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Action of urea and certain other amide reagents on crystalline porcine pancreatic amylase
ARCHIVES
OF
BIOCHEMISTRY
Action
AND
BIOPHYSICS
of Urea
on Crystalline GLORIA Depariment
of Chemistry,
Hunter
519-525 (1967)
119,
and Certain
Other
Porcine
C. TORALBALLA
Amide
Reagents
Pancreatic
Amylase’
MARY
EITINGON2
AND
College of the City University
of New York, A:ew York, New York 10021
Received July 30, 1966 The inactivation of porcine pancreatic amylase with various concentrations of urea with a fixed concentration of pancreatic amylase was investigated in one series of experiments. The changes in viscosity, absorbance in the ultraviolet, and optical rotation accompanying the loss in amylase activity were likewise determined. It was observed t,hat the rate of inactivation increased rapidly with an increase in the urea concentration. When the enzyme concentration is 1.5 mg/ml or less, the inactivation process follows first-order kinetics. This loss in activity is accompanied by an increase in reduced viscosity and in levorotation of the amylase. A very slight decrease in ultraviolet absorbance intensity was observed. In a second series of experiments, the urea concentration was fixed while the concentration of the amylase was varied. The rate of inactivation increases with a decrease in enzyme concentration. For concentrations greater than 1.5 mg/ml of amylase, the urea-amylase reaction does not follow first-order kinetics. The inactivation by urea is irreversible. Comparative inactivation studies of equimolar solutions of urea and other amide reagents show that the effectiveness in inactivating pancreatic amylase decreases in this order: guanidine > urea > 1 ,%dimethylurea > tetramethylurea.
A number of investigational approaches which have furnished valuable and pertinent information toward the elucidation of the chemical structure of crystalline procine pancreatic amylase have been carried through by Caldwell and co-workers. They reported on an improved method for the purification and crystallization of this amylase (l), on the nature and kinetics of its biological action (Z-5), on its amino acid composition (6), and on the essentiality of some of its functional groups (7-10). To date, no studies have been published on the action of urea and other amide reagents on porcine pancreatic amylase.
The present investigation was carried out to study in some detail the action of urea and certain other related denaturants on the amylase. This paper reports mainly the results of the action of urea on pancreatic amylase. To correlate any possible structural alterations in the protein molecule with loss in amylase activity, determinations were made of the effect of urea on the viscosity, the optical rotation, and the ultraviolet absorbance of the enzyme. In addition, the action of urea is compared with that of guanidine, 1,3-dimethylurea, and tetramethylurea.
1 This investigation was supported by grants GM-O8960-01 and GM-08960-02 from the National Institutes of Health, U.S. Public Health Service. A preliminary report was presented to the 142nd meeting of the American Chemical Society, Atlantic City, New Jersey, September 9-14, 1962. * Present address: Cornell Medical College, New York Hospital, New York, New York 10021.
Twice-crystallized porcine pancreatic amylase was obtained from Worthington Biochemical Corporation. The cryst,als (LOOmg) were washed repeatedly wit,h 1 cc of cold sodium phosphate buffer (0.01 M in sodium phosphate, 0.02 M in sodium chloride, pH, 7.0; temperature, 2’) until the amylase activity of the solid was found to remain constant,. Amylase stock solutions
EXPERIMENTAL
519
Materials.
PROCEDUR.ES
520
TORALBALLA
were obtained by stirring a suspension of the washed crystals in cold sodium phosphate buffer (2”) and were further purified by dialysis against a sodium phosphate buffer. The enzyme concentration of these solutions was 2-3 me/ml and the amylase activity was 13,500-14,000 maltose equivalents per milligram of solid enzyme. The concentration of each prepared amylase solution was determined spectrophotometrically at 280 rnp using a calibration curve (1). Urea (Baker, reagent grade) was crystallized twice from an ethanol-water solvent (1:l by volume). The melting point (uncorrected) of the product was 131.5”. Guanidine hydrochloride was crystallized twice from absolute ethyl alcohol. 1,3-Dimet,hylurea was crystallized twice from absolute ethanol. The product had a melting point (uncorrected) of 106.5”. Tetramethylurea, reagent grade, and purified by gas chromatography (John Deere Chemical Company), was used without further purification. Distilled water which was redistilled in an all glass distilling apparatus was used throughout. Methods. A 10 M solution of urea in sodium phosphate buffer was prepared and refrigerated until used. For each of the various urea molarities needed (i.e., 2 M, 4 M, 6 M, and 8 M) the reaction mixture was prepared by adding the required amount of the 10 M urea solution to the amylase solution. The pH of the reaction mixtures was maintained at 7.0-7.2 and the incubation temperature was 2”. This particular pH and incubation temperature were chosen because, under these conditions, porcine pancreatic amylase, by itself, does not undergo any inactivation (1, 3). Control solutions containing only the enzyme in sodium phosphate buffer were incubated at the same temperature. Aliquots of each of these urea-amylase mixtures and the controls were then removed at definite and suitable time intervals and measured simultaneously for loss in amylase activity and for changes in viscosity, optical rotation, and absorbance in the ultraviolet. Amylase activity was determined by measuring the saccharogenic action of the enzyme on a solution of Lintner soluble starch at 40” for 30 minutes according to the iodometric method of Caldwell et al. (11). This method necessitated the dilution of the urea-amylase solutions with buffered starch solutions. However, since the urea-amylase inactivation is irreversible, the value obtained for the amylase activity is not affected by the dilution. This fact was shown by comparative amylase activity measurements on diluted and undiluted urea-amylase solutions. Viscosities were measured at 2.0 f 0.1” with an Ostwald-Fehnske viscometer. Optical absorbances in the ultraviolet were determined in a
