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Hydrogen exchange on amino acids, peptides, proteins and certain related compounds
HydrogenExchangeon Amiio Acids,Peptides,Proteins andCertainRelatedCompounds’ Harold J. Morowitz From the National Heart Instifute, National Institutes of Health, Bethesda, Maryland
and Margaret W. Chapman From the National Bureau of Standards, Washington, D. C. Received $eptember 27, 1954 INTRODUCTION
The isotope-dilution technique of mixing deuterium oxide with the sample and measuring the final deuterium to hydrogen ratio provides an effective means of measuring total water content (1) in those cases where no hydrogen atoms from the nonaqueousportion of the sample exchange with the surrounding solvent. In caseswhere hydrogen exchange takes place, the measured“water content” is too high and correction must be made for the exchange.This correction depends on a knowledge of the composition of the nonaqueousmaterial and the exchange for such material under the conditions of dilution. This paper reports on the determination of the number of labile hydrogen atoms per moleculefor a seriesof amino acids, peptides, proteins, and related molecules. The number of labile hydrogens depends on experimental conditions during the exchange.Rittenberg, Keston, Schoenheimer,and Foster (2) classify amino acid hydrogens as labile (amino, carboxyl, and hydroxyl hydrogens), semilabile (carbon-bound hydrogen5 adjacent to polar groups), and stabile (carbon-bound hydrogens), Their experiments involved boiling t’he amino acids in solutions of water, mineral acids, and deuterium oxide. Stekol and Hamill (3) observedexchangesby similar 1Amino acids and peptides were obtained from Nutritional Biochemical Corporation, while the ribonuclease was supplied by Armour and Company. 110
HYDROGEN.
EXCHANGE
111
techniques. Miinzberg and Haurowitz (4) observed exchanges at semilabile positions during the enzymatic hydrolysis of casein at 38°C. Several other investigators have measured active hydrogens by means of deuterium exchange reactions (5-7). In the experiments to be discussed in this paper the material to be investigated was dissolved in 99.8 % deuterium oxide. The hydrogens of the sample material fall into two categories: labile, which exchange completely at room temperature in 20 min., and stabile, which show no significant exchange in an 8-hr. period. THEORY
If z moles of dry material containing m moles of labile hydrogen per mole are dissolved in a solvent containing g moles of DzO and b moles of HzO, the ratio of hydrogen to deuterium in the solvent before mixing, T, and the ratio after mixing, r’, will be: mx r=- b 9
r ,b+?i? =9
The factor 2 occurs in the expression for r’ since there are two moles of hydrogen per mole of water. If we take the difference of the two ratios we obtain the following relation
As x, g, and the r are measured, we may calculate m from the experimental data. EXPERIMENTAL Samplesi to be analyzed were vacuum or oven dried, weighed, and dissolved in a weighed amount of solvent of approximate composition 99.8y0 D20 and 0.2oJ, HzO. The solutions were allowed to equilibrate for 29 min. at room temperature, and the ratio of hydrogen to deuterium in the aqueous portion was measured by the optical spectroscopic method (8). In this method, the vapor from the sample to be studied is pumped through a tube where a high-frequency discharge is maintained. The emission consists largely of the Balmer series of hydrogen and deuterium, and the analysis is made by measuring the ratio of the H to the D line using a high-resolution recording spectrometer. In some instances readings were repeated periodically, as the samples equilibrated over a 6-24-hr. period, in an effort to detect a slow exchange due to semilabile hydrogen.
112
HAROLD J. MOROWITZ AND MARGARET W. CHAPMAN TABLE Hydrogen
Exchange
on Amino
Acids,
I Peptides
and Related
Compounds
I abile hydrogens per Labile h drogens per molecu 7e predicted molecule observed
Compound
3.09
Glycine Alanine Proline Serine Arginine hydrochloride Guanidine hydrochloride Creatinine hydrochloride Ethylene diaguanidine dihydrobromide Glycylglycine Glycylglycylglycine
3.04 2.04 3.89 8.00 6.20 2.90 10.1 3.95 4.91
3.0 3.0 2.0 4.0 8.0 6.0 3.0 10.0 4.0 5.0
RESULTS
All exchange observed in these experiments occurred in the 20-min. period, and subsequent readings, when carried out, showed no increase in the hydrogen to deuterium ratio. Table I lists the values obtained from a series of amino acids, peptides, and related compounds. The predicted values are based on the postulated exchange of all hydrogens not bound to carbon. From the observed amino acid and peptide values, one can calculate the number of exchangeable hydrogens for a protein of known amino acid composition. This is done by counting one labile hydrogen for each peptide bond, the appropriate number for each side chain, one for each end carboxyl, and two for each N-terminal group. Calculated values for ribonuclease and hemoglobin are 252 and 899 hydrogens per molecule based on the amino acid compositions reported by Tristram (9) and Brand (10). Exchange experiment were performed on dried crystalline ribonuclease and human red blood cells dried by vacuum TABLE Hydrogen
Exchange
II on Proteins
Material
Hemoglobin Hemoglobin Hemoglobin Hemoglobin Ribonuclease
(oven-dried red cells) (oven-dried red cells) (vacuum-desiccated red cells) (vacuum-desiccated red cells)
0 Hemoglobin hemoglobin.
values are based on the approximation
Labile hydrogens per molecule, observed”
Labile hydrogens per molecule, predicted
886 888 895 920 272
899 899 899 899 252
that the red cells are 100%
HYDROGEN EXCHANGE
113
desiccation and 105°C. heating to constant weight. Table II gives the predicted and observed values for the two materials. DISCUSSION
The close agreement between experimental and predicted values of hydrogen exchange for the compounds of Table I makes it apparent that, with respect to this type of experiment, hydrogen atoms may be classified into two broad groups, labile and stabile. This clear distinction makes it possible to predict with some accuracy, as is shown in Table II, the hydrogen exchange of protein molecules. The agreement between theory and experiment for protein molecules is further evidence, hardly necessary at this late date, of the peptide chain structure of proteins. The ability to predict the hydrogen exchange for protein makes it possible to use DzO dilution to obtain precise values of the water content of protein crystals. The accuracy of the determination of labile hydrogen is limited by the hydrogen deuterium analysis which can be performed under ideal experimental conditions with a precision of 0.1% of the H/D ratio. ACKNOWLEDGMENTS The authors wish to thank Dr. James G. Mold, Dr. William Carroll, and Dr. Herbert Broida for their assistance. SUMMARY
The numbers of hydrogens per molecule which freely exchange with hydrogen from an aqueous solvent have been determined by the isotopeexchange technique. It is found for amino acids, peptides, and proteins that all hydrogens not involved in C-H bonds may be classed as exchangeable. This information may be used to determine precisely the water content of proteins by isotope equilibrium methods using hydrogen and deuterium as water tracers. REFERENCES 1. MOROWITZ, H. J., AND BROIDA, H. P., Anal. Chem. 24,1657 (1952). 2. RITTENBERG, D., KESTON, A. S., SCHOENHEIMER,R., AND FOSTER,G. L.,J. Biol. Chem. 146, 1 (1938). 3. STEKOL, J. A., AND HAMILL, W. H., J. Riol. Chem. 120, 531 (1937). 4. MOWBERG, F. K , AND HAUROWITZ, F., 2. physiol. Chem. 266, 271 (1938). 5. ERLENMEYER, H., EPPRECHT, A., LOBECK, H., AND GARTNER, H., Helv. Chim. Acta 19, 354 (1936).
114
HAROLD
J. MOROWITZ
AND
M.4RGARET
W.
CHAPMAN
6. UCHIDA, M., J. Japan Biochem. Sot. 83,63 (1951). 7. CLARKE, H. T., JOHNSON, J. P., AND ROBINSON, R., “The Chemistry of Peniciilin,” pp. 88, 113, 289. Princeton University Press, Princeton, 1949. 8. BROIDA, H. P., MOROWITZ, H. J., AND SELGIN, M., J. Research N&Z. Bur. Standards 62, 293 (19541. 9. TRISTRAM, G. R., Advances in Protein Chem. 6, 83 (1949). 10. BRAND, E., as reported in “Crystalline Enzymes” (Northrup, Kunitz, and Herriot), p. 26. Columbia University Press, New York, 1938.
and Margaret W. Chapman From the National Bureau of Standards, Washington, D. C. Received $eptember 27, 1954 INTRODUCTION
The isotope-dilution technique of mixing deuterium oxide with the sample and measuring the final deuterium to hydrogen ratio provides an effective means of measuring total water content (1) in those cases where no hydrogen atoms from the nonaqueousportion of the sample exchange with the surrounding solvent. In caseswhere hydrogen exchange takes place, the measured“water content” is too high and correction must be made for the exchange.This correction depends on a knowledge of the composition of the nonaqueousmaterial and the exchange for such material under the conditions of dilution. This paper reports on the determination of the number of labile hydrogen atoms per moleculefor a seriesof amino acids, peptides, proteins, and related molecules. The number of labile hydrogens depends on experimental conditions during the exchange.Rittenberg, Keston, Schoenheimer,and Foster (2) classify amino acid hydrogens as labile (amino, carboxyl, and hydroxyl hydrogens), semilabile (carbon-bound hydrogen5 adjacent to polar groups), and stabile (carbon-bound hydrogens), Their experiments involved boiling t’he amino acids in solutions of water, mineral acids, and deuterium oxide. Stekol and Hamill (3) observedexchangesby similar 1Amino acids and peptides were obtained from Nutritional Biochemical Corporation, while the ribonuclease was supplied by Armour and Company. 110
HYDROGEN.
EXCHANGE
111
techniques. Miinzberg and Haurowitz (4) observed exchanges at semilabile positions during the enzymatic hydrolysis of casein at 38°C. Several other investigators have measured active hydrogens by means of deuterium exchange reactions (5-7). In the experiments to be discussed in this paper the material to be investigated was dissolved in 99.8 % deuterium oxide. The hydrogens of the sample material fall into two categories: labile, which exchange completely at room temperature in 20 min., and stabile, which show no significant exchange in an 8-hr. period. THEORY
If z moles of dry material containing m moles of labile hydrogen per mole are dissolved in a solvent containing g moles of DzO and b moles of HzO, the ratio of hydrogen to deuterium in the solvent before mixing, T, and the ratio after mixing, r’, will be: mx r=- b 9
r ,b+?i? =9
The factor 2 occurs in the expression for r’ since there are two moles of hydrogen per mole of water. If we take the difference of the two ratios we obtain the following relation
As x, g, and the r are measured, we may calculate m from the experimental data. EXPERIMENTAL Samplesi to be analyzed were vacuum or oven dried, weighed, and dissolved in a weighed amount of solvent of approximate composition 99.8y0 D20 and 0.2oJ, HzO. The solutions were allowed to equilibrate for 29 min. at room temperature, and the ratio of hydrogen to deuterium in the aqueous portion was measured by the optical spectroscopic method (8). In this method, the vapor from the sample to be studied is pumped through a tube where a high-frequency discharge is maintained. The emission consists largely of the Balmer series of hydrogen and deuterium, and the analysis is made by measuring the ratio of the H to the D line using a high-resolution recording spectrometer. In some instances readings were repeated periodically, as the samples equilibrated over a 6-24-hr. period, in an effort to detect a slow exchange due to semilabile hydrogen.
112
HAROLD J. MOROWITZ AND MARGARET W. CHAPMAN TABLE Hydrogen
Exchange
on Amino
Acids,
I Peptides
and Related
Compounds
I abile hydrogens per Labile h drogens per molecu 7e predicted molecule observed
Compound
3.09
Glycine Alanine Proline Serine Arginine hydrochloride Guanidine hydrochloride Creatinine hydrochloride Ethylene diaguanidine dihydrobromide Glycylglycine Glycylglycylglycine
3.04 2.04 3.89 8.00 6.20 2.90 10.1 3.95 4.91
3.0 3.0 2.0 4.0 8.0 6.0 3.0 10.0 4.0 5.0
RESULTS
All exchange observed in these experiments occurred in the 20-min. period, and subsequent readings, when carried out, showed no increase in the hydrogen to deuterium ratio. Table I lists the values obtained from a series of amino acids, peptides, and related compounds. The predicted values are based on the postulated exchange of all hydrogens not bound to carbon. From the observed amino acid and peptide values, one can calculate the number of exchangeable hydrogens for a protein of known amino acid composition. This is done by counting one labile hydrogen for each peptide bond, the appropriate number for each side chain, one for each end carboxyl, and two for each N-terminal group. Calculated values for ribonuclease and hemoglobin are 252 and 899 hydrogens per molecule based on the amino acid compositions reported by Tristram (9) and Brand (10). Exchange experiment were performed on dried crystalline ribonuclease and human red blood cells dried by vacuum TABLE Hydrogen
Exchange
II on Proteins
Material
Hemoglobin Hemoglobin Hemoglobin Hemoglobin Ribonuclease
(oven-dried red cells) (oven-dried red cells) (vacuum-desiccated red cells) (vacuum-desiccated red cells)
0 Hemoglobin hemoglobin.
values are based on the approximation
Labile hydrogens per molecule, observed”
Labile hydrogens per molecule, predicted
886 888 895 920 272
899 899 899 899 252
that the red cells are 100%
HYDROGEN EXCHANGE
113
desiccation and 105°C. heating to constant weight. Table II gives the predicted and observed values for the two materials. DISCUSSION
The close agreement between experimental and predicted values of hydrogen exchange for the compounds of Table I makes it apparent that, with respect to this type of experiment, hydrogen atoms may be classified into two broad groups, labile and stabile. This clear distinction makes it possible to predict with some accuracy, as is shown in Table II, the hydrogen exchange of protein molecules. The agreement between theory and experiment for protein molecules is further evidence, hardly necessary at this late date, of the peptide chain structure of proteins. The ability to predict the hydrogen exchange for protein makes it possible to use DzO dilution to obtain precise values of the water content of protein crystals. The accuracy of the determination of labile hydrogen is limited by the hydrogen deuterium analysis which can be performed under ideal experimental conditions with a precision of 0.1% of the H/D ratio. ACKNOWLEDGMENTS The authors wish to thank Dr. James G. Mold, Dr. William Carroll, and Dr. Herbert Broida for their assistance. SUMMARY
The numbers of hydrogens per molecule which freely exchange with hydrogen from an aqueous solvent have been determined by the isotopeexchange technique. It is found for amino acids, peptides, and proteins that all hydrogens not involved in C-H bonds may be classed as exchangeable. This information may be used to determine precisely the water content of proteins by isotope equilibrium methods using hydrogen and deuterium as water tracers. REFERENCES 1. MOROWITZ, H. J., AND BROIDA, H. P., Anal. Chem. 24,1657 (1952). 2. RITTENBERG, D., KESTON, A. S., SCHOENHEIMER,R., AND FOSTER,G. L.,J. Biol. Chem. 146, 1 (1938). 3. STEKOL, J. A., AND HAMILL, W. H., J. Riol. Chem. 120, 531 (1937). 4. MOWBERG, F. K , AND HAUROWITZ, F., 2. physiol. Chem. 266, 271 (1938). 5. ERLENMEYER, H., EPPRECHT, A., LOBECK, H., AND GARTNER, H., Helv. Chim. Acta 19, 354 (1936).
114
HAROLD
J. MOROWITZ
AND
M.4RGARET
W.
CHAPMAN
6. UCHIDA, M., J. Japan Biochem. Sot. 83,63 (1951). 7. CLARKE, H. T., JOHNSON, J. P., AND ROBINSON, R., “The Chemistry of Peniciilin,” pp. 88, 113, 289. Princeton University Press, Princeton, 1949. 8. BROIDA, H. P., MOROWITZ, H. J., AND SELGIN, M., J. Research N&Z. Bur. Standards 62, 293 (19541. 9. TRISTRAM, G. R., Advances in Protein Chem. 6, 83 (1949). 10. BRAND, E., as reported in “Crystalline Enzymes” (Northrup, Kunitz, and Herriot), p. 26. Columbia University Press, New York, 1938.