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A new convenient method for estimation of total cystine-cysteine in proteins
SHORT
A New
18.5
COMMUNICATIOT;S
Convenient Method Cystine-Cysteine
for Estimation in Proteins1
of Total
The most commonly used method for determination of cystine and cysteine in proteins is based on oxtdation of these amino acids to cysteic acid. Among a variety of strong oxidizing agents suitable for this reaction performic acid has been csed almost exclusively in the past (l-3). The procedure for cystine and cysteine determination thus includes the following steps: preparation of performic acid, csidation of protein and lyophilization, followed by the normal acid hydrolysis and amino acid analysis. The conversion to cysteic acid under these conditions is 85-90% (3). A more recent procedure employing HBr as a reducing agent to destroy excess performic acid before hydrolysis has given yields of 95% of cysteic acid and also permitted quantitative conversion of methionine to methionine sulfone (4). We recently found that cystine and cysteine are converted to cysteic acid when the acid hydrolysis is simply carried out in the presence of low concentrations of dimethyl sulfoxide (DMSO) . The oxidation under these conditions appears to be general, and should provide a convenient, time-saving alternative method for cystine and cysteine determination. Experimental Procedure. Standard hydrolysis procedures and amino acid analysis were used. The samples, containing either proteins or standard amino acid mixtures (equimolar quantities), were hydrolyzed in 2 ml volumes of 6 N HCl (duPont or Baker, AR) either in the absence or in the presence of DMSO (reagent grade). The samples were evacuated to less than 0.1 mm Hg and sealed. After 21 hr at 110”, the samples were taken to dryness on a flash evaporator connected to a vacuum pump. The bath temperature was maintained at 40” and the removal of HCI was completed in less than 10 min. In our experience, this step can lead to extensive destruction of some amino acids (notably methionine and cysteine) and must be carefully controlled for reproducible results. Amino acid analyses were performed with a Spinco amino acid analyzer model 12OC, with an automatic peak integrator. The integration constant for cysteic acid was determined from several runs with cysteic acid (Mann analyzed reagent) standards. In all experiments, the cysteic acid constant differed from that of aspartic acid by less than ‘Supported
by
Research
Grant
GM
15053
from
the
U. S. Public
Health
Service.
186
SHORT
COMMUNICATIONS
2%, and we have thus routinely used the aspartic acid integration constant for the quantification of both aspartic acid and cysteic acid. The identity of the product of cystine and cysteine oxidation as cysteic acid was confirmed by high-voltage paper electrophoresis with 2.5% formic acid and with pyridine/acetic acid/water (5/0.2/95, pH 6.5) solvent systems (Brinkman Pherograph) and by paper chromatography in n-butanol/acetic acid/water (3/2/l). In all systems the oxidation product showed identity to authentic cysteic acid. All the proteins used in this study were commercial preparations which were analyzed without further purification. Results. Hydrolysis in the presence of DMSO leads to destruction of several of the natural amino acids. Thus, in addition to the oxidation of cystine and cysteine, tyrosine is also quantitatively modified and is completely absent in the chromatograms, and histidine is modified to the extent that the histidine recovery is less than 10%. The methionine recovery varies considerably from protein to protein but is also very low, while serine, threonine, and proline quite consistently give a recovery value 5-10s lower than that for normal acid hydrolysis. No attempt has been made to characterize the products of the modified amino acids. It has been noted, however, that tyrosine disappearance is accompanied by the appearance of two new peaks on the chromatogram, one right in front of and one right behind the isoleucine-leucine peaks. Simple oxidative chlorination of tyrosine would give derivatives eluting much later. The remaining amino acids do not appear to be affected. by the presence of DMSO during hydrolysis, Because of this ‘destruction of several amino acids, the modified DMSO-HCI hydrolysis cannot be used by itself for amino acid determination. The complete amino acid analysis of a protein must be determined from a normal DMSO-free hydrolyzate, and the DMSO-HCl hydrolyzates furnish the analysis for cystine plus cysteine as the only unique bit of information. Because of their stability in the DMSO-HCI hydrolysis, aspartic acid and alanine provide convenient reference compounds for the cahulation of molar equivalents of cysteic acid in the unknown protein. All the data presented in this report are based on calculations of cysteic acid content relative to a known content of aspartic acid and of alanine (both values are included) and on the basis of 100% recovery of the two reference amino acids. For routine cysteic acid determinations, only the first buffer of the normal long column analyses was run, and all the necessary data for cysteic acid analysis could thus be obtained in 100 min. Quantitative amino acid analyses of standard amino acid mixtures exposed to DMSO-HCl hydrolysis are given in Table 1. In all cases a hydrolysis volume of 2 ml was used containing a total of 0.125 pmole of
SHORT
187
COMMUNICATIONS
TABLE 1 Recoveries of Cysteine as Cysteic Acid after “Hydrolysis” Amino Acid Mixtures in the Presence of DMSO DMSO
of
concentration Cysteic acid found, mM
Pi-CYS
ml/2 ml tot. vol.
