole yield. We tried to replace L-alanine by other substances. We have obtained results with the use l -aminocaproic acid (EACA) similar to those obtained with use of L-alanine. The solution of EACA should be 40%, w/v, and 0.25 ml of this solution should be added. In conclusion we consider the modified method to be useful for a large number of samples in routine protein hydroxyproline assays. REFERENCES 1. Lang, K. (1933) Hoppe-Seyler’s Z. Physiol. Chern. 219, 148. 2. Neumann, R. E., and Logan, M. A. (1950) J. Bid. Chem. 184, 299. 3. Stegemann, H. (1958) Z. Physiol. Chem. 311, 41.
HYDROXYPROLINE
DETERMINATION
555
IN PROTEINS
4. Martin, C. J., and Axelrod, A. E. (1953) Proc. Sot. Exp. Biol. Med. 83, 461. 5. Grunbaum, B. W., and Glick, D. (1956) Arch. Biochem. Biophys. 65, 260. 6. Miyada, D. S., and Tappel, A. L. (1956) Anal. Chem. 28, 909. 7. Fels, G. (1958) C/in. Chem. 4, 62. 8. Hutterer, F., and Singer, E. J. (1960) Anal. Chem. 32, 556. 9. Prockop, D. J., and Udenfriend, S. (1960) Anal. Biochem. 1, 228. 10. Le Roy, E. Carwile, Kaplan, A.,Udenfriend, S., and Sjeerdsma, A. (1964) J. Biol. Chem.
239, 3350.
11. Wyatt, G. R. (1951) Biochem. J. 48, 584. 12. Bahkowski, E., GaIasidski, W., and Rzeczycki, W. (1970) FEBS
I&t.
6, 19.
BIOCHEMISTRY
85,
550-555 (1978)
A Convenient Modification of the Method for Hydroxyproline Determination in Proteins W. GAEASI~KI,
ANNA GADEK, ANNA RATKIEWICZ,* AND W. RZECZYCKI*
Departments of General and Biochemistry,
Chemistry Medical
and *Biochemistry, Academy, 15-230
Institute Bialystok,
of Physiology Poland
Received March 8, 1977; accepted August 26, 1977 A modification of the method of hydroxyproline determination in proteins was devised. The modification consists of the hydrolysis of proteins in 72% perchloric acid of 100°C for 2 hr instead of 20 hr, autoclaving in 6 N HCl or 2 N Ba(OH)*. Determination of hydroxyproline by the modified method does not require any additional chromatographic purification, standardizes conditions of the assay, and increases the yield in a number of routine assays.
Most of the analytical methods for hydroxyproline determination are based on the method of Lang (l), Neumann and Logan (2), and Stegemann (3). Several modifications of this method have been developed (4-8), but they can be used only with relatively pure solutions. A quantitative method which can be applied for hydroxyproline determination in proteins was described by Prockop and Udenfriend (9). This method involves conversion of hydroxyproline to pyrrole, extraction into toluene, and reaction of pyrrole with Ehrlich’s reagent to yield red chromophore. The oxidation of hydroxyproline is performed in the presence of an excess of alanine to prevent the influence of the amino acids or similar substances on the yield of pyrrole. According to the method of Prockop and Udenfriend, the proteins placed in sealed tubes are hydrolyzed with a half volume of concentrated hydrochloric acid by autoclaving at 124°C for 1524 hr. In a method modified by Le Roy et al. (lo), the hydrolysis of proteins in a saturated solution of Ba(OH), by autoclaving at 124°C for 16 hr is also performed. An inconvenience of these two procedures lies in the length of time of the hydrolysis and the necessity of using the autoclave for that period. If the hydrolysis is carried out in a thermostat, the sealed tubes are very often broken. This is particularly unfortunate if, for example, the preparation of the sample for hydroxyproline determination is complicated by procedures that involve isolation, then separation with use chromatography or electrophoresis. Finding of a more reproducible and simple procedure for hydroxyproline determination in proteins was the objective of this work. Perchloric acid 0003-2697/78/0852-0550$02.00/O Copyright Q 1978 by Academic Press. Inc. All rights of reproduction in any form reserved.