AND EITINGON Cary spectrophotometer (model 14) and a Beckman DU spectrophotometer with a circulating water cooling unit. Optical rotations were measured by means of a Franz-Schmidt polarimeter and a Rudolph photoelectric polarimeter (model 200). In these determinations, the urea-amylase solutions ‘were used without further dilution. In the second series of experiments, a more concentrated solution of the amylase was obtained by dissolving it in 1 M urea solution. As described above, the reaction mixture was then prepared by mixing the appropriate amount of the 10 M urea solution and the 1 M urea amylase solution so that the urea concentration was 6 M. The amylase concentration was varied from 9.8 to 0.55 mg/ml by dilution with 6 M urea solution. The mixtures were then incubated at 2” and amylase activity determination was made at definite time intervals. Similar experiments were carried out for guanidine, 1,3-dimethylurea, and tetramethylurea to determine the inactivation of the amylase by action of these denaturants. In these experiments, only the effect on the amylase activity was measured . Renaturation experiments were carried out on the 8 M urea-amylase solutions. After the mixture had been incubated for 2% hours and the enzyme was found to be completely inactivated, the mixture was dialyzed against a sodium phosphate buffer until no urea could be detected in the dialysate. The dialyzate, which had precipitated, was then centrifuged and both the precipitate and the supernatant liquid were measured for amylase activity. In another experiment, the inactivated amylase solution was diluted 500-fold with sodium phosphate buffer and the amylase activity of this diluted solution was then determined. RESULTS
E$ect of urea concentration. The rate of inof pancreatic amylase increases with increasing urea concentration. For various urea concentrations (2 M, 4 M, 6 M, and 8 M), the amylase concentration was 1 mg/ ml. In 2 M urea solutions, pancreatic amylase is fairly stable. After 6 days of incubation, only 3 % of the amylase activity is lost. In 4 M urea solutions, 70 % of the amylase activity is lost after 6 days and in 6 M urea solutions, the amylase is completely inactive after 6 days. In 8 M urea solution, the rate of inactivation is so great that the amylase is completely inactive after 120 minutes. The kinetics of the amylase inactivation in 2 M, 4 M, and 6 M urea solutions is shown in Fig. 1, where the logarithm of the percentage activation
UREA-AMYLASE
TABLE
remaining activity was plotted against time. In each case, the plotted points have a definite rectilinear trend, indicating a reaction of the first order. Thus, the inactivation obeys a differential equation of the form dY -= dt
I
T/ME
I
VALUES OF REACTION RATE CONSTANT k FOR VARIOUS CONCENTRATIONS OF UREA Urea concentration (M) k in (days)-*
2
4
6
0.0085
0.0373
1.64
8 51.75
--kY
where y is the percentage remaining activity, t is the time in days, and k is the reaction rate constant. Precisely the same conclusions were obtained from the corresponding data on the S 11 urea-amylase inactivation. Here, however, the value of k is very much greater. The values of k for the different urea concentrations are shown in Table I. E$ect of enxywaeconcentration on the rate of inactivation in 6 M urea solutions. When various concentrations of the enzyme (9.8, 5.0, 3.0, 1.53, and 0.55 mg/ml) were incubated in 6 31 urea solutions, the rate of inactivation was found to increase with decreasing concentrations of the amylase (Fig. 2). A solution containing 9.S mg/ml of the amylase lost 50 % of its amylase activity in 2 days, a.nd one containing 0.55 mg/ml of enzyme lost 9S % of its activity in the same period. When the logarithms of percentage of remaining activity are plotted against time for the various enzyme concentrations (Fig. Z), it is seen that there is a decided linear trend when the concentration of the /
521
REACTION
I
IN
I
r
I
DA,YS
FIG. 1. Kinetics of amylase-urea interaction. Urea concentrations: 2, 4, and 6 M. Amylase concentration: 1 mg/ml; pH, 7.6-7.2; temperature, 2”.
/
amylase is 1.5 mg/ml or lower. However, for the higher concentrations of the enzyme, it appears that the linear trend does not begin at once. There is a brief period (about one day), when inactivation proceeds at a faster rate than is indicated by the later points. This seems to imply the presence of a relatively short-lived perturbing phenomenon superimposed 011 the main decay process. The linearity seen in the succeeding portion of the graph indicates that the main inactivation process is exponential. E$ect on enzyme viscosity. The change in viscosity accompanying loss of enzyme activity is indicated in Fig. 3. The measurements were made in 6 M urea solutions containing 10 mg/ml of amylase. There is a gradual increase in reduced viscosity up to the point corresponding to a loss of 80% in amylase activity. After this point, the viscosity remains sensibly constant. One further observes that the rate of increase in reduced viscosity (i.e., the slopeof the graph) steadily decreases until the reduced viscosity reaches a steady state of about 9.5 (q,&, which corresponds to a loss of SO% of amylase activity. In replicate determinations, the solutions become turbid after 12-14 days of incubation. Precipitation then followed. Effect on the absorbance in the ultraviolet. Measurements in the ultraviolet (250-350 rnp) showed a very slight decrease in the intensity of absorbance of the inactivated enzyme as compared to that of the native enzyme. No shift in the tyrosine maximum (2SO rnp) was observed. Eflect on the optical rotation. Measurements were made on 6 M urea solutions containing 10 mg/ml of amylase. The specific rotation changed from an initial value of -40” to a value of - 66”. The latter corresponded to a loss of 90 % in amylase activity. Measurements were made at 5S9 rnp (sodium D line). Irreversibility of urea inactivation. After
522
TORA’LBALLA
AND EITINGON
FIO. 2. Kinetics of amylase inactivation in 6~ urea solutions and different amylase concentrations. Enzyme concentrations: 0.55, 1.50, 3.0, 5.3, and 9.8 mg/ml; pH, 7.0-7.2; reaction temperature, 2”.
