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On the colorimetric biuret method of protein determination
.\NALYTICAL
BIOCHEMISTRY
On
the
12,
Calorimetric Protein
R. PARVIN, From
S. V. PANDE,
the Vallabhbhai
(1965)
219-229
Pate1
Chest
Received
Biuret
Method
of
Determination1 T. A. VENKITASUBRAMANIAN
AND
Institute,
University
December
7, 1964
of Delhi,
Delhi,
India
The calorimetric biuret procedure has been widely employed for estimation of protein from a variety of biological materials but studies concerning the limitations and validity of this method have been few. Pande et al. (1) noted that, for extracts of Aspergillus niger, the biuret method of Gornall et al. (2) was not as satisfactory as the phenol method of Lowry et al. (3). Khanna et al. (4) reported that the use of the biuret procedure gave misleading high values for proteins in different biological extracts. The present study confirms and amplifies the above observations. In addition, experiments pertaining to the elucidat,ion of the cause of interference in the biuret method, its removal, and the applicability of the phenol method are also described. While this manuscript was in preparation a study of similar nature appeared (5). MATERIALS
AND
METHODS
Chemicals. Sufficiently pure chemicals of either British Drug House, England, or E. Merck, Germany, were used. Bovine serum albumin (BSA) (fraction V) was a product of Sigma Chemical Co. Preparation of Cell-Free Extracts for Study: A. niger and Mycobacterium 607 were grown and harvested as described by Pande et al. (1) and Parvin et aZ. (6), respectively. These organisms were stored at -15” before use. The extracts were prepared by hand grinding with sand in glass mortar and pestle. After 10 min grinding with water to obtain about 40% (wet w/v) disrupted cell suspension, the first extract was separated by centrifugation at 10,000 X g for 10 min. The residue obtained was reground similarly but for 15 min, and the second extract was obtained by centrifugation. Further grinding of the residue for 20 min and centrifugation gave the third extract. Homogenates of animal tissues (10% wet, w/v) were prepared in either 0.25 M sucrose (rat) or water (guinea-pig) ‘Taken requirements
from for
a thesis the Ph.D.
(of R. Parvin) degree of Delhi
submitted University. 219
in
partial
fulfillment
of the
220
PARVIN,
PANDE,
AND
VENKITASUBRAMAIGIAN
by using a Potter-Elvehjem homogenizer followed by centrifugation at 600 X 9 for 20 min. Whenever cell-free preparations contained a floating fatty layer at the surface, it was removed by filtration through muslin. All homogenate preparations and centrifugations etc. were carried out between 2 and 5”. Trichloroacetic Acid Precipitation: TCA was added to homogenate to 5% final concentration and heated for 15 min at 100”. The residue obtained after cooling and brief centrifugation was washed once with 5% TCA at room temperature and dissolved in alkali either as such, or after delipidation as described below. Delipidation: The TCA precipitate was suspended well in ca. 20 vol of chloroform:methanol 2: 1 (v/v) for lipid extraction (7)) warmed (below boiling) for 5 min, and allowed to stand overnight at 4”. The residue was separated by prolonged centrifugation (at 3,000 rpm for 1 to 2 hr) and removal of solvent. It was re-extracted for lipid as above but only 1 hr contact with solvent at room temperature was allowed. The residue from this step was freed of adhering solvent by drying in vacua. For dissolution, a paste of precipitate (TCA precipitate or delipidated residue) was made with a few drops of 0.2 N NaOH by rubbing with a glass rod and more alkali was added gradually with constant mixing. If the solutions were not clear, the contents were heated for 5 min at 100”. In some cases, especially after delipidation, for complete dissolution, the alkali concentration was increased up to 1 N and the heating time prolonged up to 20 min. By dilution, the NaOH concentration was lowered to 0.1 N before performing the biuret calorimetry. For the phenol colors, these solutions were further diluted 50-fold. The small amount of alkali present in the protein solutions did not affect the calorimetry. This hot alkaline treatment considerably affected the subsequent color in both the biuret and the phenol methods, as tested with BSA. While 5 min prior heating with 0.2 N NaOH at 100” had no detectable effect, 10 min heating in 1 N NaOH lowered the biuret color by about 10% and the phenol color by about 20%. A simultaneously run standard took care of such effects to allow the necessary correction to be applied. Biuret
Method
Biuret Reagent. Four times as concentrated reagent as described by Gornall et al. (3) was prepared. Powdered 1.50 gm CuSO,*4H,O and 6.0 gm sodium potassium tartrate (NaKC4H,0s .4H,O) were dissolved in ca. 100 ml water. To this was added with constant mixing 100 ml of carbonate-free 30% NaOH. The final volume was brought to 250 ml. It was stored in an airtight polyethylene bottle and remained stable for months.
COLORIMETRIC
PROTEIK
DETERMINATION
Alkaline Tartrate Reagent. This differed from the hiuret described above in that no cupric sulfate was present.
221 reagent
Phenol Method
The procedure of Lowry et al. (2) was used. Here a marked deviation from a linear relationship between optical density (OD) and protein amount was seen; hence only those aliquots of unknown and standard which gave an OD of 0.3 I+ 0.05 were used for the calculation of E:z I\“/“). The protein colors in this method could vary with the lot of phenol reagent. In both the biuret (540 rnp) and the phenol (750 mp) methods, the OD (Bausch & Lomb calorimeter) was recorded between 45 to 90 min of color development. Kjeldahl Method
The procedure of McKenzie
and Wallace
(8) was used.
Standard Protein
A freshly prepared aqueous BSA solution was used. RESULTS
ilnalysis of Rat Tissues, Guinea-Pig Liver, and Bovine Serum Albumin.
The data obtained show (Table 1) that direct biuret calorimetry by the Gornall (3) procedure with extracts and TCA-separated protein preparations gave much higher extinction values than the BSA. The possibility of the contribution of opalescence to the biuret color measurements was considered by treating homogenates with the alkaline tartrate reagent. Tested in this way, all homogenates as well as TCA precipitates showed marked absorption. In the AOD biuret method (Table 1)) which makes allowance for such an effect, the color ext.inction values of the homogenates and the TCA precipitates fell close to that of the BSA, compared on the basis of protein nitrogen. After delipidation, the Gornall color values became comparable to the AOD values for all tissues excepting brain. For brain, the ultraviolet protein determination method is also unsuitable (9). When much of the work of this study was completed we came across the report of Kayser and Vaughn (lo), in which recognition of interference due to turbidity in the biuret procedure was realized for serum proteins. The application of the KCN method (Table 1) of these authors to guinea-pig liver showed that the values by this method were similar to the AOD biuret method, being comparable to the BSA. The phenol color values, considered on the basis of protein nitrogen,
222
PARVIN,
PANDE,
AND
VENKITASUBRAMANIAN
TABLE RELATION
6.25
1
BIUFLET COLORS, PHENOL COLORS, AND KJELDAHL IN PREPARATIONS OF RAT TISSUES, GUINEA-PM LIVER, SERUM ALBUMIN
BETWEEN
VALUES
NITROGEN
x
AND BOVINE
E~~~‘W’y) refers to the extinction of final color solutions. The weight of protein is the Kjeldahl nitrogen X 6.25. Protein nitrogen refers to TCA-separated nitrogen without correction for remaining nucleic acid nitrogen, if any. In the biuret procedure, Gornall values represent results obtained on treating protein (solution brought to 4.0 ml) with biuret reagent (1.0 ml) and recording absorbancy (3). In the AOD method, the protein solution (4.0 ml) was treated with alkaline tartrate reagent (1.0 ml) and absorbancy recorded as in the Gornall method. The difference between Gornall color value and alkaline tartrate value gave the AOD value. In the KCN method, after recording the Gornall colors a pinch of KCN (ca. 100-120 mg) was added and contents mixed; 5 min after discharge of color, absorbancy was recorded again. The difference in absorbancy before and after KCN treatment gave the KCN value (10). Original Source
and method
Bovine serum Biuret Gornall AOD Phenol %N
homogenate
Total
N
values
based
Protein
N
on After
TCA
precipitation
After delipidation
albumin:
2.94= 2.94 222 100 Percentage
Rat liver: Biuret Gornall AOD Phenol %‘oN Rat liver particulate fracttin:b Biuret Gornall AOD Phenol %‘oN Rat heart: Biuret Gornall AOD Phenol %N
2.96 222 99.4 difference obtained
2.96 220 99.4
+52
+=
+35 -7 -6 94
+37 +7 -5 94
+40 -2 -5 85.8
+47 -10 -6 85.8
100
+19 -16 -23 100
226 97.8
of E:~m’W’v) value from corresponding with bovine ccrum albumin
$33 -17 -32 100
-12 -12
2.94
-6 -2 89.1
1-46 -8 -1 89.1
value
-1 -2
-1 83.4 -6 -10 -4 x7.s
+2 +1
-4 78.4
COLORIMETRIC
PROTEIN
TABLE Original Source
and
method
1 (Continued)
homogenate
Total
N
Percentage
Rat brain: Biuret Gornall AOD Phenol % pr’ Guinea-pig liver: Binret Gornall AOD ICCN Phenol
+55 -26 -19 100
223
DETERMINATION
values
based
Protein difference obtained
N
on After TCA precipitation
After delipidation
of J?:~“w’v’ value from correspondinK with bovine serum albumin
+78 -15 -6
8i.l
c c -s s7.1
i-85 -5 +2 +5
a Average of four separate analysis. b Homogenates were centrifuged at 10,000 X g for 30 min. The separated resuspended by homogenizing with 0.25 M sucrose for use. c Since the solutions were turbid, calorimetry coldd not be performed.
value
+41 -5 - 5 $73
+:: -5 -9 +3
mat,erial
was
were about the same after TCA precipitation and delipidation as with the original homogenates. Analysis of Mycobacterium 607 and Aspergillus niger Preparations: Analysis of the successive extracts of Mycobacterium 607 (Table 2) and -4. niger (Table 3) showed that biuret calorimetry gave high extinction values as compared to the BSA values. This was either unaffected (Table 3) or slightly minimized (Table 2) by TCA precipitation. The AOD method in all these cases gave better results. After delipidation, the Gornall method gave satisfactory results with A. niger only; with M. 607 the AOD method still gave superior values. The values obtained by the KCN method were comparable (Table 2) to the AOD method. The pheno1 colors of original extracts in these cases were nearly comparable to those obtained after TCA precipitation. Delipidation did not affect the phenol color extinction values. The Kjeldahl nitrogen of these microbial extracts contained a considerable proportion of nitrogen that did not contribute to calorimetric estimations, since nearly complete rc>tcntion of the AOD biuret and the phenol colors was seen in the TCA precipitates. In the ammonium sulfate separated material also (Table 2) part of the nitrogen was removed by the TCA precipitation (perhaps due to nucleic acid removal (see ref. 6)) without affecting the phenol or AOD hiuret. values. Considered on the prot,ein nikogen basis, the Gornall
224
PARVIN,
PANDE,
AND
VENKITASUBRAMANIAK
TABLE RELATION
BETWEEN 6.25
BIURET VALUES
COLORS, PHENOL IN PREPARATIONS
2 COLORS,
.~ND KJELDAHL
Mycobacterium (Details as for Table 1) OF
NITROGEN
X
607
Percentage difference ofY(y value from corresponding value obtained with bovine serum albumin Original h;m&&
Preparation and method
First extract: Biuret Gomall AOD KCN Phenol %‘oN Second extract: Biuret Gornall AOD
KCN Phenol %‘oN Third eztract: Biuret Gornall AOD KCN Phenol %N Ammonium
sulfate
values After TCA precipitation
After delipidation
Total N
Protein N
-44 -51 -54 -48 100
+33 -9 -11 -1 52.9
+7 -37 -35 -34 100
+60 -5 +7 -1 66.S
+17 -10 -9 +5 66.8
+10 -2 -2 +3 53.2
+lQ -29 -27 -27 100
+54 -8 -5 -5 77.1
+15 -5 -4 +4 77.1
+12
0 -17 -16 -15 100
+lQ -3 -2
+1s -7 -12 +1 52.9
+10 -6 -7 0 43.8
+4 +4 +1 62.1
separated
materid:a
Biuret Gornall AOD KCN Phenol %N
0 55.4
+Q -2 -3 -3 85.4
+5 -1 -2 -1 84.6
a A separately prepared aqueous extract was brought to full saturation with ammonium sulfate and the separated material obtained after centrifugation (10 min at 10,000 X g) was dissolved in water. It was dialyzed 24 hr again& several changes of large excess of water. method here also gave high values as compared to the other methods. It must be mentioned that the microbial extracts that were obtained by centrifugation at 1000 X g for 10 min were too opalescent, for which
only the phenol method gave tolerably satisfactory results. With A. niger
(‘OLORIMETRIC
PROTEIN
TABLE RELATION
BETWEEN
BIURET
6.25
COLORS, VALUES
Original Preparation
and method
First extract: Biuret Gornall AOD Phenol %N Second extract: Biuret Gornall AOD Phenol %N Third extract: Biuret Gornall AOD Phenol %N
N
AND
OF A.