mMa
0 0.001 0.005 0.010 0.030 0.050 0.030 0.050
0 7.05 35.25 70.5 211.5 352.5 211.5 352.5
added, b mM
Cys AC
0.0625 0.0625 0.0625 0.0625 0.0625 0.0625 0.3125 0.3125
0.005 0.0282 0.0403 0.0593 0.0670 0.0624 0.2955 0.3088
% 8 45 65 95 107 100 95 99
Cys Ad
%
0.028 0.0412
44 66
0.0635 0.0627 0.299 0.311
102 100 96 99
a Calculated on basis of MW 78.14 and density (25”) 1.0958 (5). b The amino acid mixtures contained equimolar amounts of 17 amino acids, all at the level of 0.0625 m&f. In the last two experiments an additional 0.25 pmole of cysteine was added per ml hydrolysate. c Cysteic acid calculated relative to aspartic acid (0.0625 m&f). d Cysteic acid calculated relative to alanine (0.0625 m&f).
each of the common 17 amino acids. In experiments 7 and 8, an additional 0.5 pLmole of cysteine was added to bring the total of that amino acid to 0.625 pmole/2 ml. At the lower concentrations of DMSO the conversion of cysteine to cysteic acid was incomplete, but at the level of 0.2435 M DMSO the conversion was quantitative. Based on the results in Table 1, a fixed hydrolysis reagent containing 0.21 M DMSO in 6 M HCl was used in the hydrolysis of several proteins of known amino acid content. The proteins (l-2 mg) were hydrolyzed in volumes of 2 ml. The results are given in Table 2, and, allowing for normal experimental error for this type of analysis (1+3% for both cysteic acid and the reference amino acids), most of the analyses show that the conversion of cystine and cysteine to cysteic acid is quantitative. The only significant discrepancy between t,he results in Table 2 and well-established literature values is the consistently low analysis for chymotrypsinogen. In this particular case the protein purity had been established, so the deviation cannot be explained on the basis of impurities. The only known unique feature of cystine in chymotrypsinogen is the fact that one l/2-cys occupies the N-terminal position in this protein. There is no obvious reason why this should affect the DMSO oxidation of that residue, however. There does not appear to be a significant difference in the cysteic acid yield from proteins containing only cystine
(source)
a Wham b Cyst& L Cyst&c
MW
13.6 46.0 64.0 23.0 142.0 5.7 15.0 248 .o 34.0
of Several
15 32 54 22 107 3 21 281 40
Asp 12 35 46 22 157 3 12 158 16
Ala 8 6-7 34-36 10 29 6 8 22-B 6
M-Cys”
Moles amino acid/mole
Analysis
7 8 9 10 11 12 13 14 15
Ref.
italicized
8.17 6.96 33.5 8.55 29.4
CysAb
TABLE Proteins
(see 61, a range is given. The acid (moles/mole protein). (moles/mole protein).
X 10-a
Acid
discrepancies exist in the literature acid calculated relative to aspartio acid calculated relative to alanine
Ribonuclease (bovine pancreas) Ovalbumin (hen ep~) Serum albumin (bovine) Chymotrypeinosen A (bovine) Aldolase (rabbit muacle) rnsulii (bovine) Lylwyme @en BE%) catalase bovine liver) Pepsin (bovine)
Protein
Cysteic
value
102 99 93 86 101
theor.
70
Expt.
1
is used
7.62 7.47 36.0 8.92 29.2
CysAc
2 Hydrolyzed
8.22 7.07 32 6 9.1 28.7 5.95 7.4 23.5
CysAb
%
7.93 6.95 33.0 8.57 31.2 5.47 7.35 25.0
of To theory.