FIG. 3. The relationship between changes in reduced viscosity and loss in amylase activity. Urea concentration, 6 M; enzyme concentratibn, c = 1 gm/lOO ml; pH, 7.e7.2; temperature, 2’.
dialyzing out, the urea from the completely inactivated 8 M urea-amylase solution, neither the precipitate nor the supernatant liquid of the dialyzate had any amylase activity. When the inactivated urea-amylase solution was diluted (1: 5Ooj, the resulting diluted solution had no measurable amylase activity. Action of cyanate ion on pancreatic amylase. Stark et al. (12) found that urea in aqueous solutions exists in equilibrium with small amounts of ammonium cyanate (an 8 M urea solution which is more alkaline than pH 6, would be 0.02 M in cyanate). The cyanate ion is known to inactivate proteins by blocking free amino and sulfhydryl groups (12). To examine the possibility that the inactivating action of urea is due to the cyanate ion present, pancreatic amylase was reacted with a solution of potassium cyanate (cyanate concentration, 0.1 M). The rate of inactivation in potassium cyanate solution was found to be very much less than that in 8 M urea solution under identical conditions. This indicates that the denaturing action is primarily due to urea itself.
UREA-AMYLASE
FIG. 4. Effect of guanidine concentration on amylase inactivation. Guanidine concentrations: 1 and 2 M; amylase concentration, 1 mg/ml; pH 7.c7.2; temperature, 2”.
Amylase inactivation by guanidine, 1,3dimethylurea, and tetramethylurea. As expect.ed, guanidine has a stronger denaturing effect on pancreatic amylase than urea. The inactivation by guanidine (1 and 2 M) is summarized in Fig. 4. In 1 M guanidine solutions containing 1 mg/ml of amylase, it is found that in 34 days, the enzymic activity has decreased by46 %. In2 M guanidine solutions containing 1 mg/ml of amylase, the enzyme is completely inactivated in 6 days. In addition, it was found that in 4 M guanidine solutions containing 1 mg/ml of enzyme, the amylase is completely inactivated in 40 minutes. 1,3-Dimethylurea has a very slight effect on pancreatic amylase activity. In 5 M solutions of this reagent containing 1 mg/ml of enzyme, it was found that in 30 days, only 6% of the nmylase activity was lost. In 6 M solutions containing the same concentration of amylase, 15 % of the enzymic activity was lost after 30 days. In solutions of 2, 4, and 6 M tetramethylurea solutions containing in each case 1 mg/ ml of enzyme, there was no significant inactivation of the enzyme after 12 days of incubation. DISCUSSION
The denaturing effects of urea on pancreatic amylase are quite similar to effects of urea on most globular proteins (13-16). In
REACTION
5’3
solutions with identical amylase concentrations, the proportional (i.e., percentage) rate of inactivation increases rapidly with an increase in the initial concentration of urea. In 1 and 2 M urea solutions, the proportional loss in amylase activity is hardly discernible, even after long periods of incubation (e.g., 60 days). On the other hand, in S M urea solutions, the proportional rate of inactivation is so great that in 120 minutes, all of the amylase activity is lost. At concentrations less than 1.5 mg/ml of amylase, and 1-S NI urea solutions, the loss in activity follows first-order kinetics. It was reported by Caldwell and Kung (3) that concentrated solutions of pancreatic amylase are more stable than dilute ones. The protective action due to concentration has been confirmed in the reaction with urea. In the same urea concentrations, the proportional rate of inactivation is much less for solutions containing 9.S mg/ml of enzyme than for dilute solutions containing 0.55 mg/ml of enzyme. This relative stability of concentrated solutions is probably due to an association of protein molecules. The latter thus becomes less accessible to the action of urea. At concentrations greater than 1.5 mg/ml of enzyme, the loss in activity does not follow first-order kinetics. A similar observation was reported for other proteins (14, 15). The kinetics curve for concentrated solutions indicates a brief initial period (1 day) of rapid inactivation followed by a slower and longer period of decrease in amylase activity. Martin and Frazier (15) attribute this phenomenon to the presence of different states of the enzyme. In concentrated solutions of pancreatic amylase, there may be more polymeric forms than monomers. The initial stage is probably largely due to the action of urea on the monomers, and the second slower and longer stage is due to the inactivation of the associated or polymeric form of the enzyme. The solubilities of amino acids and polypeptides in urea solutions have been found to be greater than their corresponding solubilities in water (16, 17). Pancreatic amylase is also quite soluble in urea solutions (15 mg/ ml in 1 BI urea solutions compared with 2