as for Table
KJELDAHL
values
based
Protein
N -
NITROGEN
1)
on
After TCA precipitation
valuej
After delipidstion
1-79 -51 -51 100
+276 f3 f4 47.5
$243 +3 -1 47.5
+3 +1 -4 47.3
$99 -54 -55 100
1-318 -4 -3 47.4
+308 -3 -3 47.4
f7 f5
f376
+336 $12 -1 48.2
+5 +5 +1 48.2
$130 -46 -48 100
+12 +9 48.2
X
niger
difference of E:::‘““’ value from corresponding obtained with bovine serum albumin
homogenate
Total
COLORS,
PREPARATION
(Details Percentage
3
PHENOL
IN
225
DETERMINATION
+2 47
it has been found (11) that the ultraviolet protein determination method is also unsatisfactory as compared to the phenol method. Analysis of Spinach Preparation: The Gornall biuret method gave high extinction values in the homogenate as well as with the TCA precipitates (Table 4). Delipidation and acetone treatment, which also removed the pigments, lowered the Gornall color values though the AOD procedure even in these cases gave better results. With acetone-washed TCA precipitates of Cuctm also, the direct biurct calorimetry gave high values (12). In acetone treatment, complete protein precipitation (as judged by both the phenol and the AOD biuret colors which were comparable) was obtained (1) by adding a minimum of 10 vol of acetone directly to homogenate or (2) by washing the TCA precipitates with excess acetone, and or (3) by adding TCA in acetom to provide 5 vol of acetone with 575 TCA. With wheat germ, comparable protein valued were obtained by the biuret and the phenol methods after separating proteins with 30 vol of acetone (13). Both the phenol and the AOD biuret color ext,inctions were higher in spinach homogenates than those obtained after TCA precipita-
226
PARVIN,
PANDE,
AND
VENKITASUBRAMANIAN
TABLE RELATION
BETWEEN
4
BIURET COLORS, PHENOL COLORS, AND KJELDAHL 6.25 VALUES IN SPINACH LEAF PREPARATIONS
(Details of acetone treatment
Method
Biuret Gornall AOU
Phenol %N
Total N
+124 +7 -11 100
x
are described in text; other details as for Table 1)
Percentage difference of E;~:‘~‘~) value from corresponding obtained with bovine serum albumin Original h;;;~,nnte*
NITROGEN
value
values Protein
N
+186 +37 $42 78.1
After TCA precipitation
After delipidation
+ 150 -12 +2 78.1
+18 -4 fl 72.4
After acetone treatment
+15 - 3 -1 75.6
QLeaf homogenate (30% wet w/v) was obtained using a Waring Blendor and clarifying the resulting suspension by cenkifugation at 10,000 X g for 10 min.
tion. A need for the TCA precipitation of proteins prior to calorimetry was felt with Euglena also (14). DISCUSSION
The inapplicability of the direct biuret calorimetry has been realized in many instances (12-26). Thus the Cleland and Slater (17) procedure of washing TCA precipitates with ethanol before color development has been adopted by several workers (21-24). Deoxycholate (15, 18, 25, 26) and Triton (19) treatments have also been made to improve the calorimetry. However, use of the Gornall biuret method is being continued (9, 27-34), perhaps because a systematic study urging caution in the use of direct biuret calorimetry has not appeared. Since this procedure gives wrong results, as emphasized in the present study, it is a potential source of error in comparison and evaluation of results of studies in different laboratories. As the AOD or the KCN procedure improved the results, the cause of interference is the persistence of opalescence which contributes to the OD measurement (cf. ref. 5). Although carbohydrates have been considered responsible (1, 4)) TCA treatment which completely extracts glycogen (35) did not minimize interference. Removal or lowering of interference by delipidation shows that lipids are mainly responsible for this trouble. In separate experiments with the mitochondria-free fraction of rat liver preparations, it was also found that interference in the biuret procedure increased when the liver lipid content increased (cf. ref. 20). Layne (36) has recommended ether extraction of the biuret colors to
COLORIMETRIC
PROTEIN
DETERMINATIOX
227
remove interfering turbidity. We find this practice unsuitable. In many cases this minimized t.he extent of error only slightly; further, the reproducibility was poor. Also, we did not find improvement simply by the inclusion of deoxycholate (5-10 mg) 10 min before color development. In addition, in certain cases, depending upon the source, the AOD or the KCN method gave better results than did the procedure of Cleland and Slater (18) because even after delipidation the opalescence persisted. The present study reveals that with different sources, a variable degree of protein nitrogen is lost on delipidation of TCA precipitates. This emphasizes the conclusion of Munro and Downie (37) that the protein content of delipidated residue may be significantly lower than the actual protein present in the original extracts. Both in the phenol and in the AOD biuret methods, the protein colors of rat tissues and Mycobacterium 607 extracts were found to be completely retained in the TCA precipitates. This justifies the assumption that TCA-separated nitrogen is a good measure of total protein and emphasizes that, in t,he Kjeldahl method, use of the total nitrogen as an index of protein can lead to serious errors. For A. niger and more so for spinach preparations, however, the color values were higher with the initial extracts than with the TCA precipitates. Since these color reactions are not entirely specific to prot.eins, the possibility exist,s that, apart from the presence of peptides and prot,eins, nonprecipitable by TCA, other substances (1, 2, 38) could also contribute to color. The discrepancy due to this effect will be minimized by the TCA precipitation of proteins. This will also avoid interference due to (1) certain cations, such as K+ (391 Mn++ (6)) NH,+ (2,36), and (2) other substances, such as tris buffer (40)) reducing substances? (cf. ref. 41), etc. (42). When homogenates develop turbidity on standing, the KCN method appears unsatisfactory (10). This is avoided by the TCA precipitation and subsequent mild alkaline treatment which gives homogeneous protein solutions. At times this also reduced the contribution of the opalescence to the OD measurement and, therefore, made the AOD method more reliable (cf. ref. 5). This opalescence contribution could further be minimized by the inclusion of ethanol--40cjo (v/v)-in the color solutions without affecting the calorimetry. With biological extract,s, unlike with pure proteins, it is desirable to record the biuret colors without unnecessary delay since turbidities appear on standing (cf. ref. 1). In the AOD biuret method, this development of turbidity is less marked in the case of alkaline tartrate controls and hence ‘For this reason in the growth medium of A. Gger the hiuret method could not he used though, surprisingly, the phenol method served well.
