ANALYTICAL
BIOCHEMISTRY
A Simplified
69, 55-60 (197.5)
Procedure Residues
to Determine
Tryptophan
in Proteins
YASHWANT D. KARKHANIS’, DENNIS J. CARLO, AND JOHANNA ZELTNER The Merck
Institute
for
Therapeutic
Reseurch.
Rahn>ay,
Nens Jersey
07065
Received February 13, 1975: accepted May 30. 1975 A procedure is described to determine tryptophan residues in proteins using a tryptophan reagent, ?-hydroxy-5nitrobenzyl bromide. The method involves the treatment of the unfolded protein with the reagent in 9 M urea at acid pH: incubation of the mixture at room temperature for 2 hr and the removal of the excess reagent by centrifugation and gel filtration. The amount of tryptophan in a protein is determined from the optical density of the labeled protein at 280 and 410 nm, and from the known optical density of 1 mglml of the protein at 180 nm and of the reagent at 280 and 410 nm. The efficacy of the method was tested with eight proteins whose tryptophan content is known.
2-Hydroxy-5nitrobenzyl bromide is a specific reagent for tryptophan residues in proteins and peptides (I). Although it has been reported that this reagent reacts with --SH groups of proteins at higher pHs (2) this reaction is so slight that it can safely be used to modify tryptophan residues in proteins and peptides. Using this reagent, Barman and Koshland (3) developed a procedure to quantitatively determine tryptophan residues in proteins. In this procedure, a fully unfolded or carboxymethylated protein is treated with the reagent in 10 M urea at acid pH and the excess reagent is removed by gel filtration and TCA-precipitation. The amount of tryptophan is determined from the optical density of the protein at 4 10 nm and from the amount of protein determined from amino acid analysis. Because of several steps involved in this method, a large amount of protein (about 1 pmole) is required to determine tryptophan residues. This method also has a tendency to give a higher number of tryptophan residues than obtained from other procedures (4). Such results are always interpreted as due to a reaction of 2 moles of reagent with one tryptophan residue in the protein (5). In the present paper, we describe a simple procedure by which accurate determination of tryptophan residues in proteins can be made using a small amount of protein. 1 To whom inquiries about the paper should be addressed. 2 Y. D. Karkhanis, unpublished observation. 55 Copyright Q 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
56
KARKHANIS,
MATERIALS
CARLO,
AND
AND
ZELTNER
METHODS
2-Hydroxy-5nitrobenzyl bromide was a gift from British Drug House, London, England. Bio-Gel P-2 was obtained from Bio-Rad Laboratories; Sephadex G-10 and G-25 from Pharmacia Fine Chemicals. Proteins used in this assay were from the following sources: Chymotrypsinogen A, alpha-chymotrypsin, trypsin, trypsinogen, pepsin, and rabbit muscle aldolase from Worthington: rabbit muscle triose phosphate isomerase from P-L Biochemicals; hen egg lysozyme, Grade I, 3X crystallized, dialyzed, and lyophilized, from Sigma. Basic A-l protein from bovine spinal cord was a gift from Mr. Jesse Jackson of our laboratory. Carboxymethylation of lysozyme was carried out according to Crestfield, Moore, and Stein (9). Determination of extinction coeficient of the reagent. Stock solution of the reagent (1.3 X 10-4~) was made by dissolving the reagent in a few drops of methanol and diluting to 100 ml in a volumetric flask with 0.015 M NH,OH in 8 M urea, pH 10.2. Different dilutions of this stock solution were made and were read at 280 and 410 nm. From the slope of the curve, optical density versus concentration, extinction coefficients were determined. This experiment was repeated five times. Assay Procedure 1. Dissolve l-2 mg of protein in 400 ~1 of 10 M urea whose pH is brought below 5 by adding a few drops of acetic acid. Incubate this mixture at room temperature until the protein is completely unfolded; if the protein requires carboxymethylation or aminoethylation, such step should be carried out prior to unfolding. 2. Add 50 ~1 of methanol, mix well, and add 2 mg of reagent. Shake the mixture and allow it to stand at room temperature for 10 min. 3. After two more additions of the reagent every 10 min, incubate the mixture at room temperature for 2 hr. During this interval, occasionally shake the reaction mixture. The reagent reacts with the tryptophan residues in the protein and also undergoes hydrolysis. The hydrolysis gives rise to an insoluble hydroxide derivative of the reagent and a proton; because of proton release, the pH of the reaction mixture decreases. 4. After 2 hr of incubation, centrifuge the reaction mixture in a clinical centrifuge at maximum speed for 2 min. 5. Apply the supernatant to either a Bio-Gel P-2, Sephadex G-10, or G-25 column which is equilibrated with 0.015 N NH,OH in 8 M urea with at least ten times the volume of the column. Incomplete equilibration leads to higher values of tryptophans due to an absorbed reagent on the protein surface. 6. Two bands are seen on the column. The front yellow band which is