550
HYDROXYPROLINE
DETERMINATION
IN PROTEINS
551
(PCA), 72% was satisfactorily used for DNA hydrolysis (11). DNA incubated with PCA is split into its four main bases with no distinct influence on the structures of the bases. We employed PCA reagent in the hydrolysis of hydroxyproline-containing proteins with good results. MATERIALS
AND METHODS
Materials
Plasma globulins were obtained from the defibrinated bovine plasma as described previously (12); p-dimethylaminebenzaldehyde (Sigma) for preparation of Ehrlich’s reagent was freshly recrystallized from ethanol. L-Hydroxyproline and L-alanine were from Calbiochem, Dowex 50-W was from Sigma, and other reagents were obtained from POCH Gliwice, Poland. The Hydrolysis
of Proteins
I. Prockop and Udenfriend’s method (9). About 8 mg of dried collagen was placed in a tube, and 4 ml of 6 N HCl were added; the tube was then sealed and autoclaved at 120°C for 16 hr. The hydrolysate was neutralized by the addition of 6 N NaOH with the use of phenolphthaleine as an indicator. The volume was made up to 200 ml with water in a volumetric flask. Two milliliters of this solution was made up to 8 ml with water, and the hydroxyproline contents were assayed by the method of Prockop and Udenfriend (9). 2. Le Roy et al.‘s method (IO). In this method 35-70 mg of plasma proteins were placed in a tube, then 2 ml of distilled water and 4 ml of a saturated solution of Ba(OH)* were added. The tube was sealed and autoclaved at 120°C for 16 hr. The hydrolysate was neutralized with 6 N H,SO, with the use phenolphthalein as an indicator. To the neutral solution, 0.2 ml of 6 N HCl were added, and the BaSO, precipitate was removed by centrifugation at 1OOOgfor 10 min. The supernatant solution was purified on a Dowex 50-W column (1 x 5 cm) equilibrated with 1 N HCl. The purified fraction of hydroxyproline eluted with 20 ml of 1 N HCl was neutralized with 1 N NaOH to pH 8.5 using phenolphthalein as an indicator. It was then concentrated to a volume of 8 ml, and hydroxyproline was assayed by the method of Prockop and Udenfriend (9). 3. The modified method. About 8 mg of dried collagen or 35 mg of dried plasma proteins (for instance, fibrinogen) were placed in a small tube (2-3 ml in volume), then 0.25 ml of 72% PCA was added. The tube was sealed with stopper and heated at 100°C for 2 hr. The hydrolysate was dark brown, but it did not influence the successive steps of hydroxyproline determination. To the hydrolysate, 2 ml of water were added, and this mixture was neutralized with 6 N NaOH using phenolphthalein as an indicator. Next, the hydrolysate was quantitatively transferred to a SO-ml centrifuge tube,
552
GAEASIfiSKI
ET
AL.
and the volume was made up to 8 ml with water. The hydroxyproline contents were assayed by the method of Prockop and Udenfriend (9) with some modifications which standardized the determination procedure. Complete determination can be carried out in a single centrifuge tube without transfer. It was also established that suitable saturations can be made if 3.5 g of KC1 are added before the oxidation step and 2 g of KC1 are added at the moment when the oxidation is stopped by the addition of 3.6 M sodium thiosulfate. To the mixture, 10 ml of toluene were added, then the tube was stoppered and shaken vigorously for 2 min in an electricshaker, instead of shaking by hand 100 times. The mixture was centrifuged at 3000 r-pm for 5 min, and the toluene was discarded. The tube was then capped and heated in a boiling-water bath for 30 min, after which the mixture was cooled and 10 ml of toluene were added, and the tube was stoppered, shaken in electric shaker, and centrifuged as above. Two parallel 2.5-ml portions of the toluene phase were placed in separate tubes, 1 ml of Ehrlich’s reagent was added with stirring, and after 15 min, E&&, was read on a Beckman-Du spectrophotometer using toluene as a blank. The contents of hydroxyproline were calculated from the standard curve or from the parallel assay of the hydroxyproline standard sample. RESULTS
In the preliminary experiments, the effect of hydrolysis conditions with PCA on the yielding capacity of hydroxyproline was studied. The results presented in Table 1 show no losses of added hydroxyproline and no increases in hydroxyproline content after the addition of proline to the sample. The following experiments examined the influence of interfering material in the method of Le Roy ef al. (10) and in our modified method. From Fig. 1 we can see that after hydrolysis of fibrinogen in a Ba(OH), solution (IO), absorbance at 450 nm is much higher in the sample assayed without ion exchange purification, then in that purified on the column. This is in agreement with Le Roy et al.‘8 results (10). After the hydrolysis of fibrinogen in PCA, no differences in absorbances at 450 nm were observed. Contrary to Le Roy ef al.‘s results (lo), the spectra of total plasma globulin PCA hydrolysates passed through the cation exchanger, then eluted with 1 and 3 N HCl showed very low levels of interfering material, and a second peak of absorbance at 560 nm was not observed. The results of hydroxyproline determinations in collagen and gelatin performed after hydrolysis in 6 N HCl or in PCA were quite similar when obtained either by Prockop’s and Udenfiiend’s method (collagen, 7.64 + 0.7%; gelatin, 9.00 ? 0.25%) or by the modified method (collagen, 7.60 + 0.3%; gelatin, 8.76 -C 0.7%). Our results, obtained by the modified method with or without use of the column, are similar to those obtained by Le Roy et af.‘s method with column purification. The hydroxyproline contents in fibrinogen hydro-