228
PARVIN,
PANDE,
AND
VENKITASUBRAMANIAh-
this becomes all the more necessary. This is also unavoidable when alcohol, acetone, or deoxycholate is present. In such cases, development of yellowish color on standing, probably due to Cu++ reduction, invalidates the calorimetry. Finally, whereas the chromogenicity of individual proteins differs in the biuret procedure (3) and more so in the phenol method (2, 43), for mixtures of proteins in biological extracts the chromogenicity was not found to be markedly different, as this was similar to that of the BSA. In such cases, therefore, the use of BSA as reference standard for colorimetric protein determination methods appears adequate. SUMMARY
A study of the applicability of a calorimetric biuret procedure for protein determination to a variety of biological preparation has revealed that the practice of using this procedure is undesirable because markedly apparent high values are obtained in this way. In some cases, this interference was mainly due to lipids. Use of suitable controls to account for the interfering opalescence in biuret color measurements considerably improved the method. Conditions affecting the reliability of the procedure are also described. For crude extracts the calorimetric phenol method was found to be more reliable. However, a prior separation of protein by TCA is often advantageous not alone for Kjeldahl analysis but also for calorimetric estimations. The actual chromogenicity of the protein mixtures of different biological extracts studied was found to be comparable to that of the commonly employed standard protein, bovine serum albumin. ACKNOWLEDGMENTS This work was supported in part by funds from the Indian Council of Medical Research, New Delhi, and Grant No. E-3427, National Institute of Allergy and Infectious Diseases, U. S. P. H. S. We are grateful to Dr. R. Viswanathan, Director of this 1nstitut.e for his interest in this work. The technical assistance of Mr. R. K. Bhatnagar is gratefully acknowledged. REFERENCES S. V., TEWARI, K. K., AND KRISHNAN, P. S., Arch. Mikrobiol. 39, 343 (1961). 2. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193,265 (1951). 3. GORNALL, A. G., BARADAWILL, C. J., AND DAVID, M. M., J. Biol. Chem. 177, 751 (1949). 4. KHANNA, R., TEWARI, K. K., AND KRISHNAN, P. S., Arch. Mikrobiol. 44, 352 (1962). 5. INCHIOSA, M. A., J. Lab. Clin. Med. 63, 319 (1964). 1. PANDE,
(‘OLORIMETRIC
PROTEIN
DETERMINATION
229
6. PARVIN, R., PANDE, S. V., AND VENKITASUBRAMASIAN, T. A., Biochim. Biophys. Acta !&I, 260 (1964). 7. FOLCH, J., Lms, M., AND STANLEY, G. H. S., J. Biol. Chem. 226, 497 (1957). 8. MCKENZIE, H. A., AND W.~LL.~CE, H. S., Australian J. Chem. 7, 55 (1954). 9. MANSOOR, M., KALYANKAR, G. D., AND TALWAR, G. P., Biochim. Biophys. Actn 77, 307 (1963). 10. KEYSER, J. W., AND VAUGHX, J., Biochem. J. 44, xxii (1949). Il. LI, L. H., AND KING, K. W., Appl. Microbial. 11, 320 (1963). 12. MUKERJI, S. K., S.ww.u,, G. G., ASD KRIRHNAN, P. S., Indian J. Biochem. 1, 36 (1964). 13. JOYCE,B. K., AND GRISOLIA, S., J. Biol. Chem. 235, 2278 (1960). 14. BRAWERMAN, G., AND KONIGSBERG, N., Biochim. Biophys. Acta 43, 374 (1960). 15. RINCLER, R. L., MINAKAMI, S., AND SISGER, T. P., J. Riol. Chem. 238, 801 (1963). 16. FEINSTEIN, R. N., Radiation Research 4, 217 (1956). 17. CLELAND, K. W., AND SLATER, E. C., Biochem. J. 53, 547 (1953). 18. JACOBS, E. E., JACOB, M.. SANADI, D. R., AND BRADLEY, 1,. B., J. Biol. Chem. 223, 147 (1956). 19. NORDLIE, R. C., AND LAHDY, H. A., Biochim. Biophys. Acta 50Y 189 (1961). 20. FREEDLAND, R. A., AND HARPER, A. E., J. Biol. Chem. 228,743 (1957). 21. VAN ROSSUM, G. D. V., Biochim. Biophys. Acta 74, 15 (1963). 22. JOBSIS, F. F., Biochim. Biophys. Acta 74, 60 (1963). 23. BORST, P., Biochim. Biophys. Acta 57, 256 (1962). 24. CHRIST, E. J., AND HULSMANN, W. C., Biochim. Biophys. Acta 66, 72 (1962). 25. CAMERINO, P. W., AND SMITH, L., J. Biol. Chem. 239,2345 (1964). 26. KIMURA, T., AND TOBARI, J., Biochim. Biophys. Acta 73, 399 (1963). 27. GOLDMAN, D. S., J. Bact. 71, 732 (1956). 28. GOLDMAN, D. S., J. Bact. 86,30 (1963). 29. HOMMES, F. A., Biochim. Biophys. Acta 77, 173 (1963). 30. ABRAHAM, S., MATTHEE.. K. J., AND CHAIKOFF, I. L., J. Biol. Chem. 235, 2551 (1960). 31. ABRAHAM, S., MATTHES, K. J., APED CHAIKOFF, I. L., Biochim. Biophys. Acta 76, 357 (1963). 32. HESS, R., AND PEARSE, G. E., Biochim. Biophys. Acta 71, 285 (1963). 33. BORTZ, W., ABRAHAM, S., AND CHAIKOFF, I. L., J. Biol. Chem. 238, 1266 (1963). 34. PYNADATH, T. I., AND KUMAR, S., Biochim. Biophys. Acta $4, 251 (1964). 35. KEMP, A.. AND KITSVAN HEIJNINGEN, A. J. M., Biochem. J. 56, 646 (1954). 36. LAYNE, E., in “Method in Enzymology” (Colowick, S. P., and Kaplan, N. O., eds.), Vol. III, p. 447. Academic Press, Sew York, 1957. 37. MUNRO, H. N., AND DOWNIE, E. D., Arch. Biochem. Biophys. 106, 516 (1964). 38. UPDIKE, S. S., AND DOUNCE, A. L., Anal. Biochem. 6, 1 (1963). 39. SERBAN, M., TANASESCU, D., AND VASU, S., Acud. Rep. Popdare Romine &u&i Cercetari Biochim. 6, 115 (1963). 40. MYODA, T., AND WERKMAN. C. H., Iowa &ate J. sci. 35, 463 (1961). 41. RACUSEN, D., AND JOHNSTONE, D. B., Nature 191,492 (1961). 42. JANECEK, J., AND RICKENBERG, H. V., Biochim. Biophys. Actu 81, 108 (1964). 43. MORTENSON, L. E., Biochim. Biophys. Acta 81, 71 (1964).
