Inhibition of enzymic hydrolysis of plasma albumin by detergents

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 93, 538--541

(1961)

Inhibition of Enzymic Hydrolysis of Plasma Albumin by Detergents SAMUEL

I. E P S T E I N

AND P A U L A. P O S S I C K

From the Department of Chemistry, ~ Tufts University, Medjord, Massachusetts Received January 25, 1961 Digestion of bovine plasma albumin by either trypsin or chymotrypsin is markedly diminished by sodium octyl sulfate, yet enzymic hydrolysis of simple substances is hardly affected by this detergent. Tryptic digestion of bovine albumin is similarly inhibited by sodium decyl and dodecyl sulfates, while digestion of e-globulin is nearly unaffected. Implications of the inhibition are discussed. INTRODUCTION M e t h y l o r a n g e (1) a n d c e r t a i n f a t t y acids (2) r e t a r d e n z y m i c h y d r o l y s i s of p l a s m a albumins. W e n o w find t h a t c e r t a i n s o d i u m a l k y l sulfates m a r k e d l y d i m i n i s h o v e r - a l l digestion of b o v i n e p l a s m a a l b m n i n ( B P A ) . T h e s e results are p e r t i n e n t to curr e n t concepts of p r o t e o ly s is a n d of t he s t r u c t u r e of p l a s m a albumins. EXPERIMENTAL Crystallized BPA was obtained from Armour and Nutritional Biochemieals (N), 10× crystallized bovine mercaptalbumin (MA) from N, bovine a-globulin (Fraction IV) from N, 2× crystallized salt-free trypsin (T) from N and Worthington Biochemical (W), and 3× crystallized a-chymotrypsin (CT) from W. The sodium octyl ($8), decyl ($10), and dodecyl ($12) sulfates, purified (assay 99.0% or higher), were the generous gift of E. I. du Font de Nemours and Co. Dialyzed solutions of the proteins were diluted using these optical factors (280 mt~, Beckman DU): BPA and MA, 1.52 (3); CT, 0.53 (4); T, 0.64 (4); a-globulin, 0.94 (dry weight determination). Fatty acid contamination (5) of BPA and MA was ignored. Solutions were stored at 5°C. The absorption (280 mt~) of trichloroacetic acid (TCA)-soluble products was the usual measure of hydrolysis. Digestion was started by mixing 1 ml. of a concentrated solution of T or CT in 0.001 1 Contribution No. 268. This study was supported by grants E1704 and E1704 (C1) of the National Institute of Allergy and Infectious Diseases and by a grant from the Cyrus M. Warren fund of the American Academy of Arts and Sciences.

M HC1 with the other components of the digestion mixture, which were contained in 32 ml. of pH 8.2-8.3, ionic strength 0.1 buffer (made by titrating 0.1 M NaCI, 0.1 M H3BO~ with 0.1 M NaOH) (6). The digestion mixture contained 0.30% substrate (4.4 × 10-5 M BPA, assuming molecular weight of 69,000), 0.0097% T (or 0.0107% CT), and binding agent, when present, at the following mole ratios (all mole ratios cited are relative to BPA): $8, generally 20; $10, 20; $12, 20. The digestion temperature was 35°C. To 4 ml. of digest was added 3 ml. of 10% TCA. Blanks were prepared by adding enzyme after TCA (enzyme autolysis was ignored). Assuming the above optical factor for BPA, an optical density of 1.13 would imply 100% digestion. Some digestions were followed by a manual pHstar method (Beckman model G) at 25.3°C. The digestion mixture composition was the above, except that the solvent was essentially 0.1 M NaC1. Mixtures were brought to pH 8.30 with 0.102 N NaOH, which was also used to maintain the pH after addition of enzyme (0.001 M HC1). In one trial, an identical rate was observed using 0.0102 N NaOtI. Nitrogen was passed through soda-lime and, after washing, over the digest. Readings were usually taken at 2-rain. intervals. If the pK of the liberated amino groups is 7.6 (7), each microliter of NaOH used corresponds to 0.084 bonds per BPA molecule. Hydrolysis of 0.00098 M benzoyl-L-arginine amide (BAA, Mann Research Labs.) by T (0.0097%) and 0.00097 M methyl hippurate (MI-I, H. M. Chemical Co.) by CT (0.0104%) was tracked by the change in optical density (AO.D.) at 260 and 257.5 m~, respectively. The buffer medium was identical with that for the TCA method. A temperatm'e of 25°C. was maintained using Beckman Thermospacers.

538

INHIBITION OF ENZYMIC HYDROLYSIS RESULTS

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T h a t hydrolysis of B P A by either T or C T is retarded by $8 is shown in Figs. 1-3. A pair of digestions were run simultaneously by the T C A method, one with and one without detergent, assuring enzyme and substrate of identical condition. The ratiO of absorption readings, at equal times, refleets the extent of retardation. Generally, for such a paired run, this ratio was nearly independent of time. The average of such ratios, for four or more times during a run, ranged between 0.10 and 0.35 for thirteen runs. Among these thirteen were r e p r e ' sented three lots of T and four of albumin. The measurement of initial rates by the p H - s t a t method confirms the marked inhibition. The minor influence of $8 on T activity agrees with findings for the system under other conditions (8). Inhibition of either T or C T in albumin digests is still smaller because of binding of $8 to albumin. Prior incubation of $8 with T for 5 rain., rather than with BPA, had no significant effect on the extent of digestion. T h a t proteetion develops rapidly presumably reflects the speed of S8-BPA association. The result also indicates t h a t T is unharmed by brief exposure to these amounts of $8. When mixtures of C T and BPA, containing various mole ratios of $8, were digested

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Fro. 1. Effect of S8 ( f i l l e d symbols, mole r a t i o 20) on digestion of 0.30% a l b u m i n b y 0.0097% T, p H 8.2-8.3. T h r e e different lots each of a l b u m i n and trypsin are represented. O t h e r conditions in text.

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Ft0.2. Effect of $8 (filled symbols, at indicated mote ratios) on digestion of 0.30% BPA (Armour, lot T68204) by 0.0107% CT (W, lot 537-40), pH 8.3. Digestions performed simultaneously have symbols of sa~e shape. Other conditions in text, except that the digestion temperature was 25.3°C. 0,4-

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FIo. 3. Effect of $8 (filled symbols, 8.8 X 10 ~ M, as in digests of mole ratio 20) on digestion of simple substrates: 0.00098 M BAA by 0.0097% T and 0.00097 M MH by 0.0104% CT, pH 8.2. Enzyme absorption has been deducted, AO.D. of 0.70 (MH, 257.5 mt~) and 0.48 (BAA, 260 mt~) would imply 100% hydrolysis. Other conditions in text. for 2 hr., the optical densities of TCA-soluble products were (mole ratio, then O.D. are listed) : 20, 0.023; 6.7, 0.030; 3.3, 0.058; 1.1, 0.140; 0, 0.250. The average mole ratios of bound $8 are about 8, 6.5, and 5, for total

540

EPSTEIN AND POSSICK

mole ratios of 20, 10, and 6.7, respectively [Fig. 1 of Ref. (9)]. At lower ratios, over 90% binding is expected. In making these estimates, we assumed identical binding at pH's 8.3 and 6.1. Inhibition at pH 7.1 in t h e B P A - S 8 - T system is comparable to that at pH 8.3, as this assumption suggests. The important fact is that the mole ratio of total as well as bound $8 is relatively small, yet inhibition is marked. S10 and S12, at a mole ratio of 20 [average bound detergent mole ratios of 10-12 (9)], reduced digestion of BPA (TCA method) to about 20% of the detergent-free rate. $8, $10, and $12 at the same weight concentration as in the BPA digests had little effect on 0.30% a-globulin digestion; S12, in fact, increased digestion by 10%. Since the a-globulin findings may be a resultant of substrate denaturation and T inactivation, measurements at lower S10 and $12 concentrations would be desirable. Considering the $8 results, it seems likely that considerable inhibition would still obtain. DISCUSSION Not only do suitable additives prevent acceleration of digestion, induced by a denaturant (10), but they also may reduce the digestion rate of plasma albumins below that of the ordinary protein. The common property of alkyl sulfates'and the previously reported retardants is that all bind tightly to albumins (11). We might infer that all tightly bound substances can retard BPA digestion. However, the binding sites may not be independent: either methyl orange or S12 can bind to some sites (12). Several causes may contribute to inhibition. Thus, increased net negative charge on BPA, due to binding, may inhibit action by CT, which is isoelectric at pH ~5. Steric hindrance, particularly of T, may ensue from binding to cationic sites of BPA. However, many organic anions, including fatty acid anions which are structurally similar to the sodium alkyl sulfates, also inhibit the heat denaturation of plasma albmnin (13). Thus a broader explanation seems necessary. For the analogous case of the protection of BPA against proteolysis (14) and heat denaturation by Ca ++, a susceptible form,