524
TORALBALLA
in water at 2”). The 1 M urea solutions of amylase, when kept at 2”, suffered no detectable loss in amylase activity after 14 days. Apparently, the process of solution is not intimately correlated with the process of inactivation. Increments in viscosity as well as increments in levorotation generally reflect alterations in both the secondary and tertiary structures of a protein molecule (13, 18, 19). The changes observed in the viscosity and optical rotation accompanying the inactivation of pancreatic amylase therefore reflect alterations in the tertiary and secondary structures of this enzyme. The decrease in intensity of absorbance in the ultraviolet of the inactivated enzyme also indicates a change in the secondary and tertiary structures (20-22). The inactivation of the enzyme cannot be reversed either by dialyses of the urea or by diluting the urea-amylase solutions. Our results concerning the relative effectiveness of urea and other amide reagents as denaturants are in agreement with the findings of other investigators (16). In equimolar concentrations, the order of effectiveness in inactivating pancreatic amylase is guanidine > urea > 1,3-dimenthylurea > tetramethylurea. This, in general, is the case for proteins which, like pancreatic amylase, are sensitive to the “urea-amide” type of denaturants as contrasted to the “hydrophobic” type of denaturing agents. The fact that here urea is more effective as an inactivating agent than either 1,3-dimethylurea or tetramethylurea confirms earlier findings (23) that methyl substitution in the urea molecule decreases denaturing effectiveness. A number of alternate mechanisms have been proposed to explain the inactivating effect of urea and similar amide reagents on proteins (13, 16, 24, 25). Prominent among these ideas are exchange hydrogen bonding, disruption of hydrophobic forces, and destruction of the so-called iceberg lattice. Our own results show that a change in secondary and tertiary structures occurs in the inactivation of pancreatic amylase by urea. These results do not allow us, however, to make any conclusion as to the mechanism of the action of urea on pancreatic mg/ml
AND
EITINGON
amylase. Experiments are in progress to extend and provide us with more definite information. ACKNOWLEDGMENT Part of the experimental work was done at the Chemistry Department of Barnard College, Columbia University, New York, New York. REFERENCES 1. CALDWELL, M. L., ADAMS, MILDRED, KUNQ, JO-FEN TUNG, AND TORALBALLA, GLORIA C., J. Am. Chem. Sot. 74, 4033 (1952). 2. KUNG, JO-FEN TUNG, HANRAHAN, VIRGINIA M., AND CALDWELL, M. L., J. Am. Chem. sot. 76,5548 (1953). 3. KUNG, JO-FEN TUNG, AND CALDWELL, M. L., J. Am. Chem. Sot. 76, 3132 (1953). 4. VAN DYK, J. W., KUNG, JO-FEN TUNG, AND CALDWELL, M. L., J. Am. Chem. Sot. ‘78, 3343 (1956). 5. VAN DYK, J. W., AND CALDWELL, M. L., J. Am. Chem. Sot. 78, 3345 (1956). 6. CALDWELL, M. L., DICKEY, EMMA S., HANRAHAN, VIRGINIA M., KUNG, H. C., KUNG, JO-FEN TUNG, AND MISKO, MARY, J. Am. Chem. Sot. 76, 143 (1954). 7. LITTLE, JOHN E., AND CALDWELL, M. L., J. Biol. Chem. 142, 585 (1942). 8. LITTLE, JOHN E., AND CALDWELL, M. L., J. Biol. Chem. 147, 229 (1943). 9. CALDWELL, M. L., WEILL, C. E., AND WEIL, RUTH S., J. Am. Chem. Sot. 67, 1079 (1945). 10. RADICHEVICH, ILDIKO, BECKER, MARY M., EITINGON, MARY, GETTLER, VIRGINIA H., TORALBALLA, GLORIA C., AND CALDWELL, M. L., J. Am. Chem. Sot. 81, 2845 (1959). 11. CALDWELL, M. L., DOEBBELING, S. E., AND MANIAN, S. II., Ind. Eng. Chem., Anal. Ed. 8, 181 (1936). 12. STARK, G. R., STEIN, W. H., AND MOORE, S., J. Biol. Chem. 236, 3177 (1960). 13. KAUZMANN, W., Advan. Protein Chem. 14, 1 (1959). 14. KAUZMANN, W., AND SIMPSON, R. B., J. Am. Chem. Sot. 76, 5139 (1953). 15. MARTIN, C. J., AND FRAZIER, A. R., J. BioE. Chem. 238, 3869 (1963). 16. GORDON, J. A., AND JENCKS, W. P., Biochemistry 2, 47 (1963). 17. WHITNEY, P. L., AND TANFORD, C., J. Biol. Chem. 237, PC1735 (1962). 18. YANG, JEN TSI, Advan. Protein Chem. 16, 323 (1961). 19. URNES, PETER, AND DOTY, PAUL, Advan. Protein Chem. 16, 401 (1961).
UREA-AMYLASE 20. YANARI, SAM, AND BOVEY, F. A., J. Biol. Chem. 236, 2818 (1960). 21. LEACH, S. J., AND SCHERAGA, HAROLD A., J. Biol. Chem. 236, 2827 (1960). 22. WETLAUFER, D., J. Biol. Chem. 233, 142 (1958).
REACTION
525
23. ROBINSON, D. R., AND JENCKS, W. P., J. Biol. Chem. 238, PC1558 (1963). 24. NOZAKI, YASUHICO, AND TANFORD, C., J. Bid. Chem. 238, 4074 (1963). 25. KLOTZ, I., AND STIZYKER, V., J. Am. Chem. Sot. 82, 5169 (1960).