BIOCHEMISTRY
On
the
12,
Calorimetric Protein
R. PARVIN, From
S. V. PANDE,
the Vallabhbhai
(1965)
219-229
Pate1
Chest
Received
Biuret
Method
of
Determination1 T. A. VENKITASUBRAMANIAN
AND
Institute,
University
December
7, 1964
of Delhi,
Delhi,
India
The calorimetric biuret procedure has been widely employed for estimation of protein from a variety of biological materials but studies concerning the limitations and validity of this method have been few. Pande et al. (1) noted that, for extracts of Aspergillus niger, the biuret method of Gornall et al. (2) was not as satisfactory as the phenol method of Lowry et al. (3). Khanna et al. (4) reported that the use of the biuret procedure gave misleading high values for proteins in different biological extracts. The present study confirms and amplifies the above observations. In addition, experiments pertaining to the elucidat,ion of the cause of interference in the biuret method, its removal, and the applicability of the phenol method are also described. While this manuscript was in preparation a study of similar nature appeared (5). MATERIALS
AND
METHODS
Chemicals. Sufficiently pure chemicals of either British Drug House, England, or E. Merck, Germany, were used. Bovine serum albumin (BSA) (fraction V) was a product of Sigma Chemical Co. Preparation of Cell-Free Extracts for Study: A. niger and Mycobacterium 607 were grown and harvested as described by Pande et al. (1) and Parvin et aZ. (6), respectively. These organisms were stored at -15” before use. The extracts were prepared by hand grinding with sand in glass mortar and pestle. After 10 min grinding with water to obtain about 40% (wet w/v) disrupted cell suspension, the first extract was separated by centrifugation at 10,000 X g for 10 min. The residue obtained was reground similarly but for 15 min, and the second extract was obtained by centrifugation. Further grinding of the residue for 20 min and centrifugation gave the third extract. Homogenates of animal tissues (10% wet, w/v) were prepared in either 0.25 M sucrose (rat) or water (guinea-pig) ‘Taken requirements
from for
a thesis the Ph.D.
(of R. Parvin) degree of Delhi
submitted University. 219
in
partial
fulfillment
of the
220
PARVIN,
PANDE,
AND
VENKITASUBRAMAIGIAN
by using a Potter-Elvehjem homogenizer followed by centrifugation at 600 X 9 for 20 min. Whenever cell-free preparations contained a floating fatty layer at the surface, it was removed by filtration through muslin. All homogenate preparations and centrifugations etc. were carried out between 2 and 5”. Trichloroacetic Acid Precipitation: TCA was added to homogenate to 5% final concentration and heated for 15 min at 100”. The residue obtained after cooling and brief centrifugation was washed once with 5% TCA at room temperature and dissolved in alkali either as such, or after delipidation as described below. Delipidation: The TCA precipitate was suspended well in ca. 20 vol of chloroform:methanol 2: 1 (v/v) for lipid extraction (7)) warmed (below boiling) for 5 min, and allowed to stand overnight at 4”. The residue was separated by prolonged centrifugation (at 3,000 rpm for 1 to 2 hr) and removal of solvent. It was re-extracted for lipid as above but only 1 hr contact with solvent at room temperature was allowed. The residue from this step was freed of adhering solvent by drying in vacua. For dissolution, a paste of precipitate (TCA precipitate or delipidated residue) was made with a few drops of 0.2 N NaOH by rubbing with a glass rod and more alkali was added gradually with constant mixing. If the solutions were not clear, the contents were heated for 5 min at 100”. In some cases, especially after delipidation, for complete dissolution, the alkali concentration was increased up to 1 N and the heating time prolonged up to 20 min. By dilution, the NaOH concentration was lowered to 0.1 N before performing the biuret calorimetry. For the phenol colors, these solutions were further diluted 50-fold. The small amount of alkali present in the protein solutions did not affect the calorimetry. This hot alkaline treatment considerably affected the subsequent color in both the biuret and the phenol methods, as tested with BSA. While 5 min prior heating with 0.2 N NaOH at 100” had no detectable effect, 10 min heating in 1 N NaOH lowered the biuret color by about 10% and the phenol color by about 20%. A simultaneously run standard took care of such effects to allow the necessary correction to be applied. Biuret
Method
Biuret Reagent. Four times as concentrated reagent as described by Gornall et al. (3) was prepared. Powdered 1.50 gm CuSO,*4H,O and 6.0 gm sodium potassium tartrate (NaKC4H,0s .4H,O) were dissolved in ca. 100 ml water. To this was added with constant mixing 100 ml of carbonate-free 30% NaOH. The final volume was brought to 250 ml. It was stored in an airtight polyethylene bottle and remained stable for months.
COLORIMETRIC
PROTEIK
DETERMINATION
Alkaline Tartrate Reagent. This differed from the hiuret described above in that no cupric sulfate was present.
221 reagent
Phenol Method
The procedure of Lowry et al. (2) was used. Here a marked deviation from a linear relationship between optical density (OD) and protein amount was seen; hence only those aliquots of unknown and standard which gave an OD of 0.3 I+ 0.05 were used for the calculation of E:z I\“/“). The protein colors in this method could vary with the lot of phenol reagent. In both the biuret (540 rnp) and the phenol (750 mp) methods, the OD (Bausch & Lomb calorimeter) was recorded between 45 to 90 min of color development. Kjeldahl Method
The procedure of McKenzie
and Wallace
(8) was used.
Standard Protein
A freshly prepared aqueous BSA solution was used. RESULTS
ilnalysis of Rat Tissues, Guinea-Pig Liver, and Bovine Serum Albumin.