TRYPTOPHAN
DETERMINATION
IN
57
PROTEINS
the labeled protein is collected in a test tube: the second smeared band, the hydrolysis product, is discarded.3 Calculations Oh of the yellow label = 18 9 OD,,,O. 14 x OD,,,, Molarity of the protein = MW of protein X E,,,“.“+ Number of tryptophanes = OD,,, X MW of protein X E,,2.1CC Mole of protein 18,700 (OQ,,J - 0. I4 x OD,,, 1 ’
Molarity
RESULTS
The extinction coefficients of the reagent at 280 and 410 nm were 2500 M-‘cm-’ and 18,000 M-‘cm-‘, respectively. This shows that in a protein modified with 2-hydroxy-Snitrobenzyl bromide, there is a substantial contribution due to yellow label at 280 nm; therefore, during calculations such contribution was taken into consideration. 100
075
0.25
Tube
number
on P-2 column I. Elution of tryptophan-labeled yellow chymotrypsinogen (20 x I cm). The reservoir solution contained 0.015 M NH,OH in 8 M urea, pH 10.2. The flow rate was 12 ml/hr. Each fraction was 0.5 ml in volume. After mixing with 0.5 ml reservoir solution, the optical density of the solution at 280 and 410 nm was read. Tube number 6, on dilution, gave optical densities 0.309 and 0.613 at 280 and 410 nm, respectively. Tubes I l-14 which contained excess reagent gave optical density above 3 at both these wavelengths. FIG.
B A third yellow trobenzyl hydroxide finally merges with
band is obtained which is due to a small amount of 2-hydroxy-Sniwhich settles at the top of the column and is eluted slowly. This band the second band.
58
KARKHANIS,
CARLO,
AND
TABLE TRYPTOPHAN
ANALYSIS
Protein
OF PROTEINS
ZELTNER
1 OF KNOWN
TRYPTOPHAN
Authors’ method 3.2 6.1 4.1 4.2 7.9 8.1 7.9 5.8 11.8 1.0
Lysozyme Carboxymethyl lysozyme Trypsin Trypsinogen Chymotrypsin Chymotrypsinogen Triose phosphate isomerase Pepsin Aldolase Basic A-l protein
CONTENT
Literature 6 6 4 4 8 8 8 6 12 1.0
Figure 1 shows the elution profile of tryptophan-labeled chymotrypsinogen. It can be seen that there is a good separation between the labeled protein and excess reagent. The advantage of using ammonium hydroxide in 8 M urea was that the protein band could be seen and collected in a test tube. Another advantage of this chromatography is that the reagent is eluted very slowly at high pH and facilitates excellent separation. A large volume of the reservoir buffer is needed to elute all the excess reagent. Table 1 shows the application of the method to the proteins whose tryptophan content is known. Only lysozyme gave a lower value because of incomplete unfolding. This was shown by the fact that after carboxymethylation was carried out, the correct number of tryptophans were obtained. DISCUSSION
We have developed a simplified procedure to determine tryptophan residues in proteins. This procedure differs from that of Barman and Koshland (3) in that our procedure involves only three steps: (1) labeling the protein with the reagent; (2) separation of the labeled protein from excess reagent by centrifugation and gel filtration; and (3) reading the optical density of the labeled protein at 280 and 410 nm. The tryptophan content of the protein is determined from the known extinction coefficient of the reagent at 280 and 410 nm and from the known optical density of the protein at 280 nm at a 1 mg/ml concentration. On the other hand, the Barman and Koshland procedure (3), which uses this reagent, involves not only several steps but requires higher amounts of protein for tryptophan determination. In our procedure the tryptophan content was determined directly from the optical density; typically only l-2 mg of protein is needed for a tryptophan determination. A single tryptophan
TRYPTOPHAN
DETERMINATION
IN
PROTEINS
59
