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Inactivation of the protease inhibitor phenylmethylsulfonyl fluoride in buffers
ANALYTICAL
BIOCHEMISTRY
Inactivation
86,
574-579 (1978)
of the Protease inhibitor Phenylmethylsulfonyl Fluoride in Buffers GORDON
Department
of Neurology.
University
T. JAMES
of Colorado
Medico1
Center,
Denver,
Colorado
80262
Received April 20, 1977; accepted December 12. 1977 Aqueous preparations ofphenylmethylsulfonyl fluoride (PMSF) become inactive toward proteases unless promptly brought into contact with protease. Inactivation of PMSF increases with increased pH and temperature. Half-lives ofthe inhibitor at 25°C are approximately I10,55, and 35 min at pH 7.0,7.5, and 8.0. respectively. At pH 8, 100 /.LM PMSF is almost completely inactivated within 1 hr at 25°C or within 22 hr at 4°C. Stock solutions of PMSF in 100% isopropanol are stable at 25°C for months if not longer. Reactivation of PMSF-inhibited chymotrypsin did not occur within 1 week at 25°C at pH 7.0.
Inhibition of proteases has become important not only for well-defined biochemical studies of pure proteases but also for prevention of proteolytic degradation of other proteins in biological preparations. The protease inhibitor phenylmethylsulfonyl fluoride (PMSF) is now widely used for these purposes. Fahrney and Gold (1) described the kinetics of inhibition of the reaction of PMSF and analogs with trypsin and chymotrypsin. It has been shown that the active site serine residue reacts with PMSF (2). A number of workers have used PMSF to prevent artifactual proteolytic modification of various proteins during purification or during other work (3-8). Chong et al. (9) found that a neutral chromatin protease was inhibited by PMSF. Besides proteases PMSF sometimes inhibits other types of enzymes, usually esterases, e.g., acetylcholinesterase (1). Kumar (IO) found that palmityl coenzyme A deacylase was selectively inhibited by PMSF within a pigeon liver fatty acid synthetase complex, and Gaertner and Cole (7) noted an inhibition of some of the biosynthetic enzymes in the aromatic complex of Neurospora, as well as an effective inhibition of a proteolytic contaminant. Human liver arylsulfatase A was partially inhibited by PMSF at or near the optimal pH for enzyme activity (11). The above reports show that PMSF is widely employed in protein chemistry and molecular biology. However little definitive data is available on the stability of this inhibitor under various conditions. This paper provides such data.
0003-?697178/0862-0574$02.00/O Copyright All rights
B 1978 by Academic Press, Inc. of reproduction in any form reserved.
574
PHENYLMETHYLSULFONYL MATERIALS
FLUORIDE
575
AND METHODS
Bovine trypsin, a-chymotrypsin, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma Co. Pronase, azocoll, and N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes) were from Calbiochem. Stock solutions of 10 and 100 mM PMSF in 100% 2-propanol were made and stored at room temperature. Proteases were dissolved in cold 10e3 N HCl at 5 mg/lOO ~1 and kept in an ice bath. PMSF stability at 100 PM in buffers was studied by adding 1~1 of 100 mM PMSF stock solution to 1.0 ml of the buffer under study. Then 10 ~1 of protease solution (0.5 mg) was added immediately for zero-time samples or optimum inhibition samples. Otherwise 10 ~1 of protease was added at a timed interval per sample so that preincubation of PMSF in buffer could occur before coming in contact with protease. Each timed sample also consisted of a control tube, i.e., 1 ~1 of 2-propanol was added rather than the PMSF stock solution; protease was added when appropriate. Some experiments were also performed with 20 pM PMSF in buffer, with an appropriate aliquot of 10 mM PMSF stock solution (see Discussion). After addition of protease to samples with or without PMSF, incubation was carried out for 5 min to allow reaction with PMSF. Then 4.0 ml of the buffer under study was added and mixed, and 1.O ml of the resulting 5-ml solution was transferred to a tube containing 5 mg of solid azocoll. The optical density of solubilized peptides containing the azo dye was measured at 520 nm after centrifugation of remaining insoluble azocoll(12). Blank controls consisted of above components minus proteases. The calculated molar ratio of PMSF to chymotrypsin under the above routine conditions was either 5 to I or 1 to 1, as noted in the Results. These would be the actual ratios for zero-time samples and for PMSFpreincubated samples where no hydrolysis or inactivation of PMSF had occurred. Stability studies of PMSF in 2-propanol were performed by taking aliquots of the stock solutions at monthly intervals into buffer followed immediately by addition of chymotrypsin as described above. RESULTS
Production of soluble azo-peptides by chymotryptic cleavage of azocoll was nearly linear for 2 hr. Blanks with no protease with or without PMSF or carrier solvent gave zero absorbancies at 520 nm. Therefore the method of assay was valid. Assays with pronase or trypsin gave similar results except that when PMSF was used it did not inhibit trypsin and especially pronase as efficiently as chymotrypsin. Stock solutions of either 10 or 100 mM PMSF in 2-propanol at 25°C were fully active for many months after their preparation. At a 1 to 1 molar ratio
576
GORDON
T. JAMES
30 60 PREINCUBATION
FIG. I. Inactivation of PMSF preincubated at 25°C addition of chymotrypsin. Buffers used were 10 mM in chloride: (0) sodium phosphate, pH 7.0; (A) Hepes, PMSF concentration: 100 PM. Fully active PMSF (I chymotrypsin.
90 TIME
120 (min)
in buffers as a function of time prior to buffering species and 150 mM in sodium pH 7.5: (W) Tris-HCI. pH 8.0. Initial .O) is defined by complete inhibition of
of inhibitor to enzyme, complete inhibition of chymotrypsin was found at all intervals tested: 1, 2, 3, 5, and 9 months after preparation of PMSF in 100% 2-propanol. Approximate lifetimes of 100 PM PMSF in various buffers at 25°C are shown in Fig. I, relative to inhibition of 20 PM chymotrypsin. At pH 7.0 the inhibitor is still in excess of 20 PM concentration after 2 hr of incubation in buffer, since the enzyme was fully inhibited. However, almost complete inactivation of PMSF occurred after 45-60 min of preincubation at pH 8.0. When PMSF was preincubated at 20 p.~ in buffers at 25°C and then checked with 20 PM chymotrypsin, it was shown that the inhibitor is inactivated at pH 7.0 (Fig. 2). This also occurred at pH 6.0 (data not shown) but was considerably slower than at pH 7.0. As seen in Fig. 2, half-lives of 20 PM PMSF are about 110, 55, and 35 min at pH 7.0, 7.5, and 8.0, respectively. When 100 PM PMSF was preincubated in buffers at 4°C rather than 25°C and then tested with chymotrypsin, complete loss of inhibitory activity occurred within 30, 22, and 10 hr at pH 7.6, 8.0, and 8.6, respectively (Fig. 3).
PREINCUBATION
TIME
(mid
FIG. 2. Same as Fig. 1 except that in the assay with chymotrypsin, the molar ratio of PMSF to enzyme was 1 to 1 rather than 5 to 1. using 20 pM inhibitor preincubated in the buffer (see Discussion).
PHENYLMETHYLSULFONYL
FLUORIDE
577
FIG. 3. Decrease in protease inhibition as in Fig. 1 except that PMSF was preincubated at 4°C. Buffers employed had the same compositions as for Fig. I. The pH values given are those measured at 4°C: (A) