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N-14 magnetic resonance study of the interaction of urea and guanidine hydrochloride with albumins
135
BIOCHIMICA ET BIOPHYSICA ACTA
BBA
26561
N-I 4 MAGNETIC RESONANCE STUDY OF T H E I N T E R A C T I O N OF U R E A AND G U A N I D I N E H Y D R O C H L O R I D E W I T H ALBUMINS ]3. M. F U N G AND S U S A N G. S A R N E Y
Department of Chemistry, Tufts University, Medford, Mass. o22:55 ( U.S.A .) (Received N o v e m b e r i 3 t h , 197 o)
SUMMARY
N-I 4 nuclear magnetic resonance of urea and guanidine hydrochloride in aqueous solutions was studied. It was found t h a t the line width increases in the presence of albumins but does not change with the addition of polylysine.
In recent years nuclear magnetic resonance (NMR) has been successfully used to study the interactions of biological systems with small molecules or ions. The binding of penicillin I and sulfonamides 2 to bovine serum albumin was studied b y proton NMR. Works on m a n y other systems have been reviewed recently b y ROBERTS AND J A R D E T Z K Y 3.
The denaturation of bovine serum albumin by urea and guanidine hydrochloride has been studied with respect to changes in viscosity4, optical rotatory dispersion a, ultraviolet absorption 6, t r y p t o p h a n fluorescence 7, and by other techniques s-lo. In all these studies the structure or shape of the protein molecule was the probe. We would like to propose investigating the denaturation problem by nitrogen-I 4 resonance of urea and guanidine hydrochloride in bovine serum albumin solutions. This method differs from the previous methods in probing the denaturing agent itself. The aqueous solutions of bovine serum albumin show no detectable nitrogen-i 4 resonance at 4.0 MHz and room temperature. This is probably due to the large size and low concentration of the protein. Consequently, the study of the N-I 4 resonance of urea and guanidine hydrochloride in the presence of protein is uncomplicated and convenient. Bovine serum albumin is slightly acidic in aqueous solution and the addition of urea or guanidine hydrochloride changes the pH. Therefore, the solution was buffered with 0.05 M phosphate buffer (I :i, NaH2PO4/NaHPO4). 1-6 M urea solutions had a p H range of 6.9o-7.15, and 0.5-2.0 M guanidine hydrochloride solutions had a p H range of 6.30-5.7 o. For a fixed concentration of urea or guanidine the p H of the solution stayed essentially constant regardless of the bovine serum albumin concentration which varied from 2-20 mg/ml in our experiment. The N-I 4 resonance experiment showed that the N-I 4 line width increased only very slightly with the concentration of urea or guanidine in the absence of bovine serum albumin. In the presence of bovine serum albumin, the line width increased Biochim. Biophys. Aeta, 237 (1971) I 3 5 - I 3 7
136
B.M. FUNG, S. G. SARNEY
significantly as the bovine serum albumin concentration was increased (Fig. I). Similar changes in the line width of urea and guanidine were also observed when egg albumin and human serum albumin were added into the solutions. On the contrary, the N-I 4 line width did not change within experimental error with the concentration of poly-Llysine (tool. wt. approx. 106000) at pH 7 (Fig. I).
• 500
-J
" "}BSA "
=-~
Z~o o
•
I I sine
O O
'o
3o
,b
go ram,
Concentrat I on
Fig. I. N-14 resonance line widths as fimctions of bovine serum albumin (BSA) and polylysine concentration. ~ , ~k, 2 M guanidine; O, i M guanidine; [], 6 M urea; &, ~ , 4 M urea; G, 2 M urea.
The increase in the line width reflects an increase in the correlation time of the denaturant molecules. For the present systems, clearly the change in external viscosity is not the cause in the correlation time, because the viscosity of the polylysine solutions increased as much as 5-fold in the concentration range studied, but the N-I 4 line width of urea and guanidine stayed essentially constant. On the other hand, the largest viscosity change was only about 15 % for the bovine serum albumin solutions, while the N-I 4 line width increased about 30%. The very small change in the line width with the increase of urea and guanidine concentration suggests that urea-urea and urea-water (or guanidine-guanidine and guanidine-water) interactions are not the major cause in the change of the correlation time. The most likely cause of the increase in the N-I 4 line width is the binding of urea and guanidine to the albumins. It was shown that the proton relaxation time of small drug molecules that are bound to the bovine serum albumin is several thousand times those of the free molecules at the binding site 1,~, because the very large albumin molecules have long correlation times due to slow motion. The equilibrium constant of the binding Call be calculated from the line width data1, ~. For urea and guanidine, the increase in line width is relatively small (approx. 30%) compared to sulfonamides and penicillin (3o-4o-fold). Because of the small change and rather large inaccuracy (5 %) in the N-I 4 line width, it is not possible to give meaningful quantitative calculation on the present systems. However, the N-I4 resonance study does indicate that there is a definite interaction between urea and guanidine and the albumins. The details of the interaction and the equilibrium constants have to be studied further. Biochim. Biophys. Acta, 237 (1971) 135-137
NMR
STUDY OF ALBUMIN INTERACTIONS
137
ACKNOWLEDGMENT
This work was supported by the National Institutes of Health. Helpful discussion with Dr. C. D. Jardetzky is acknowledged. REFERENCES i J. J. FISCHER AND O. JARDETZKY, J. Am. Chem. Soc., 87 (1965) 3237. JARDETZKY AND N. G. WADE-JARDETZKY, Mol. Pharmacol., I (1965) 214. 3 G. C. K. ROBERTS AND O. JARDETZKY, Advan. Protein Chem., 24 (197 o) 4474 H. K. FRENSDORFF, M. T. WATSON AND W. I~AUZMANN,J. Am. Chem. Sot., 75 (1953) 5167 • 5 R. B. SIMPSON AND W. KAUZMANN, J. Am. Chem. Soc., 75 (I953) 5154 • 6 A. N. GLAZER, H. A. McKENZlE AND R. G. WAKE, Biochim. Biophys. Acta, 69 (1963) 240. 7 T. R. HOPKINS AND J. D. SPIKES, Biochem. Biophys. Res. Commun., 3° (1968) 54 o. 8 F. J. GUTTER, E. A. PETERSON AND H. A. SOBER, Arch. Biochem. Biophys., 72 (1957) 194. 9 S. I(ATZ AND T. G. FERRIS, Biochemistry, 5 (1966) 3246. 1o T. G. FERRIS AND K. KATZ, Nature, 211 (1966) 586.
20.
Biochim. Biophys. Acta, 237 (1971) 135-137
BBA 26555 (Na+-K+)-ATPase IN THE K I D N E Y OF NORMAL AND CASTRATED MICE N E B O J ~ A A V D A L O V I ~ AND G E O R G E SACHS
Department of Physiology, Faculty of Medicine, University of Zagreb, Zagreb (Yugoslavia) and Division of Gastroenterology, Department of Medicine, University of Alabama in Birmingham, Ala. 35233 (U.S.A.) (Received November i3th, 197 o)
SUMMARY
It was found that castration in mice resulted in (I) a decrease of urine output; (2) an increase of sodium concentration in urine without a change in potassium concentration; (3) a decrease in oxygen consumption of the mouse kidney tissue; (4) a significant increase of the specific activity of (Na+-K+)-ATPase. The values of all parameters studied returned almost to normal values after 7-days treatment with testosterone propionate.