residue present in the basic protein (6) was easily determined using this procedure. Whether the tryptophan content of a protein is high or low it can easily be determined from the uv absorbance. The unfolding of the protein prior to labeling is very important and varies with different proteins. Lysozyme could not be labeled completely; only after carboxymethylation was the correct number of tryptophans obtained. The resistance of lysozyme to unfolding has been reported by Tanford, Pain, and Otchin (7). With the other proteins used in this study, 10 M urea at acid pH was sufficient for unfolding. Under the experimental conditions mentioned, proteins having free -SH groups did not show any reaction of its -SH groups with the reagent. The use of ammonium hydroxide in 8 M urea as an eluent has the advantage in that one can see the yellow protein band moving in front of the excess reagent. This eliminates the use of a fraction collector and is advantageous in making an appropriate cut. Another advantage of elution at pH 10 is that the hydrolyzed reagent is highly ionized and has a tendency to stick to gel matrix. This gives excellent separation between the labeled protein and excess reagent. This effect of binding of the reagent to gel matrix is more pronounced when Sephadex G-l 0 and G-25 are used. Three additions of the reagent to the reaction mixture and shorter incubation, rather than one large addition and longer incubation, deserves comment. When the latter condition was used, we found a large amount of reagent absorbed on the surface which could not be removed after chromatography, TCA precipitation, ethanol washing, and extensive dialysis. Whenever it was possible to remove this excess reagent, we found that the labeling was incomplete. This is due to faster hydrolysis of the reagent. Since the reagent undergoes two reactions, hydrolysis and reaction with tryptophan residues, it is better to add it three times to achieve complete labeling of the protein rather than making one large addition. With stepwise addition a shorter incubation period is required and the reaction with tryptophan residues is complete. The amino acid analysis of the protein to determine the amount of protein was not necessary, since for most of the purified proteins data on optical density of 1 mg/ml solution is either available or can be accurately determined. An extra step of amino acid analysis for protein estimation can introduce errors in the correct determination of tryptophan residues in proteins since several steps are involved in amino acid analysis. We have applied this procedure to triose phosphate isomerase and obtained eight residues of tryptophan per mole of the protein: this is consistent with the results obtained by Miller and Waley (4) using other methods. Using the procedure of Barman and Koshland (3), these workers obtained 12 residues of tryptophan. The labeling of the protein
60
KARKHANIS,
CARLO,
AND ZELTNER
was done under similar conditions as ours with the exception of a single addition of the reagent and longer incubation. Since no reaction with -SH groups was shown, we think that the higher number of tryptophan residues obtained must be due to absorption of the reagent on the surface of the protein. The interpretation of disubstituted product given by the authors does not explain why 16 tryptophans were not obtained. With our procedure, we found no evidence of disubstituted product formation (8) #ith any of the proteins tested, as shown in Table 1. The disubstitution would have led to the determination of higher number of tryptophan residues in this protein. Because of simplicity and rapidity of the method, using our procedure, one should be able to determine the number of tryptophan residues in a protein in less than 3 hr after the unfolding stage. Not only can this method be used for tryptophan analysis, but also may be used to label tryptophan residues in proteins and peptides for active site and sequence studies. REFERENCES 1. Koshland, D. E., Karkhanis, Y. D., and Latham, H. G. (1964) J. Amer. Chem. Sot. 86, 1448. 2. Horton, H. R., and Koshland, D. E., Jr. (1965) J. Amer. Chem. Sot. 87, 1126. 3. Barman, T. E., and Koshland, D. E. (1967) J. Biol. Chem. 242, 5771. 4. Miller, J. C., and Waley, S. G. (1971) Biochem. J. 122, 209. 5. Scoffone, E., and Fontana, A. (1970) in Protein Sequence Determination (Needleman, S., ed.), Vol. 8, pp. 185-210, Springer-Verlag, New York. 6. Karkhanis, Y. D., Carlo, D. J., Brostoff, S. W., and Eylar, E. H. (1975) J. Biol. Chem. 250, 1718. 7. Tanford, C., Pain, R. H., and Otchin, N. S. (1966) J. Mol. Biol. 15, 489. 8. Louden, G. M., and Koshland, D. E. (1970) J. Biol. Chem. 245, 2267. 9. Crestfield, A. M., Moore, S., and Stein, W. H. (1963) /. Biol. Chem. 238, 622.