OF
BIOCHEMISTRY
Action
AND
BIOPHYSICS
of Urea
on Crystalline GLORIA Depariment
of Chemistry,
Hunter
519-525 (1967)
119,
and Certain
Other
Porcine
C. TORALBALLA
Amide
Reagents
Pancreatic
Amylase’
MARY
EITINGON2
AND
College of the City University
of New York, A:ew York, New York 10021
Received July 30, 1966 The inactivation of porcine pancreatic amylase with various concentrations of urea with a fixed concentration of pancreatic amylase was investigated in one series of experiments. The changes in viscosity, absorbance in the ultraviolet, and optical rotation accompanying the loss in amylase activity were likewise determined. It was observed t,hat the rate of inactivation increased rapidly with an increase in the urea concentration. When the enzyme concentration is 1.5 mg/ml or less, the inactivation process follows first-order kinetics. This loss in activity is accompanied by an increase in reduced viscosity and in levorotation of the amylase. A very slight decrease in ultraviolet absorbance intensity was observed. In a second series of experiments, the urea concentration was fixed while the concentration of the amylase was varied. The rate of inactivation increases with a decrease in enzyme concentration. For concentrations greater than 1.5 mg/ml of amylase, the urea-amylase reaction does not follow first-order kinetics. The inactivation by urea is irreversible. Comparative inactivation studies of equimolar solutions of urea and other amide reagents show that the effectiveness in inactivating pancreatic amylase decreases in this order: guanidine > urea > 1 ,%dimethylurea > tetramethylurea.
A number of investigational approaches which have furnished valuable and pertinent information toward the elucidation of the chemical structure of crystalline procine pancreatic amylase have been carried through by Caldwell and co-workers. They reported on an improved method for the purification and crystallization of this amylase (l), on the nature and kinetics of its biological action (Z-5), on its amino acid composition (6), and on the essentiality of some of its functional groups (7-10). To date, no studies have been published on the action of urea and other amide reagents on porcine pancreatic amylase.
The present investigation was carried out to study in some detail the action of urea and certain other related denaturants on the amylase. This paper reports mainly the results of the action of urea on pancreatic amylase. To correlate any possible structural alterations in the protein molecule with loss in amylase activity, determinations were made of the effect of urea on the viscosity, the optical rotation, and the ultraviolet absorbance of the enzyme. In addition, the action of urea is compared with that of guanidine, 1,3-dimethylurea, and tetramethylurea.
1 This investigation was supported by grants GM-O8960-01 and GM-08960-02 from the National Institutes of Health, U.S. Public Health Service. A preliminary report was presented to the 142nd meeting of the American Chemical Society, Atlantic City, New Jersey, September 9-14, 1962. * Present address: Cornell Medical College, New York Hospital, New York, New York 10021.
Twice-crystallized porcine pancreatic amylase was obtained from Worthington Biochemical Corporation. The cryst,als (LOOmg) were washed repeatedly wit,h 1 cc of cold sodium phosphate buffer (0.01 M in sodium phosphate, 0.02 M in sodium chloride, pH, 7.0; temperature, 2’) until the amylase activity of the solid was found to remain constant,. Amylase stock solutions
EXPERIMENTAL
519
Materials.
PROCEDUR.ES
520
TORALBALLA
were obtained by stirring a suspension of the washed crystals in cold sodium phosphate buffer (2”) and were further purified by dialysis against a sodium phosphate buffer. The enzyme concentration of these solutions was 2-3 me/ml and the amylase activity was 13,500-14,000 maltose equivalents per milligram of solid enzyme. The concentration of each prepared amylase solution was determined spectrophotometrically at 280 rnp using a calibration curve (1). Urea (Baker, reagent grade) was crystallized twice from an ethanol-water solvent (1:l by volume). The melting point (uncorrected) of the product was 131.5”. Guanidine hydrochloride was crystallized twice from absolute ethyl alcohol. 1,3-Dimet,hylurea was crystallized twice from absolute ethanol. The product had a melting point (uncorrected) of 106.5”. Tetramethylurea, reagent grade, and purified by gas chromatography (John Deere Chemical Company), was used without further purification. Distilled water which was redistilled in an all glass distilling apparatus was used throughout. Methods. A 10 M solution of urea in sodium phosphate buffer was prepared and refrigerated until used. For each of the various urea molarities needed (i.e., 2 M, 4 M, 6 M, and 8 M) the reaction mixture was prepared by adding the required amount of the 10 M urea solution to the amylase solution. The pH of the reaction mixtures was maintained at 7.0-7.2 and the incubation temperature was 2”. This particular pH and incubation temperature were chosen because, under these conditions, porcine pancreatic amylase, by itself, does not undergo any inactivation (1, 3). Control solutions containing only the enzyme in sodium phosphate buffer were incubated at the same temperature. Aliquots of each of these urea-amylase mixtures and the controls were then removed at definite and suitable time intervals and measured simultaneously for loss in amylase activity and for changes in viscosity, optical rotation, and absorbance in the ultraviolet. Amylase activity was determined by measuring the saccharogenic action of the enzyme on a solution of Lintner soluble starch at 40” for 30 minutes according to the iodometric method of Caldwell et al. (11). This method necessitated the dilution of the urea-amylase solutions with buffered starch solutions. However, since the urea-amylase inactivation is irreversible, the value obtained for the amylase activity is not affected by the dilution. This fact was shown by comparative amylase activity measurements on diluted and undiluted urea-amylase solutions. Viscosities were measured at 2.0 f 0.1” with an Ostwald-Fehnske viscometer. Optical absorbances in the ultraviolet were determined in a