The data obtained show (Table 1) that direct biuret calorimetry by the Gornall (3) procedure with extracts and TCA-separated protein preparations gave much higher extinction values than the BSA. The possibility of the contribution of opalescence to the biuret color measurements was considered by treating homogenates with the alkaline tartrate reagent. Tested in this way, all homogenates as well as TCA precipitates showed marked absorption. In the AOD biuret method (Table 1)) which makes allowance for such an effect, the color ext.inction values of the homogenates and the TCA precipitates fell close to that of the BSA, compared on the basis of protein nitrogen. After delipidation, the Gornall color values became comparable to the AOD values for all tissues excepting brain. For brain, the ultraviolet protein determination method is also unsuitable (9). When much of the work of this study was completed we came across the report of Kayser and Vaughn (lo), in which recognition of interference due to turbidity in the biuret procedure was realized for serum proteins. The application of the KCN method (Table 1) of these authors to guinea-pig liver showed that the values by this method were similar to the AOD biuret method, being comparable to the BSA. The phenol color values, considered on the basis of protein nitrogen,
222
PARVIN,
PANDE,
AND
VENKITASUBRAMANIAN
TABLE RELATION
6.25
1
BIUFLET COLORS, PHENOL COLORS, AND KJELDAHL IN PREPARATIONS OF RAT TISSUES, GUINEA-PM LIVER, SERUM ALBUMIN
BETWEEN
VALUES
NITROGEN
x
AND BOVINE
E~~~‘W’y) refers to the extinction of final color solutions. The weight of protein is the Kjeldahl nitrogen X 6.25. Protein nitrogen refers to TCA-separated nitrogen without correction for remaining nucleic acid nitrogen, if any. In the biuret procedure, Gornall values represent results obtained on treating protein (solution brought to 4.0 ml) with biuret reagent (1.0 ml) and recording absorbancy (3). In the AOD method, the protein solution (4.0 ml) was treated with alkaline tartrate reagent (1.0 ml) and absorbancy recorded as in the Gornall method. The difference between Gornall color value and alkaline tartrate value gave the AOD value. In the KCN method, after recording the Gornall colors a pinch of KCN (ca. 100-120 mg) was added and contents mixed; 5 min after discharge of color, absorbancy was recorded again. The difference in absorbancy before and after KCN treatment gave the KCN value (10). Original Source
and method
Bovine serum Biuret Gornall AOD Phenol %N
homogenate
Total
N
values
based
Protein
N
on After
TCA
precipitation
After delipidation
albumin:
2.94= 2.94 222 100 Percentage
Rat liver: Biuret Gornall AOD Phenol %‘oN Rat liver particulate fracttin:b Biuret Gornall AOD Phenol %‘oN Rat heart: Biuret Gornall AOD Phenol %N
2.96 222 99.4 difference obtained
2.96 220 99.4
+52
+=
+35 -7 -6 94
+37 +7 -5 94
+40 -2 -5 85.8
+47 -10 -6 85.8
100
+19 -16 -23 100
226 97.8
of E:~m’W’v) value from corresponding with bovine ccrum albumin
$33 -17 -32 100
-12 -12
2.94
-6 -2 89.1
1-46 -8 -1 89.1
value
-1 -2
-1 83.4 -6 -10 -4 x7.s
+2 +1
-4 78.4
COLORIMETRIC
PROTEIN
TABLE Original Source
and
method
1 (Continued)
homogenate
Total
N
Percentage
Rat brain: Biuret Gornall AOD Phenol % pr’ Guinea-pig liver: Binret Gornall AOD ICCN Phenol
+55 -26 -19 100
223
DETERMINATION
values
based
Protein difference obtained
N
on After TCA precipitation
After delipidation
of J?:~“w’v’ value from correspondinK with bovine serum albumin
+78 -15 -6
8i.l
c c -s s7.1
i-85 -5 +2 +5
a Average of four separate analysis. b Homogenates were centrifuged at 10,000 X g for 30 min. The separated resuspended by homogenizing with 0.25 M sucrose for use. c Since the solutions were turbid, calorimetry coldd not be performed.
value
+41 -5 - 5 $73
+:: -5 -9 +3
mat,erial
was
were about the same after TCA precipitation and delipidation as with the original homogenates. Analysis of Mycobacterium 607 and Aspergillus niger Preparations: Analysis of the successive extracts of Mycobacterium 607 (Table 2) and -4. niger (Table 3) showed that biuret calorimetry gave high extinction values as compared to the BSA values. This was either unaffected (Table 3) or slightly minimized (Table 2) by TCA precipitation. The AOD method in all these cases gave better results. After delipidation, the Gornall method gave satisfactory results with A. niger only; with M. 607 the AOD method still gave superior values. The values obtained by the KCN method were comparable (Table 2) to the AOD method. The pheno1 colors of original extracts in these cases were nearly comparable to those obtained after TCA precipitation. Delipidation did not affect the phenol color extinction values. The Kjeldahl nitrogen of these microbial extracts contained a considerable proportion of nitrogen that did not contribute to calorimetric estimations, since nearly complete rc>tcntion of the AOD biuret and the phenol colors was seen in the TCA precipitates. In the ammonium sulfate separated material also (Table 2) part of the nitrogen was removed by the TCA precipitation (perhaps due to nucleic acid removal (see ref. 6)) without affecting the phenol or AOD hiuret. values. Considered on the prot,ein nikogen basis, the Gornall
224
PARVIN,
PANDE,
AND
VENKITASUBRAMANIAK
TABLE RELATION
BETWEEN 6.25
BIURET VALUES
COLORS, PHENOL IN PREPARATIONS
2 COLORS,
.~ND KJELDAHL
Mycobacterium (Details as for Table 1) OF
NITROGEN
X
607
Percentage difference ofY(y value from corresponding value obtained with bovine serum albumin Original h;m&&
Preparation and method
First extract: Biuret Gomall AOD KCN Phenol %‘oN Second extract: Biuret Gornall AOD
KCN Phenol %‘oN Third eztract: Biuret Gornall AOD KCN Phenol %N Ammonium
sulfate
values After TCA precipitation
After delipidation
Total N
Protein N
-44 -51 -54 -48 100
+33 -9 -11 -1 52.9
+7 -37 -35 -34 100
+60 -5 +7 -1 66.S
+17 -10 -9 +5 66.8
+10 -2 -2 +3 53.2
+lQ -29 -27 -27 100
+54 -8 -5 -5 77.1
+15 -5 -4 +4 77.1
+12
0 -17 -16 -15 100
+lQ -3 -2
+1s -7 -12 +1 52.9
+10 -6 -7 0 43.8
+4 +4 +1 62.1
separated
materid:a
Biuret Gornall AOD KCN Phenol %N
0 55.4
+Q -2 -3 -3 85.4
+5 -1 -2 -1 84.6
a A separately prepared aqueous extract was brought to full saturation with ammonium sulfate and the separated material obtained after centrifugation (10 min at 10,000 X g) was dissolved in water. It was dialyzed 24 hr again& several changes of large excess of water. method here also gave high values as compared to the other methods. It must be mentioned that the microbial extracts that were obtained by centrifugation at 1000 X g for 10 min were too opalescent, for which
only the phenol method gave tolerably satisfactory results. With A. niger
(‘OLORIMETRIC
PROTEIN
TABLE RELATION
BETWEEN
BIURET
6.25
COLORS, VALUES
Original Preparation
and method
First extract: Biuret Gornall AOD Phenol %N Second extract: Biuret Gornall AOD Phenol %N Third extract: Biuret Gornall AOD Phenol %N
N
AND
OF A.