103 101 91 91 99 99 92 84
2
found
CysAc
acid
of DMSO
Expt.
theor.
Cyst&c
Presence
for calculation
95 107 100 89 101
theor.
%
in the
%
99 99 92 86 108 91 92 89
theor.
%
95 107 99 99 104
30.9 5.95 7.9 6.22
95
theor.
34.3
7.57
CysAa
Expt.
6.18
7.88
27.9 5.77
35.0
7.92
CysAc
3 %
103
99
96 96
97
99
theor.
F :!
2
SHORT
COMMUNICATIONS
189
(ribonuciease, bovine serum albumin, insulin, lysozyme) a.nd from those containing primarily cysteine (aldolase and ovalbumin) , nor does the presence of carbohydrate in ovalbumin affect the analysis. Based on these results, it would appear that the simple procedure of hydrolyzing proteins in the presence of DMSO will give values for total cystine and cysteine content as good as those obtained after performic acid oxidation. Discussion. We have made no attempt to elucidate the chemical mechanisms involved in the amino acid modification by DMSO. The oxidative properties of DMSO are well documented (16, 17) and the acidcatalyzed thiomethoxymethylation of aromatic alcohols is also well established (18, 19). There should therefore be no problem in explaining the observed modifications in terms of known DMSO reactions. It may be of interest to note that in the experiments in Table 1 where the yield of cyst&c acid was low (at low levels of DMSO) the cysteine peak was completely absent, indicating the some intermediate may be formed in the oxidation. Summary. Hydrolysis of proteins in 6 N HCl containing 0.2-0.3M dimethyl sulfoxide leads to quantitative oxidation of cystine and cysteine to cysteic acid, which can be determined by direct amino analysis. Because of alteration of several other amino acids under these hydrolysis conditions, the rest of the amino acids must be determined from parallel hydrolyzates carried out in the conventional manner in the absence of dimcthyl sulfoxide. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10.
11. 12.
13. 14. 15. 16.
17.
SANGEB, F., HIRS, C. H.
Biochem. J. 44, 126 (1949). W., .l. BioZ. Chem. 219,611 (1956) HIRS, C. H. W., Method-s in Enzymol. 11, 197 (1967). MOORE, S., J. Biol. Chem. 238, 235 (1963). MACGREGOR, W. S., Ann, N. Y. Acad. Sci. 141, Article 1, 3 (1967). CECIL, R., AND MCPHEE, J. R., Advan. Prot. Chem. 14, 255 (1959). SIUTH, D. G., STEIN, W. G., AND MOORE, S., J. Bill. Chem. 238, 227 (1963). LEWIS, J. C.. SNELL. T\T. S., HIHSCHMAN, D. J., AND FRAENKEL-CONRAT, H., J. Biol. Chem. 186, 23 (1950). SPAHR, P. F., AND EDSALL, J. T., J. Biol. Chem. 239,850 (1964). WILCOX, P. E., COHEN, E., END TAN, W., J. Biot. Chem. 228, 999 (1957). SHI~ZU, H., AND OZAWJ, H., Biochim. Biophys. Acta 133, 195 (1967). RYLE, A. P., S.~NGER, F., SMITH, L. F., AND KITAI, R., Biochem. J. 60, 541 (1955). CANFIELD, R. E., AND LIU, A. K., J. Biol. Chem. 240, 1997 (1965). R.ADHAKRISHNAN, T. H., AND SARMA, P. S., B&hem. J. 97, 827 (1965). RAJAOOPAL.AN, T. G., MOORE, S., AND STEIN, W. H., J. Biol. Chem. 241,494O (1966). PFITZNER, K. E., AND MOETATT, J. G.. J. Am. Chem. Sot. 87, 5661 (1965). ALBRIGHT, J. D., AND GOLDMAN, L., J. Am. Chem. Sot. 87, 4214 (1965).
190
SHORT
18. BURDON, 19. PFPZNER, WtW.
COMMUNICATIONS
M. G., AND MOFTATT, J. G., K. E., MARINO, J. P., AND
J. A’m. Chem. Sot. 87, 4656 (1965). OLOFSON, R. A., J. Am. Chem. Sot. 87, 4658
L.