AND EITINGON Cary spectrophotometer (model 14) and a Beckman DU spectrophotometer with a circulating water cooling unit. Optical rotations were measured by means of a Franz-Schmidt polarimeter and a Rudolph photoelectric polarimeter (model 200). In these determinations, the urea-amylase solutions ‘were used without further dilution. In the second series of experiments, a more concentrated solution of the amylase was obtained by dissolving it in 1 M urea solution. As described above, the reaction mixture was then prepared by mixing the appropriate amount of the 10 M urea solution and the 1 M urea amylase solution so that the urea concentration was 6 M. The amylase concentration was varied from 9.8 to 0.55 mg/ml by dilution with 6 M urea solution. The mixtures were then incubated at 2” and amylase activity determination was made at definite time intervals. Similar experiments were carried out for guanidine, 1,3-dimethylurea, and tetramethylurea to determine the inactivation of the amylase by action of these denaturants. In these experiments, only the effect on the amylase activity was measured . Renaturation experiments were carried out on the 8 M urea-amylase solutions. After the mixture had been incubated for 2% hours and the enzyme was found to be completely inactivated, the mixture was dialyzed against a sodium phosphate buffer until no urea could be detected in the dialysate. The dialyzate, which had precipitated, was then centrifuged and both the precipitate and the supernatant liquid were measured for amylase activity. In another experiment, the inactivated amylase solution was diluted 500-fold with sodium phosphate buffer and the amylase activity of this diluted solution was then determined. RESULTS
E$ect of urea concentration. The rate of inof pancreatic amylase increases with increasing urea concentration. For various urea concentrations (2 M, 4 M, 6 M, and 8 M), the amylase concentration was 1 mg/ ml. In 2 M urea solutions, pancreatic amylase is fairly stable. After 6 days of incubation, only 3 % of the amylase activity is lost. In 4 M urea solutions, 70 % of the amylase activity is lost after 6 days and in 6 M urea solutions, the amylase is completely inactive after 6 days. In 8 M urea solution, the rate of inactivation is so great that the amylase is completely inactive after 120 minutes. The kinetics of the amylase inactivation in 2 M, 4 M, and 6 M urea solutions is shown in Fig. 1, where the logarithm of the percentage activation
UREA-AMYLASE
TABLE
remaining activity was plotted against time. In each case, the plotted points have a definite rectilinear trend, indicating a reaction of the first order. Thus, the inactivation obeys a differential equation of the form dY -= dt
I
T/ME
I
VALUES OF REACTION RATE CONSTANT k FOR VARIOUS CONCENTRATIONS OF UREA Urea concentration (M) k in (days)-*
2
4
6
0.0085
0.0373
1.64
8 51.75
--kY
where y is the percentage remaining activity, t is the time in days, and k is the reaction rate constant. Precisely the same conclusions were obtained from the corresponding data on the S 11 urea-amylase inactivation. Here, however, the value of k is very much greater. The values of k for the different urea concentrations are shown in Table I. E$ect of enxywaeconcentration on the rate of inactivation in 6 M urea solutions. When various concentrations of the enzyme (9.8, 5.0, 3.0, 1.53, and 0.55 mg/ml) were incubated in 6 31 urea solutions, the rate of inactivation was found to increase with decreasing concentrations of the amylase (Fig. 2). A solution containing 9.S mg/ml of the amylase lost 50 % of its amylase activity in 2 days, a.nd one containing 0.55 mg/ml of enzyme lost 9S % of its activity in the same period. When the logarithms of percentage of remaining activity are plotted against time for the various enzyme concentrations (Fig. Z), it is seen that there is a decided linear trend when the concentration of the /
521
REACTION
I
IN
I
r
I
DA,YS
FIG. 1. Kinetics of amylase-urea interaction. Urea concentrations: 2, 4, and 6 M. Amylase concentration: 1 mg/ml; pH, 7.6-7.2; temperature, 2”.
/
amylase is 1.5 mg/ml or lower. However, for the higher concentrations of the enzyme, it appears that the linear trend does not begin at once. There is a brief period (about one day), when inactivation proceeds at a faster rate than is indicated by the later points. This seems to imply the presence of a relatively short-lived perturbing phenomenon superimposed 011 the main decay process. The linearity seen in the succeeding portion of the graph indicates that the main inactivation process is exponential. E$ect on enzyme viscosity. The change in viscosity accompanying loss of enzyme activity is indicated in Fig. 3. The measurements were made in 6 M urea solutions containing 10 mg/ml of amylase. There is a gradual increase in reduced viscosity up to the point corresponding to a loss of 80% in amylase activity. After this point, the viscosity remains sensibly constant. One further observes that the rate of increase in reduced viscosity (i.e., the slopeof the graph) steadily decreases until the reduced viscosity reaches a steady state of about 9.5 (q,&, which corresponds to a loss of SO% of amylase activity. In replicate determinations, the solutions become turbid after 12-14 days of incubation. Precipitation then followed. Effect on the absorbance in the ultraviolet. Measurements in the ultraviolet (250-350 rnp) showed a very slight decrease in the intensity of absorbance of the inactivated enzyme as compared to that of the native enzyme. No shift in the tyrosine maximum (2SO rnp) was observed. Eflect on the optical rotation. Measurements were made on 6 M urea solutions containing 10 mg/ml of amylase. The specific rotation changed from an initial value of -40” to a value of - 66”. The latter corresponded to a loss of 90 % in amylase activity. Measurements were made at 5S9 rnp (sodium D line). Irreversibility of urea inactivation. After
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FIO. 2. Kinetics of amylase inactivation in 6~ urea solutions and different amylase concentrations. Enzyme concentrations: 0.55, 1.50, 3.0, 5.3, and 9.8 mg/ml; pH, 7.0-7.2; reaction temperature, 2”.