common to both processes has been suggested (15). One effect ascribed to the additive is the displacement of an equilibrium toward the unsusceptible form. Reduction of the concentration of the intermediate lowers the rate. Such a mechanism is attractive here, because of evidence for two interconvertible forms of plasma albumin ( l l ) . This mechanism implies that any products of digestion would be independent of the presence of additive. Inhibition may be explained qualitatively without postulating a susceptible form. The transition state for a process, starting with the inhibitor-albumin complex, might be essentially that for the albumin, but with the additive considerably removed from its site. A portion of the free energy of dissociation would then add to the ordinary free energy of activation. It has been suggested that the positive AS for the binding of alkyl sulfates (9) and other substances to albumin derives from a release of local strains; these strains may stem from awkward tertiary linkages between polypeptide chains (16). Strained portions of a protein molecule would probably contain weakened bonds, susceptible to various reactions. Release of strain would improve stability. That a small number of molecules markedly diminish digestion suggests that there are few strained regions. This conclusion is compatible, too, with the relative resistance of BPA to digestion. A comparison of the products of the inhibited and uninhibited reactions would illumine the mechanism of inhibition. It remains possible that protection of specific portions of the protein molecules occurs, which might be of value in isolation of active protein fragments. ACKNOWLEDGEMENT We thank Nancy A. Hunt and Earl B. ttoyt, Jr., for careful technical assistance with part of this work. REFERENCES 1. LABEYRIE, F., AND NASLIN, L., Biochim. et Biophys. Acta 27, 564 (1958). 2. Kol'~Do, M., J. Biochem. (Tokyo) 45, 705

(1959).

I N H I B I T I O N OF ENZYMIC HYDROLYSIS 3. CoH~, E. J., HUGHES, W. L., Jr., AND WEARE, J. H., J. Am. Chem. Soc. 69, 1753 (1947). 4. Worthington Enzymes, Descriptive Manual No. 8, Worthington Biochemical Corp., Freehold, N. J., 1954. 5. GOOI)MAI'~,D. S., Y. Am. Chem. Soc. 80~ 3892 (1958). 6. SRI RAM, J., AlX:DMAURER,P. ~I., Arch. Biochem. Biophys. 70, 185 (1957). 7. COHN, E. J., AND ]~DSALL, J. T. "Proteins, Amino Acids and Peptides," p. 85 Reinhold, New York, 1943. 8. VISWAR~ATttA,T., PALLANSCI-I,M. J., AND LIEDER, I. E., Y. Biol. Chem. 212, 301 (1955). 9. KARUSIt, F., AND SOI'~EI~BERG~M., J. Am. Chem. Soc. 71, 1369 (1949).

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10. LUCK, J. M., J. Phys. & Colloid. Chem. 51, 229 (1947). 11. FOSTER,J. F., /n "The Plasma Proteins" (F. W. Putnam, ed.), Vol. I. Academic Press, New York, 1960. 12. KLOTZ, I. M., TRIWVSH, H., A~D WALKER, F. M., Y. Am. Chem. Soc. 70, 2935 (1948). 13. BOWER,P. D., Lu~, F. G., BALLOU,G. A., LucK, J. M., A~D RICE, 1%. G., J. Biol. Chem. 162, 181 (1946). 14. GORINI, L., AND AUDRAIN,L., Biochim. et Biophys. Acta 9, 180 (1952). 15. GURD, F. R. N., AND WILCOX, P. E., Advances inProtein Chem. 11, 311 (1956). 16. SCI-IELLMAN, J. A., LUMRY, R., AND SAMUELS, L. T., J. Am. Chem. Soe. 76, 2808 (1954).