RICHARD FINN
of Biochemistry University of Minnesota Medical Minneapolis, Minnesota 66.&6 Received May 6,1&W
WOLD
Department
’
National Institute
of General
School
Medical
Sciences
Postdoctoral
Fellow
SPENCER~
A New
18.5
COMMUNICATIOT;S
Convenient Method Cystine-Cysteine
for Estimation in Proteins1
of Total
The most commonly used method for determination of cystine and cysteine in proteins is based on oxtdation of these amino acids to cysteic acid. Among a variety of strong oxidizing agents suitable for this reaction performic acid has been csed almost exclusively in the past (l-3). The procedure for cystine and cysteine determination thus includes the following steps: preparation of performic acid, csidation of protein and lyophilization, followed by the normal acid hydrolysis and amino acid analysis. The conversion to cysteic acid under these conditions is 85-90% (3). A more recent procedure employing HBr as a reducing agent to destroy excess performic acid before hydrolysis has given yields of 95% of cysteic acid and also permitted quantitative conversion of methionine to methionine sulfone (4). We recently found that cystine and cysteine are converted to cysteic acid when the acid hydrolysis is simply carried out in the presence of low concentrations of dimethyl sulfoxide (DMSO) . The oxidation under these conditions appears to be general, and should provide a convenient, time-saving alternative method for cystine and cysteine determination. Experimental Procedure. Standard hydrolysis procedures and amino acid analysis were used. The samples, containing either proteins or standard amino acid mixtures (equimolar quantities), were hydrolyzed in 2 ml volumes of 6 N HCl (duPont or Baker, AR) either in the absence or in the presence of DMSO (reagent grade). The samples were evacuated to less than 0.1 mm Hg and sealed. After 21 hr at 110”, the samples were taken to dryness on a flash evaporator connected to a vacuum pump. The bath temperature was maintained at 40” and the removal of HCI was completed in less than 10 min. In our experience, this step can lead to extensive destruction of some amino acids (notably methionine and cysteine) and must be carefully controlled for reproducible results. Amino acid analyses were performed with a Spinco amino acid analyzer model 12OC, with an automatic peak integrator. The integration constant for cysteic acid was determined from several runs with cysteic acid (Mann analyzed reagent) standards. In all experiments, the cysteic acid constant differed from that of aspartic acid by less than ‘Supported
by
Research
Grant
GM
15053
from
the
U. S. Public
Health
Service.
186
SHORT
COMMUNICATIONS
2%, and we have thus routinely used the aspartic acid integration constant for the quantification of both aspartic acid and cysteic acid. The identity of the product of cystine and cysteine oxidation as cysteic acid was confirmed by high-voltage paper electrophoresis with 2.5% formic acid and with pyridine/acetic acid/water (5/0.2/95, pH 6.5) solvent systems (Brinkman Pherograph) and by paper chromatography in n-butanol/acetic acid/water (3/2/l). In all systems the oxidation product showed identity to authentic cysteic acid. All the proteins used in this study were commercial preparations which were analyzed without further purification. Results. Hydrolysis in the presence of DMSO leads to destruction of several of the natural amino acids. Thus, in addition to the oxidation of cystine and cysteine, tyrosine is also quantitatively modified and is completely absent in the chromatograms, and histidine is modified to the extent that the histidine recovery is less than 10%. The methionine recovery varies considerably from protein to protein but is also very low, while serine, threonine, and proline quite consistently give a recovery value 5-10s lower than that for normal acid hydrolysis. No attempt has been made to characterize the products of the modified amino acids. It has been noted, however, that tyrosine disappearance is accompanied by the appearance of two new peaks on the chromatogram, one right in front of and one right behind the isoleucine-leucine peaks. Simple oxidative chlorination of tyrosine would give derivatives eluting much later. The remaining amino acids do not appear to be affected. by the presence of DMSO during hydrolysis, Because of this ‘destruction of several amino acids, the modified DMSO-HCI hydrolysis cannot be used by itself for amino acid determination. The complete amino acid analysis of a protein must be determined from a normal DMSO-free hydrolyzate, and the DMSO-HCl hydrolyzates furnish the analysis for cystine plus cysteine as the only unique bit of information. Because of their stability in the DMSO-HCI hydrolysis, aspartic acid and alanine provide convenient reference compounds for the cahulation of molar equivalents of cysteic acid in the unknown protein. All the data presented in this report are based on calculations of cysteic acid content relative to a known content of aspartic acid and of alanine (both values are included) and on the basis of 100% recovery of the two reference amino acids. For routine cysteic acid determinations, only the first buffer of the normal long column analyses was run, and all the necessary data for cysteic acid analysis could thus be obtained in 100 min. Quantitative amino acid analyses of standard amino acid mixtures exposed to DMSO-HCl hydrolysis are given in Table 1. In all cases a hydrolysis volume of 2 ml was used containing a total of 0.125 pmole of
SHORT
187
COMMUNICATIONS
TABLE 1 Recoveries of Cysteine as Cysteic Acid after “Hydrolysis” Amino Acid Mixtures in the Presence of DMSO DMSO
of
concentration Cysteic acid found, mM
Pi-CYS
ml/2 ml tot. vol.