FIG. 3. The relationship between changes in reduced viscosity and loss in amylase activity. Urea concentration, 6 M; enzyme concentratibn, c = 1 gm/lOO ml; pH, 7.e7.2; temperature, 2’.
dialyzing out, the urea from the completely inactivated 8 M urea-amylase solution, neither the precipitate nor the supernatant liquid of the dialyzate had any amylase activity. When the inactivated urea-amylase solution was diluted (1: 5Ooj, the resulting diluted solution had no measurable amylase activity. Action of cyanate ion on pancreatic amylase. Stark et al. (12) found that urea in aqueous solutions exists in equilibrium with small amounts of ammonium cyanate (an 8 M urea solution which is more alkaline than pH 6, would be 0.02 M in cyanate). The cyanate ion is known to inactivate proteins by blocking free amino and sulfhydryl groups (12). To examine the possibility that the inactivating action of urea is due to the cyanate ion present, pancreatic amylase was reacted with a solution of potassium cyanate (cyanate concentration, 0.1 M). The rate of inactivation in potassium cyanate solution was found to be very much less than that in 8 M urea solution under identical conditions. This indicates that the denaturing action is primarily due to urea itself.
UREA-AMYLASE
FIG. 4. Effect of guanidine concentration on amylase inactivation. Guanidine concentrations: 1 and 2 M; amylase concentration, 1 mg/ml; pH 7.c7.2; temperature, 2”.
Amylase inactivation by guanidine, 1,3dimethylurea, and tetramethylurea. As expect.ed, guanidine has a stronger denaturing effect on pancreatic amylase than urea. The inactivation by guanidine (1 and 2 M) is summarized in Fig. 4. In 1 M guanidine solutions containing 1 mg/ml of amylase, it is found that in 34 days, the enzymic activity has decreased by46 %. In2 M guanidine solutions containing 1 mg/ml of amylase, the enzyme is completely inactivated in 6 days. In addition, it was found that in 4 M guanidine solutions containing 1 mg/ml of enzyme, the amylase is completely inactivated in 40 minutes. 1,3-Dimethylurea has a very slight effect on pancreatic amylase activity. In 5 M solutions of this reagent containing 1 mg/ml of enzyme, it was found that in 30 days, only 6% of the nmylase activity was lost. In 6 M solutions containing the same concentration of amylase, 15 % of the enzymic activity was lost after 30 days. In solutions of 2, 4, and 6 M tetramethylurea solutions containing in each case 1 mg/ ml of enzyme, there was no significant inactivation of the enzyme after 12 days of incubation. DISCUSSION
The denaturing effects of urea on pancreatic amylase are quite similar to effects of urea on most globular proteins (13-16). In
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solutions with identical amylase concentrations, the proportional (i.e., percentage) rate of inactivation increases rapidly with an increase in the initial concentration of urea. In 1 and 2 M urea solutions, the proportional loss in amylase activity is hardly discernible, even after long periods of incubation (e.g., 60 days). On the other hand, in S M urea solutions, the proportional rate of inactivation is so great that in 120 minutes, all of the amylase activity is lost. At concentrations less than 1.5 mg/ml of amylase, and 1-S NI urea solutions, the loss in activity follows first-order kinetics. It was reported by Caldwell and Kung (3) that concentrated solutions of pancreatic amylase are more stable than dilute ones. The protective action due to concentration has been confirmed in the reaction with urea. In the same urea concentrations, the proportional rate of inactivation is much less for solutions containing 9.S mg/ml of enzyme than for dilute solutions containing 0.55 mg/ml of enzyme. This relative stability of concentrated solutions is probably due to an association of protein molecules. The latter thus becomes less accessible to the action of urea. At concentrations greater than 1.5 mg/ml of enzyme, the loss in activity does not follow first-order kinetics. A similar observation was reported for other proteins (14, 15). The kinetics curve for concentrated solutions indicates a brief initial period (1 day) of rapid inactivation followed by a slower and longer period of decrease in amylase activity. Martin and Frazier (15) attribute this phenomenon to the presence of different states of the enzyme. In concentrated solutions of pancreatic amylase, there may be more polymeric forms than monomers. The initial stage is probably largely due to the action of urea on the monomers, and the second slower and longer stage is due to the inactivation of the associated or polymeric form of the enzyme. The solubilities of amino acids and polypeptides in urea solutions have been found to be greater than their corresponding solubilities in water (16, 17). Pancreatic amylase is also quite soluble in urea solutions (15 mg/ ml in 1 BI urea solutions compared with 2