BIOCHEMISTRY
A Simplified
69, 55-60 (197.5)
Procedure Residues
to Determine
Tryptophan
in Proteins
YASHWANT D. KARKHANIS’, DENNIS J. CARLO, AND JOHANNA ZELTNER The Merck
Institute
for
Therapeutic
Reseurch.
Rahn>ay,
Nens Jersey
07065
Received February 13, 1975: accepted May 30. 1975 A procedure is described to determine tryptophan residues in proteins using a tryptophan reagent, ?-hydroxy-5nitrobenzyl bromide. The method involves the treatment of the unfolded protein with the reagent in 9 M urea at acid pH: incubation of the mixture at room temperature for 2 hr and the removal of the excess reagent by centrifugation and gel filtration. The amount of tryptophan in a protein is determined from the optical density of the labeled protein at 280 and 410 nm, and from the known optical density of 1 mglml of the protein at 180 nm and of the reagent at 280 and 410 nm. The efficacy of the method was tested with eight proteins whose tryptophan content is known.
2-Hydroxy-5nitrobenzyl bromide is a specific reagent for tryptophan residues in proteins and peptides (I). Although it has been reported that this reagent reacts with --SH groups of proteins at higher pHs (2) this reaction is so slight that it can safely be used to modify tryptophan residues in proteins and peptides. Using this reagent, Barman and Koshland (3) developed a procedure to quantitatively determine tryptophan residues in proteins. In this procedure, a fully unfolded or carboxymethylated protein is treated with the reagent in 10 M urea at acid pH and the excess reagent is removed by gel filtration and TCA-precipitation. The amount of tryptophan is determined from the optical density of the protein at 4 10 nm and from the amount of protein determined from amino acid analysis. Because of several steps involved in this method, a large amount of protein (about 1 pmole) is required to determine tryptophan residues. This method also has a tendency to give a higher number of tryptophan residues than obtained from other procedures (4). Such results are always interpreted as due to a reaction of 2 moles of reagent with one tryptophan residue in the protein (5). In the present paper, we describe a simple procedure by which accurate determination of tryptophan residues in proteins can be made using a small amount of protein. 1 To whom inquiries about the paper should be addressed. 2 Y. D. Karkhanis, unpublished observation. 55 Copyright Q 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
56
KARKHANIS,
MATERIALS
CARLO,
AND
AND
ZELTNER
METHODS
2-Hydroxy-5nitrobenzyl bromide was a gift from British Drug House, London, England. Bio-Gel P-2 was obtained from Bio-Rad Laboratories; Sephadex G-10 and G-25 from Pharmacia Fine Chemicals. Proteins used in this assay were from the following sources: Chymotrypsinogen A, alpha-chymotrypsin, trypsin, trypsinogen, pepsin, and rabbit muscle aldolase from Worthington: rabbit muscle triose phosphate isomerase from P-L Biochemicals; hen egg lysozyme, Grade I, 3X crystallized, dialyzed, and lyophilized, from Sigma. Basic A-l protein from bovine spinal cord was a gift from Mr. Jesse Jackson of our laboratory. Carboxymethylation of lysozyme was carried out according to Crestfield, Moore, and Stein (9). Determination of extinction coeficient of the reagent. Stock solution of the reagent (1.3 X 10-4~) was made by dissolving the reagent in a few drops of methanol and diluting to 100 ml in a volumetric flask with 0.015 M NH,OH in 8 M urea, pH 10.2. Different dilutions of this stock solution were made and were read at 280 and 410 nm. From the slope of the curve, optical density versus concentration, extinction coefficients were determined. This experiment was repeated five times. Assay Procedure 1. Dissolve l-2 mg of protein in 400 ~1 of 10 M urea whose pH is brought below 5 by adding a few drops of acetic acid. Incubate this mixture at room temperature until the protein is completely unfolded; if the protein requires carboxymethylation or aminoethylation, such step should be carried out prior to unfolding. 2. Add 50 ~1 of methanol, mix well, and add 2 mg of reagent. Shake the mixture and allow it to stand at room temperature for 10 min. 3. After two more additions of the reagent every 10 min, incubate the mixture at room temperature for 2 hr. During this interval, occasionally shake the reaction mixture. The reagent reacts with the tryptophan residues in the protein and also undergoes hydrolysis. The hydrolysis gives rise to an insoluble hydroxide derivative of the reagent and a proton; because of proton release, the pH of the reaction mixture decreases. 4. After 2 hr of incubation, centrifuge the reaction mixture in a clinical centrifuge at maximum speed for 2 min. 5. Apply the supernatant to either a Bio-Gel P-2, Sephadex G-10, or G-25 column which is equilibrated with 0.015 N NH,OH in 8 M urea with at least ten times the volume of the column. Incomplete equilibration leads to higher values of tryptophans due to an absorbed reagent on the protein surface. 6. Two bands are seen on the column. The front yellow band which is
TRYPTOPHAN
DETERMINATION
IN
57
PROTEINS
the labeled protein is collected in a test tube: the second smeared band, the hydrolysis product, is discarded.3 Calculations Oh of the yellow label = 18 9 OD,,,O. 14 x OD,,,, Molarity of the protein = MW of protein X E,,,“.“+ Number of tryptophanes = OD,,, X MW of protein X E,,2.1CC Mole of protein 18,700 (OQ,,J - 0. I4 x OD,,, 1 ’
Molarity
RESULTS
The extinction coefficients of the reagent at 280 and 410 nm were 2500 M-‘cm-’ and 18,000 M-‘cm-‘, respectively. This shows that in a protein modified with 2-hydroxy-Snitrobenzyl bromide, there is a substantial contribution due to yellow label at 280 nm; therefore, during calculations such contribution was taken into consideration. 100
075
0.25
Tube
number
on P-2 column I. Elution of tryptophan-labeled yellow chymotrypsinogen (20 x I cm). The reservoir solution contained 0.015 M NH,OH in 8 M urea, pH 10.2. The flow rate was 12 ml/hr. Each fraction was 0.5 ml in volume. After mixing with 0.5 ml reservoir solution, the optical density of the solution at 280 and 410 nm was read. Tube number 6, on dilution, gave optical densities 0.309 and 0.613 at 280 and 410 nm, respectively. Tubes I l-14 which contained excess reagent gave optical density above 3 at both these wavelengths. FIG.
B A third yellow trobenzyl hydroxide finally merges with
band is obtained which is due to a small amount of 2-hydroxy-Sniwhich settles at the top of the column and is eluted slowly. This band the second band.
58
KARKHANIS,
CARLO,
AND
TABLE TRYPTOPHAN
ANALYSIS
Protein
OF PROTEINS
ZELTNER
1 OF KNOWN
TRYPTOPHAN
Authors’ method 3.2 6.1 4.1 4.2 7.9 8.1 7.9 5.8 11.8 1.0
Lysozyme Carboxymethyl lysozyme Trypsin Trypsinogen Chymotrypsin Chymotrypsinogen Triose phosphate isomerase Pepsin Aldolase Basic A-l protein
CONTENT
Literature 6 6 4 4 8 8 8 6 12 1.0
Figure 1 shows the elution profile of tryptophan-labeled chymotrypsinogen. It can be seen that there is a good separation between the labeled protein and excess reagent. The advantage of using ammonium hydroxide in 8 M urea was that the protein band could be seen and collected in a test tube. Another advantage of this chromatography is that the reagent is eluted very slowly at high pH and facilitates excellent separation. A large volume of the reservoir buffer is needed to elute all the excess reagent. Table 1 shows the application of the method to the proteins whose tryptophan content is known. Only lysozyme gave a lower value because of incomplete unfolding. This was shown by the fact that after carboxymethylation was carried out, the correct number of tryptophans were obtained. DISCUSSION
We have developed a simplified procedure to determine tryptophan residues in proteins. This procedure differs from that of Barman and Koshland (3) in that our procedure involves only three steps: (1) labeling the protein with the reagent; (2) separation of the labeled protein from excess reagent by centrifugation and gel filtration; and (3) reading the optical density of the labeled protein at 280 and 410 nm. The tryptophan content of the protein is determined from the known extinction coefficient of the reagent at 280 and 410 nm and from the known optical density of the protein at 280 nm at a 1 mg/ml concentration. On the other hand, the Barman and Koshland procedure (3), which uses this reagent, involves not only several steps but requires higher amounts of protein for tryptophan determination. In our procedure the tryptophan content was determined directly from the optical density; typically only l-2 mg of protein is needed for a tryptophan determination. A single tryptophan
TRYPTOPHAN
DETERMINATION
IN
PROTEINS
59
residue present in the basic protein (6) was easily determined using this procedure. Whether the tryptophan content of a protein is high or low it can easily be determined from the uv absorbance. The unfolding of the protein prior to labeling is very important and varies with different proteins. Lysozyme could not be labeled completely; only after carboxymethylation was the correct number of tryptophans obtained. The resistance of lysozyme to unfolding has been reported by Tanford, Pain, and Otchin (7). With the other proteins used in this study, 10 M urea at acid pH was sufficient for unfolding. Under the experimental conditions mentioned, proteins having free -SH groups did not show any reaction of its -SH groups with the reagent. The use of ammonium hydroxide in 8 M urea as an eluent has the advantage in that one can see the yellow protein band moving in front of the excess reagent. This eliminates the use of a fraction collector and is advantageous in making an appropriate cut. Another advantage of elution at pH 10 is that the hydrolyzed reagent is highly ionized and has a tendency to stick to gel matrix. This gives excellent separation between the labeled protein and excess reagent. This effect of binding of the reagent to gel matrix is more pronounced when Sephadex G-l 0 and G-25 are used. Three additions of the reagent to the reaction mixture and shorter incubation, rather than one large addition and longer incubation, deserves comment. When the latter condition was used, we found a large amount of reagent absorbed on the surface which could not be removed after chromatography, TCA precipitation, ethanol washing, and extensive dialysis. Whenever it was possible to remove this excess reagent, we found that the labeling was incomplete. This is due to faster hydrolysis of the reagent. Since the reagent undergoes two reactions, hydrolysis and reaction with tryptophan residues, it is better to add it three times to achieve complete labeling of the protein rather than making one large addition. With stepwise addition a shorter incubation period is required and the reaction with tryptophan residues is complete. The amino acid analysis of the protein to determine the amount of protein was not necessary, since for most of the purified proteins data on optical density of 1 mg/ml solution is either available or can be accurately determined. An extra step of amino acid analysis for protein estimation can introduce errors in the correct determination of tryptophan residues in proteins since several steps are involved in amino acid analysis. We have applied this procedure to triose phosphate isomerase and obtained eight residues of tryptophan per mole of the protein: this is consistent with the results obtained by Miller and Waley (4) using other methods. Using the procedure of Barman and Koshland (3), these workers obtained 12 residues of tryptophan. The labeling of the protein
60
KARKHANIS,
CARLO,
AND ZELTNER
was done under similar conditions as ours with the exception of a single addition of the reagent and longer incubation. Since no reaction with -SH groups was shown, we think that the higher number of tryptophan residues obtained must be due to absorption of the reagent on the surface of the protein. The interpretation of disubstituted product given by the authors does not explain why 16 tryptophans were not obtained. With our procedure, we found no evidence of disubstituted product formation (8) #ith any of the proteins tested, as shown in Table 1. The disubstitution would have led to the determination of higher number of tryptophan residues in this protein. Because of simplicity and rapidity of the method, using our procedure, one should be able to determine the number of tryptophan residues in a protein in less than 3 hr after the unfolding stage. Not only can this method be used for tryptophan analysis, but also may be used to label tryptophan residues in proteins and peptides for active site and sequence studies. REFERENCES 1. Koshland, D. E., Karkhanis, Y. D., and Latham, H. G. (1964) J. Amer. Chem. Sot. 86, 1448. 2. Horton, H. R., and Koshland, D. E., Jr. (1965) J. Amer. Chem. Sot. 87, 1126. 3. Barman, T. E., and Koshland, D. E. (1967) J. Biol. Chem. 242, 5771. 4. Miller, J. C., and Waley, S. G. (1971) Biochem. J. 122, 209. 5. Scoffone, E., and Fontana, A. (1970) in Protein Sequence Determination (Needleman, S., ed.), Vol. 8, pp. 185-210, Springer-Verlag, New York. 6. Karkhanis, Y. D., Carlo, D. J., Brostoff, S. W., and Eylar, E. H. (1975) J. Biol. Chem. 250, 1718. 7. Tanford, C., Pain, R. H., and Otchin, N. S. (1966) J. Mol. Biol. 15, 489. 8. Louden, G. M., and Koshland, D. E. (1970) J. Biol. Chem. 245, 2267. 9. Crestfield, A. M., Moore, S., and Stein, W. H. (1963) /. Biol. Chem. 238, 622.