Testosterone administration to intact and castrated mice has a demonstrable renotrophic effect 1. The increase in renal mass coincides with the change in renal blood flow 2. Although this renotrophic action of testosterone has been demonstrated both in mice and rats, its role in regulating sodium and potassium transport in the Biochim. Biophys. Acta, 237 (1971) 137 14o
BIOCHIMICA ET BIOPHYSICA ACTA
BBA
26561
N-I 4 MAGNETIC RESONANCE STUDY OF T H E I N T E R A C T I O N OF U R E A AND G U A N I D I N E H Y D R O C H L O R I D E W I T H ALBUMINS ]3. M. F U N G AND S U S A N G. S A R N E Y
Department of Chemistry, Tufts University, Medford, Mass. o22:55 ( U.S.A .) (Received N o v e m b e r i 3 t h , 197 o)
SUMMARY
N-I 4 nuclear magnetic resonance of urea and guanidine hydrochloride in aqueous solutions was studied. It was found t h a t the line width increases in the presence of albumins but does not change with the addition of polylysine.
In recent years nuclear magnetic resonance (NMR) has been successfully used to study the interactions of biological systems with small molecules or ions. The binding of penicillin I and sulfonamides 2 to bovine serum albumin was studied b y proton NMR. Works on m a n y other systems have been reviewed recently b y ROBERTS AND J A R D E T Z K Y 3.
The denaturation of bovine serum albumin by urea and guanidine hydrochloride has been studied with respect to changes in viscosity4, optical rotatory dispersion a, ultraviolet absorption 6, t r y p t o p h a n fluorescence 7, and by other techniques s-lo. In all these studies the structure or shape of the protein molecule was the probe. We would like to propose investigating the denaturation problem by nitrogen-I 4 resonance of urea and guanidine hydrochloride in bovine serum albumin solutions. This method differs from the previous methods in probing the denaturing agent itself. The aqueous solutions of bovine serum albumin show no detectable nitrogen-i 4 resonance at 4.0 MHz and room temperature. This is probably due to the large size and low concentration of the protein. Consequently, the study of the N-I 4 resonance of urea and guanidine hydrochloride in the presence of protein is uncomplicated and convenient. Bovine serum albumin is slightly acidic in aqueous solution and the addition of urea or guanidine hydrochloride changes the pH. Therefore, the solution was buffered with 0.05 M phosphate buffer (I :i, NaH2PO4/NaHPO4). 1-6 M urea solutions had a p H range of 6.9o-7.15, and 0.5-2.0 M guanidine hydrochloride solutions had a p H range of 6.30-5.7 o. For a fixed concentration of urea or guanidine the p H of the solution stayed essentially constant regardless of the bovine serum albumin concentration which varied from 2-20 mg/ml in our experiment. The N-I 4 resonance experiment showed that the N-I 4 line width increased only very slightly with the concentration of urea or guanidine in the absence of bovine serum albumin. In the presence of bovine serum albumin, the line width increased Biochim. Biophys. Aeta, 237 (1971) I 3 5 - I 3 7
136
B.M. FUNG, S. G. SARNEY
significantly as the bovine serum albumin concentration was increased (Fig. I). Similar changes in the line width of urea and guanidine were also observed when egg albumin and human serum albumin were added into the solutions. On the contrary, the N-I 4 line width did not change within experimental error with the concentration of poly-Llysine (tool. wt. approx. 106000) at pH 7 (Fig. I).
• 500
-J
" "}BSA "
=-~
Z~o o
•
I I sine
O O
'o
3o
,b
go ram,
Concentrat I on
Fig. I. N-14 resonance line widths as fimctions of bovine serum albumin (BSA) and polylysine concentration. ~ , ~k, 2 M guanidine; O, i M guanidine; [], 6 M urea; &, ~ , 4 M urea; G, 2 M urea.
The increase in the line width reflects an increase in the correlation time of the denaturant molecules. For the present systems, clearly the change in external viscosity is not the cause in the correlation time, because the viscosity of the polylysine solutions increased as much as 5-fold in the concentration range studied, but the N-I 4 line width of urea and guanidine stayed essentially constant. On the other hand, the largest viscosity change was only about 15 % for the bovine serum albumin solutions, while the N-I 4 line width increased about 30%. The very small change in the line width with the increase of urea and guanidine concentration suggests that urea-urea and urea-water (or guanidine-guanidine and guanidine-water) interactions are not the major cause in the change of the correlation time. The most likely cause of the increase in the N-I 4 line width is the binding of urea and guanidine to the albumins. It was shown that the proton relaxation time of small drug molecules that are bound to the bovine serum albumin is several thousand times those of the free molecules at the binding site 1,~, because the very large albumin molecules have long correlation times due to slow motion. The equilibrium constant of the binding Call be calculated from the line width data1, ~. For urea and guanidine, the increase in line width is relatively small (approx. 30%) compared to sulfonamides and penicillin (3o-4o-fold). Because of the small change and rather large inaccuracy (5 %) in the N-I 4 line width, it is not possible to give meaningful quantitative calculation on the present systems. However, the N-I4 resonance study does indicate that there is a definite interaction between urea and guanidine and the albumins. The details of the interaction and the equilibrium constants have to be studied further. Biochim. Biophys. Acta, 237 (1971) 135-137
NMR
STUDY OF ALBUMIN INTERACTIONS
137
ACKNOWLEDGMENT
This work was supported by the National Institutes of Health. Helpful discussion with Dr. C. D. Jardetzky is acknowledged. REFERENCES i J. J. FISCHER AND O. JARDETZKY, J. Am. Chem. Soc., 87 (1965) 3237. JARDETZKY AND N. G. WADE-JARDETZKY, Mol. Pharmacol., I (1965) 214. 3 G. C. K. ROBERTS AND O. JARDETZKY, Advan. Protein Chem., 24 (197 o) 4474 H. K. FRENSDORFF, M. T. WATSON AND W. I~AUZMANN,J. Am. Chem. Sot., 75 (1953) 5167 • 5 R. B. SIMPSON AND W. KAUZMANN, J. Am. Chem. Soc., 75 (I953) 5154 • 6 A. N. GLAZER, H. A. McKENZlE AND R. G. WAKE, Biochim. Biophys. Acta, 69 (1963) 240. 7 T. R. HOPKINS AND J. D. SPIKES, Biochem. Biophys. Res. Commun., 3° (1968) 54 o. 8 F. J. GUTTER, E. A. PETERSON AND H. A. SOBER, Arch. Biochem. Biophys., 72 (1957) 194. 9 S. I(ATZ AND T. G. FERRIS, Biochemistry, 5 (1966) 3246. 1o T. G. FERRIS AND K. KATZ, Nature, 211 (1966) 586.
20.
Biochim. Biophys. Acta, 237 (1971) 135-137
BBA 26555 (Na+-K+)-ATPase IN THE K I D N E Y OF NORMAL AND CASTRATED MICE N E B O J ~ A A V D A L O V I ~ AND G E O R G E SACHS
Department of Physiology, Faculty of Medicine, University of Zagreb, Zagreb (Yugoslavia) and Division of Gastroenterology, Department of Medicine, University of Alabama in Birmingham, Ala. 35233 (U.S.A.) (Received November i3th, 197 o)
SUMMARY
It was found that castration in mice resulted in (I) a decrease of urine output; (2) an increase of sodium concentration in urine without a change in potassium concentration; (3) a decrease in oxygen consumption of the mouse kidney tissue; (4) a significant increase of the specific activity of (Na+-K+)-ATPase. The values of all parameters studied returned almost to normal values after 7-days treatment with testosterone propionate.
Testosterone administration to intact and castrated mice has a demonstrable renotrophic effect 1. The increase in renal mass coincides with the change in renal blood flow 2. Although this renotrophic action of testosterone has been demonstrated both in mice and rats, its role in regulating sodium and potassium transport in the Biochim. Biophys. Acta, 237 (1971) 137 14o