mMa
0 0.001 0.005 0.010 0.030 0.050 0.030 0.050
0 7.05 35.25 70.5 211.5 352.5 211.5 352.5
added, b mM
Cys AC
0.0625 0.0625 0.0625 0.0625 0.0625 0.0625 0.3125 0.3125
0.005 0.0282 0.0403 0.0593 0.0670 0.0624 0.2955 0.3088
% 8 45 65 95 107 100 95 99
Cys Ad
%
0.028 0.0412
44 66
0.0635 0.0627 0.299 0.311
102 100 96 99
a Calculated on basis of MW 78.14 and density (25”) 1.0958 (5). b The amino acid mixtures contained equimolar amounts of 17 amino acids, all at the level of 0.0625 m&f. In the last two experiments an additional 0.25 pmole of cysteine was added per ml hydrolysate. c Cysteic acid calculated relative to aspartic acid (0.0625 m&f). d Cysteic acid calculated relative to alanine (0.0625 m&f).
each of the common 17 amino acids. In experiments 7 and 8, an additional 0.5 pLmole of cysteine was added to bring the total of that amino acid to 0.625 pmole/2 ml. At the lower concentrations of DMSO the conversion of cysteine to cysteic acid was incomplete, but at the level of 0.2435 M DMSO the conversion was quantitative. Based on the results in Table 1, a fixed hydrolysis reagent containing 0.21 M DMSO in 6 M HCl was used in the hydrolysis of several proteins of known amino acid content. The proteins (l-2 mg) were hydrolyzed in volumes of 2 ml. The results are given in Table 2, and, allowing for normal experimental error for this type of analysis (1+3% for both cysteic acid and the reference amino acids), most of the analyses show that the conversion of cystine and cysteine to cysteic acid is quantitative. The only significant discrepancy between t,he results in Table 2 and well-established literature values is the consistently low analysis for chymotrypsinogen. In this particular case the protein purity had been established, so the deviation cannot be explained on the basis of impurities. The only known unique feature of cystine in chymotrypsinogen is the fact that one l/2-cys occupies the N-terminal position in this protein. There is no obvious reason why this should affect the DMSO oxidation of that residue, however. There does not appear to be a significant difference in the cysteic acid yield from proteins containing only cystine
(source)
a Wham b Cyst& L Cyst&c
MW
13.6 46.0 64.0 23.0 142.0 5.7 15.0 248 .o 34.0
of Several
15 32 54 22 107 3 21 281 40
Asp 12 35 46 22 157 3 12 158 16
Ala 8 6-7 34-36 10 29 6 8 22-B 6
M-Cys”
Moles amino acid/mole
Analysis
7 8 9 10 11 12 13 14 15
Ref.
italicized
8.17 6.96 33.5 8.55 29.4
CysAb
TABLE Proteins
(see 61, a range is given. The acid (moles/mole protein). (moles/mole protein).
X 10-a
Acid
discrepancies exist in the literature acid calculated relative to aspartio acid calculated relative to alanine
Ribonuclease (bovine pancreas) Ovalbumin (hen ep~) Serum albumin (bovine) Chymotrypeinosen A (bovine) Aldolase (rabbit muacle) rnsulii (bovine) Lylwyme @en BE%) catalase bovine liver) Pepsin (bovine)
Protein
Cysteic
value
102 99 93 86 101
theor.
70
Expt.
1
is used
7.62 7.47 36.0 8.92 29.2
CysAc
2 Hydrolyzed
8.22 7.07 32 6 9.1 28.7 5.95 7.4 23.5
CysAb
%
7.93 6.95 33.0 8.57 31.2 5.47 7.35 25.0
of To theory.
103 101 91 91 99 99 92 84
2
found
CysAc
acid
of DMSO
Expt.
theor.