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in water at 2”). The 1 M urea solutions of amylase, when kept at 2”, suffered no detectable loss in amylase activity after 14 days. Apparently, the process of solution is not intimately correlated with the process of inactivation. Increments in viscosity as well as increments in levorotation generally reflect alterations in both the secondary and tertiary structures of a protein molecule (13, 18, 19). The changes observed in the viscosity and optical rotation accompanying the inactivation of pancreatic amylase therefore reflect alterations in the tertiary and secondary structures of this enzyme. The decrease in intensity of absorbance in the ultraviolet of the inactivated enzyme also indicates a change in the secondary and tertiary structures (20-22). The inactivation of the enzyme cannot be reversed either by dialyses of the urea or by diluting the urea-amylase solutions. Our results concerning the relative effectiveness of urea and other amide reagents as denaturants are in agreement with the findings of other investigators (16). In equimolar concentrations, the order of effectiveness in inactivating pancreatic amylase is guanidine > urea > 1,3-dimenthylurea > tetramethylurea. This, in general, is the case for proteins which, like pancreatic amylase, are sensitive to the “urea-amide” type of denaturants as contrasted to the “hydrophobic” type of denaturing agents. The fact that here urea is more effective as an inactivating agent than either 1,3-dimethylurea or tetramethylurea confirms earlier findings (23) that methyl substitution in the urea molecule decreases denaturing effectiveness. A number of alternate mechanisms have been proposed to explain the inactivating effect of urea and similar amide reagents on proteins (13, 16, 24, 25). Prominent among these ideas are exchange hydrogen bonding, disruption of hydrophobic forces, and destruction of the so-called iceberg lattice. Our own results show that a change in secondary and tertiary structures occurs in the inactivation of pancreatic amylase by urea. These results do not allow us, however, to make any conclusion as to the mechanism of the action of urea on pancreatic mg/ml
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amylase. Experiments are in progress to extend and provide us with more definite information. ACKNOWLEDGMENT Part of the experimental work was done at the Chemistry Department of Barnard College, Columbia University, New York, New York. REFERENCES 1. CALDWELL, M. L., ADAMS, MILDRED, KUNQ, JO-FEN TUNG, AND TORALBALLA, GLORIA C., J. Am. Chem. Sot. 74, 4033 (1952). 2. KUNG, JO-FEN TUNG, HANRAHAN, VIRGINIA M., AND CALDWELL, M. L., J. Am. Chem. sot. 76,5548 (1953). 3. KUNG, JO-FEN TUNG, AND CALDWELL, M. L., J. Am. Chem. Sot. 76, 3132 (1953). 4. VAN DYK, J. W., KUNG, JO-FEN TUNG, AND CALDWELL, M. L., J. Am. Chem. Sot. ‘78, 3343 (1956). 5. VAN DYK, J. W., AND CALDWELL, M. L., J. Am. Chem. Sot. 78, 3345 (1956). 6. CALDWELL, M. L., DICKEY, EMMA S., HANRAHAN, VIRGINIA M., KUNG, H. C., KUNG, JO-FEN TUNG, AND MISKO, MARY, J. Am. Chem. Sot. 76, 143 (1954). 7. LITTLE, JOHN E., AND CALDWELL, M. L., J. Biol. Chem. 142, 585 (1942). 8. LITTLE, JOHN E., AND CALDWELL, M. L., J. Biol. Chem. 147, 229 (1943). 9. CALDWELL, M. L., WEILL, C. E., AND WEIL, RUTH S., J. Am. Chem. Sot. 67, 1079 (1945). 10. RADICHEVICH, ILDIKO, BECKER, MARY M., EITINGON, MARY, GETTLER, VIRGINIA H., TORALBALLA, GLORIA C., AND CALDWELL, M. L., J. Am. Chem. Sot. 81, 2845 (1959). 11. CALDWELL, M. L., DOEBBELING, S. E., AND MANIAN, S. II., Ind. Eng. Chem., Anal. Ed. 8, 181 (1936). 12. STARK, G. R., STEIN, W. H., AND MOORE, S., J. Biol. Chem. 236, 3177 (1960). 13. KAUZMANN, W., Advan. Protein Chem. 14, 1 (1959). 14. KAUZMANN, W., AND SIMPSON, R. B., J. Am. Chem. Sot. 76, 5139 (1953). 15. MARTIN, C. J., AND FRAZIER, A. R., J. BioE. Chem. 238, 3869 (1963). 16. GORDON, J. A., AND JENCKS, W. P., Biochemistry 2, 47 (1963). 17. WHITNEY, P. L., AND TANFORD, C., J. Biol. Chem. 237, PC1735 (1962). 18. YANG, JEN TSI, Advan. Protein Chem. 16, 323 (1961). 19. URNES, PETER, AND DOTY, PAUL, Advan. Protein Chem. 16, 401 (1961).
UREA-AMYLASE 20. YANARI, SAM, AND BOVEY, F. A., J. Biol. Chem. 236, 2818 (1960). 21. LEACH, S. J., AND SCHERAGA, HAROLD A., J. Biol. Chem. 236, 2827 (1960). 22. WETLAUFER, D., J. Biol. Chem. 233, 142 (1958).
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23. ROBINSON, D. R., AND JENCKS, W. P., J. Biol. Chem. 238, PC1558 (1963). 24. NOZAKI, YASUHICO, AND TANFORD, C., J. Bid. Chem. 238, 4074 (1963). 25. KLOTZ, I., AND STIZYKER, V., J. Am. Chem. Sot. 82, 5169 (1960).