HYDROXYPROLINE
DETERMINATION TABLE
THE EFFECT
Expt
OF ADDING HYDROXYPROLINE OF HYDROXYPROLINE
Weight of sample bg)
Protein
1
OR PROLINE TO THE SAMPLE DETERMINED IN PROTEINS
Total hydroxyproline in sample (/a)
Added to examined protein
2 3 4 5
Collagen Collagen Collagen Collagen Collagen
8 8 8 8 8
Hydroxyproline Hydroxyproline Hydroxyproline Hydroxyproline
(500 (500 (500 (500
1 2 3 4
Fibrinogen Fibrinogen Fibrinogen Fibrinogen
35 35 35 35
Hydroxyproline Hydroxyproline Hydroxyproline
(5 kg) (5 pg) (5 pg)
1 2
Fibrinogen Fibrinogen
35 35
Proline (1 mg)
1
553
IN PROTEINS
Yield of added hydroxyproline 043)
(%)
570 530 480 510
110 105 96 102
8.9 13.8 13.6 13.8
-
98 94 98
9.9 9.6
-
670 1240 1200 1150 1180
pg) pg) pg) pg)
ON THE YIELD
4.9 4.7 4.9
-
lyzed in PCA were 22.80 -+ 1.85% (without column) and 21.90 k 1.83% (with column) and in Ba(OH), were 26.20% + 1.3% (without column) and 22.00 + 1.3% (with column). Similar results were also obtained by Le Roy et al.‘s method (without column, 31.20 + 1.2%; with column, 28.80 OD
-
BOG
wth
-
Ea(OH)2
without
cdumn column
-
PCA
with
o-+
PCA
wlthout
column column
FIG. 1. The spectra of fibrinogen hydrolysates in Ba(OH), and in PCA, with and without purification on the cation exchanger.
554
GAILASIfiSKI
ET AL.
+ 3.0%) and the modified method (without column, 28.8 + 1.4%) for plasma globulins.
column,
29.2 + 1.75%; with
DISCUSSION
The findings presented in this paper show the modified method described here can be used for hydroxyproline determination in proteins with quite good results. The advantages of this modification are the much shorter protein hydrolysis time and the fact that the autoclave is not required. Heating sealed tubes in a thermostat at 124°C instead the use of an autoclave, causes tube breakage. Moreover, the transfer of hydrolysate from tube to tube creates the possibility of losses. The purification of the hydrolysate on the ion exchange column chromatography is not necessary in our modified method. The higher standardization of the hydroxyproline assays makes this method quite reproducible. The performance of the total assay procedure in one centrifuge tube and shaking in an electric shaker for 2 min instead by hand 100 times makes this method much more efficient. Many samples can be assayed in parallel; thus, using the modified method, 10 samples can be assayed in 6 hr, whereas only one sample could be assayed in 4 hr before. As can be seen from the results presented, the reproducibilities of the modified method and other methods (9,lO) are similar. No losses of hydroxyproline during the hydrolysis with PCA and no excess hydroxyproline content after addition of proline to the sample in the assay conditions were noted. The results presented in Fig. 1 show that absorption spectra of chromophores from hydroxyproline determined in proteins by the PCA method are similar with and without purification on cation exchange resin. But in the Ba(OH), method (IO), OD,,, ,,,,, of the sample measured without purification is much higher in unpurified material. This means the interfering material is separated during hydrolysis with PCA. In Prockop’s and Udenfriend’s method the hydroxyproline is oxidized in the presence of an excess of L-alanine in order to prevent the influence of varying amounts of amino acids or similar substances on the pyrrole yield. We tried to replace L-alanine by other substances. We have obtained results with the use l -aminocaproic acid (EACA) similar to those obtained with use of L-alanine. The solution of EACA should be 40%, w/v, and 0.25 ml of this solution should be added. In conclusion we consider the modified method to be useful for a large number of samples in routine protein hydroxyproline assays. REFERENCES 1. Lang, K. (1933) Hoppe-Seyler’s Z. Physiol. Chern. 219, 148. 2. Neumann, R. E., and Logan, M. A. (1950) J. Bid. Chem. 184, 299. 3. Stegemann, H. (1958) Z. Physiol. Chem. 311, 41.
HYDROXYPROLINE
DETERMINATION
555
IN PROTEINS
4. Martin, C. J., and Axelrod, A. E. (1953) Proc. Sot. Exp. Biol. Med. 83, 461. 5. Grunbaum, B. W., and Glick, D. (1956) Arch. Biochem. Biophys. 65, 260. 6. Miyada, D. S., and Tappel, A. L. (1956) Anal. Chem. 28, 909. 7. Fels, G. (1958) C/in. Chem. 4, 62. 8. Hutterer, F., and Singer, E. J. (1960) Anal. Chem. 32, 556. 9. Prockop, D. J., and Udenfriend, S. (1960) Anal. Biochem. 1, 228. 10. Le Roy, E. Carwile, Kaplan, A.,Udenfriend, S., and Sjeerdsma, A. (1964) J. Biol. Chem.
239, 3350.
11. Wyatt, G. R. (1951) Biochem. J. 48, 584. 12. Bahkowski, E., GaIasidski, W., and Rzeczycki, W. (1970) FEBS
I&t.
6, 19.