as for Table
KJELDAHL
values
based
Protein
N -
NITROGEN
1)
on
After TCA precipitation
valuej
After delipidstion
1-79 -51 -51 100
+276 f3 f4 47.5
$243 +3 -1 47.5
+3 +1 -4 47.3
$99 -54 -55 100
1-318 -4 -3 47.4
+308 -3 -3 47.4
f7 f5
f376
+336 $12 -1 48.2
+5 +5 +1 48.2
$130 -46 -48 100
+12 +9 48.2
X
niger
difference of E:::‘““’ value from corresponding obtained with bovine serum albumin
homogenate
Total
COLORS,
PREPARATION
(Details Percentage
3
PHENOL
IN
225
DETERMINATION
+2 47
it has been found (11) that the ultraviolet protein determination method is also unsatisfactory as compared to the phenol method. Analysis of Spinach Preparation: The Gornall biuret method gave high extinction values in the homogenate as well as with the TCA precipitates (Table 4). Delipidation and acetone treatment, which also removed the pigments, lowered the Gornall color values though the AOD procedure even in these cases gave better results. With acetone-washed TCA precipitates of Cuctm also, the direct biurct calorimetry gave high values (12). In acetone treatment, complete protein precipitation (as judged by both the phenol and the AOD biuret colors which were comparable) was obtained (1) by adding a minimum of 10 vol of acetone directly to homogenate or (2) by washing the TCA precipitates with excess acetone, and or (3) by adding TCA in acetom to provide 5 vol of acetone with 575 TCA. With wheat germ, comparable protein valued were obtained by the biuret and the phenol methods after separating proteins with 30 vol of acetone (13). Both the phenol and the AOD biuret color ext,inctions were higher in spinach homogenates than those obtained after TCA precipita-
226
PARVIN,
PANDE,
AND
VENKITASUBRAMANIAN
TABLE RELATION
BETWEEN
4
BIURET COLORS, PHENOL COLORS, AND KJELDAHL 6.25 VALUES IN SPINACH LEAF PREPARATIONS
(Details of acetone treatment
Method
Biuret Gornall AOU
Phenol %N
Total N
+124 +7 -11 100
x
are described in text; other details as for Table 1)
Percentage difference of E;~:‘~‘~) value from corresponding obtained with bovine serum albumin Original h;;;~,nnte*
NITROGEN
value
values Protein
N
+186 +37 $42 78.1
After TCA precipitation
After delipidation
+ 150 -12 +2 78.1
+18 -4 fl 72.4
After acetone treatment
+15 - 3 -1 75.6
QLeaf homogenate (30% wet w/v) was obtained using a Waring Blendor and clarifying the resulting suspension by cenkifugation at 10,000 X g for 10 min.
tion. A need for the TCA precipitation of proteins prior to calorimetry was felt with Euglena also (14). DISCUSSION
The inapplicability of the direct biuret calorimetry has been realized in many instances (12-26). Thus the Cleland and Slater (17) procedure of washing TCA precipitates with ethanol before color development has been adopted by several workers (21-24). Deoxycholate (15, 18, 25, 26) and Triton (19) treatments have also been made to improve the calorimetry. However, use of the Gornall biuret method is being continued (9, 27-34), perhaps because a systematic study urging caution in the use of direct biuret calorimetry has not appeared. Since this procedure gives wrong results, as emphasized in the present study, it is a potential source of error in comparison and evaluation of results of studies in different laboratories. As the AOD or the KCN procedure improved the results, the cause of interference is the persistence of opalescence which contributes to the OD measurement (cf. ref. 5). Although carbohydrates have been considered responsible (1, 4)) TCA treatment which completely extracts glycogen (35) did not minimize interference. Removal or lowering of interference by delipidation shows that lipids are mainly responsible for this trouble. In separate experiments with the mitochondria-free fraction of rat liver preparations, it was also found that interference in the biuret procedure increased when the liver lipid content increased (cf. ref. 20). Layne (36) has recommended ether extraction of the biuret colors to
COLORIMETRIC
PROTEIN
DETERMINATIOX
227
remove interfering turbidity. We find this practice unsuitable. In many cases this minimized t.he extent of error only slightly; further, the reproducibility was poor. Also, we did not find improvement simply by the inclusion of deoxycholate (5-10 mg) 10 min before color development. In addition, in certain cases, depending upon the source, the AOD or the KCN method gave better results than did the procedure of Cleland and Slater (18) because even after delipidation the opalescence persisted. The present study reveals that with different sources, a variable degree of protein nitrogen is lost on delipidation of TCA precipitates. This emphasizes the conclusion of Munro and Downie (37) that the protein content of delipidated residue may be significantly lower than the actual protein present in the original extracts. Both in the phenol and in the AOD biuret methods, the protein colors of rat tissues and Mycobacterium 607 extracts were found to be completely retained in the TCA precipitates. This justifies the assumption that TCA-separated nitrogen is a good measure of total protein and emphasizes that, in t,he Kjeldahl method, use of the total nitrogen as an index of protein can lead to serious errors. For A. niger and more so for spinach preparations, however, the color values were higher with the initial extracts than with the TCA precipitates. Since these color reactions are not entirely specific to prot.eins, the possibility exist,s that, apart from the presence of peptides and prot,eins, nonprecipitable by TCA, other substances (1, 2, 38) could also contribute to color. The discrepancy due to this effect will be minimized by the TCA precipitation of proteins. This will also avoid interference due to (1) certain cations, such as K+ (391 Mn++ (6)) NH,+ (2,36), and (2) other substances, such as tris buffer (40)) reducing substances? (cf. ref. 41), etc. (42). When homogenates develop turbidity on standing, the KCN method appears unsatisfactory (10). This is avoided by the TCA precipitation and subsequent mild alkaline treatment which gives homogeneous protein solutions. At times this also reduced the contribution of the opalescence to the OD measurement and, therefore, made the AOD method more reliable (cf. ref. 5). This opalescence contribution could further be minimized by the inclusion of ethanol--40cjo (v/v)-in the color solutions without affecting the calorimetry. With biological extract,s, unlike with pure proteins, it is desirable to record the biuret colors without unnecessary delay since turbidities appear on standing (cf. ref. 1). In the AOD biuret method, this development of turbidity is less marked in the case of alkaline tartrate controls and hence ‘For this reason in the growth medium of A. Gger the hiuret method could not he used though, surprisingly, the phenol method served well.