Cyst&c
Presence
for calculation
95 107 100 89 101
theor.
%
in the
%
99 99 92 86 108 91 92 89
theor.
%
95 107 99 99 104
30.9 5.95 7.9 6.22
95
theor.
34.3
7.57
CysAa
Expt.
6.18
7.88
27.9 5.77
35.0
7.92
CysAc
3 %
103
99
96 96
97
99
theor.
F :!
2
SHORT
COMMUNICATIONS
189
(ribonuciease, bovine serum albumin, insulin, lysozyme) a.nd from those containing primarily cysteine (aldolase and ovalbumin) , nor does the presence of carbohydrate in ovalbumin affect the analysis. Based on these results, it would appear that the simple procedure of hydrolyzing proteins in the presence of DMSO will give values for total cystine and cysteine content as good as those obtained after performic acid oxidation. Discussion. We have made no attempt to elucidate the chemical mechanisms involved in the amino acid modification by DMSO. The oxidative properties of DMSO are well documented (16, 17) and the acidcatalyzed thiomethoxymethylation of aromatic alcohols is also well established (18, 19). There should therefore be no problem in explaining the observed modifications in terms of known DMSO reactions. It may be of interest to note that in the experiments in Table 1 where the yield of cyst&c acid was low (at low levels of DMSO) the cysteine peak was completely absent, indicating the some intermediate may be formed in the oxidation. Summary. Hydrolysis of proteins in 6 N HCl containing 0.2-0.3M dimethyl sulfoxide leads to quantitative oxidation of cystine and cysteine to cysteic acid, which can be determined by direct amino analysis. Because of alteration of several other amino acids under these hydrolysis conditions, the rest of the amino acids must be determined from parallel hydrolyzates carried out in the conventional manner in the absence of dimcthyl sulfoxide. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
9. 10.
11. 12.
13. 14. 15. 16.
17.
SANGEB, F., HIRS, C. H.
Biochem. J. 44, 126 (1949). W., .l. BioZ. Chem. 219,611 (1956) HIRS, C. H. W., Method-s in Enzymol. 11, 197 (1967). MOORE, S., J. Biol. Chem. 238, 235 (1963). MACGREGOR, W. S., Ann, N. Y. Acad. Sci. 141, Article 1, 3 (1967). CECIL, R., AND MCPHEE, J. R., Advan. Prot. Chem. 14, 255 (1959). SIUTH, D. G., STEIN, W. G., AND MOORE, S., J. Bill. Chem. 238, 227 (1963). LEWIS, J. C.. SNELL. T\T. S., HIHSCHMAN, D. J., AND FRAENKEL-CONRAT, H., J. Biol. Chem. 186, 23 (1950). SPAHR, P. F., AND EDSALL, J. T., J. Biol. Chem. 239,850 (1964). WILCOX, P. E., COHEN, E., END TAN, W., J. Biot. Chem. 228, 999 (1957). SHI~ZU, H., AND OZAWJ, H., Biochim. Biophys. Acta 133, 195 (1967). RYLE, A. P., S.~NGER, F., SMITH, L. F., AND KITAI, R., Biochem. J. 60, 541 (1955). CANFIELD, R. E., AND LIU, A. K., J. Biol. Chem. 240, 1997 (1965). R.ADHAKRISHNAN, T. H., AND SARMA, P. S., B&hem. J. 97, 827 (1965). RAJAOOPAL.AN, T. G., MOORE, S., AND STEIN, W. H., J. Biol. Chem. 241,494O (1966). PFITZNER, K. E., AND MOETATT, J. G.. J. Am. Chem. Sot. 87, 5661 (1965). ALBRIGHT, J. D., AND GOLDMAN, L., J. Am. Chem. Sot. 87, 4214 (1965).
190
SHORT
18. BURDON, 19. PFPZNER, WtW.
COMMUNICATIONS
M. G., AND MOFTATT, J. G., K. E., MARINO, J. P., AND
J. A’m. Chem. Sot. 87, 4656 (1965). OLOFSON, R. A., J. Am. Chem. Sot. 87, 4658
L.
RICHARD FINN
of Biochemistry University of Minnesota Medical Minneapolis, Minnesota 66.&6 Received May 6,1&W
WOLD
Department
’
National Institute
of General
School
Medical
Sciences
Postdoctoral
Fellow
SPENCER~