228
PARVIN,
PANDE,
AND
VENKITASUBRAMANIAh-
this becomes all the more necessary. This is also unavoidable when alcohol, acetone, or deoxycholate is present. In such cases, development of yellowish color on standing, probably due to Cu++ reduction, invalidates the calorimetry. Finally, whereas the chromogenicity of individual proteins differs in the biuret procedure (3) and more so in the phenol method (2, 43), for mixtures of proteins in biological extracts the chromogenicity was not found to be markedly different, as this was similar to that of the BSA. In such cases, therefore, the use of BSA as reference standard for colorimetric protein determination methods appears adequate. SUMMARY
A study of the applicability of a calorimetric biuret procedure for protein determination to a variety of biological preparation has revealed that the practice of using this procedure is undesirable because markedly apparent high values are obtained in this way. In some cases, this interference was mainly due to lipids. Use of suitable controls to account for the interfering opalescence in biuret color measurements considerably improved the method. Conditions affecting the reliability of the procedure are also described. For crude extracts the calorimetric phenol method was found to be more reliable. However, a prior separation of protein by TCA is often advantageous not alone for Kjeldahl analysis but also for calorimetric estimations. The actual chromogenicity of the protein mixtures of different biological extracts studied was found to be comparable to that of the commonly employed standard protein, bovine serum albumin. ACKNOWLEDGMENTS This work was supported in part by funds from the Indian Council of Medical Research, New Delhi, and Grant No. E-3427, National Institute of Allergy and Infectious Diseases, U. S. P. H. S. We are grateful to Dr. R. Viswanathan, Director of this 1nstitut.e for his interest in this work. The technical assistance of Mr. R. K. Bhatnagar is gratefully acknowledged. REFERENCES S. V., TEWARI, K. K., AND KRISHNAN, P. S., Arch. Mikrobiol. 39, 343 (1961). 2. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J., J. Biol. Chem. 193,265 (1951). 3. GORNALL, A. G., BARADAWILL, C. J., AND DAVID, M. M., J. Biol. Chem. 177, 751 (1949). 4. KHANNA, R., TEWARI, K. K., AND KRISHNAN, P. S., Arch. Mikrobiol. 44, 352 (1962). 5. INCHIOSA, M. A., J. Lab. Clin. Med. 63, 319 (1964). 1. PANDE,
(‘OLORIMETRIC
PROTEIN
DETERMINATION
229
6. PARVIN, R., PANDE, S. V., AND VENKITASUBRAMASIAN, T. A., Biochim. Biophys. Acta !&I, 260 (1964). 7. FOLCH, J., Lms, M., AND STANLEY, G. H. S., J. Biol. Chem. 226, 497 (1957). 8. MCKENZIE, H. A., AND W.~LL.~CE, H. S., Australian J. Chem. 7, 55 (1954). 9. MANSOOR, M., KALYANKAR, G. D., AND TALWAR, G. P., Biochim. Biophys. Actn 77, 307 (1963). 10. KEYSER, J. W., AND VAUGHX, J., Biochem. J. 44, xxii (1949). Il. LI, L. H., AND KING, K. W., Appl. Microbial. 11, 320 (1963). 12. MUKERJI, S. K., S.ww.u,, G. G., ASD KRIRHNAN, P. S., Indian J. Biochem. 1, 36 (1964). 13. JOYCE,B. K., AND GRISOLIA, S., J. Biol. Chem. 235, 2278 (1960). 14. BRAWERMAN, G., AND KONIGSBERG, N., Biochim. Biophys. Acta 43, 374 (1960). 15. RINCLER, R. L., MINAKAMI, S., AND SISGER, T. P., J. Riol. Chem. 238, 801 (1963). 16. FEINSTEIN, R. N., Radiation Research 4, 217 (1956). 17. CLELAND, K. W., AND SLATER, E. C., Biochem. J. 53, 547 (1953). 18. JACOBS, E. E., JACOB, M.. SANADI, D. R., AND BRADLEY, 1,. B., J. Biol. Chem. 223, 147 (1956). 19. NORDLIE, R. C., AND LAHDY, H. A., Biochim. Biophys. Acta 50Y 189 (1961). 20. FREEDLAND, R. A., AND HARPER, A. E., J. Biol. Chem. 228,743 (1957). 21. VAN ROSSUM, G. D. V., Biochim. Biophys. Acta 74, 15 (1963). 22. JOBSIS, F. F., Biochim. Biophys. Acta 74, 60 (1963). 23. BORST, P., Biochim. Biophys. Acta 57, 256 (1962). 24. CHRIST, E. J., AND HULSMANN, W. C., Biochim. Biophys. Acta 66, 72 (1962). 25. CAMERINO, P. W., AND SMITH, L., J. Biol. Chem. 239,2345 (1964). 26. KIMURA, T., AND TOBARI, J., Biochim. Biophys. Acta 73, 399 (1963). 27. GOLDMAN, D. S., J. Bact. 71, 732 (1956). 28. GOLDMAN, D. S., J. Bact. 86,30 (1963). 29. HOMMES, F. A., Biochim. Biophys. Acta 77, 173 (1963). 30. ABRAHAM, S., MATTHEE.. K. J., AND CHAIKOFF, I. L., J. Biol. Chem. 235, 2551 (1960). 31. ABRAHAM, S., MATTHES, K. J., APED CHAIKOFF, I. L., Biochim. Biophys. Acta 76, 357 (1963). 32. HESS, R., AND PEARSE, G. E., Biochim. Biophys. Acta 71, 285 (1963). 33. BORTZ, W., ABRAHAM, S., AND CHAIKOFF, I. L., J. Biol. Chem. 238, 1266 (1963). 34. PYNADATH, T. I., AND KUMAR, S., Biochim. Biophys. Acta $4, 251 (1964). 35. KEMP, A.. AND KITSVAN HEIJNINGEN, A. J. M., Biochem. J. 56, 646 (1954). 36. LAYNE, E., in “Method in Enzymology” (Colowick, S. P., and Kaplan, N. O., eds.), Vol. III, p. 447. Academic Press, Sew York, 1957. 37. MUNRO, H. N., AND DOWNIE, E. D., Arch. Biochem. Biophys. 106, 516 (1964). 38. UPDIKE, S. S., AND DOUNCE, A. L., Anal. Biochem. 6, 1 (1963). 39. SERBAN, M., TANASESCU, D., AND VASU, S., Acud. Rep. Popdare Romine &u&i Cercetari Biochim. 6, 115 (1963). 40. MYODA, T., AND WERKMAN. C. H., Iowa &ate J. sci. 35, 463 (1961). 41. RACUSEN, D., AND JOHNSTONE, D. B., Nature 191,492 (1961). 42. JANECEK, J., AND RICKENBERG, H. V., Biochim. Biophys. Actu 81, 108 (1964). 43. MORTENSON, L. E., Biochim. Biophys. Acta 81, 71 (1964).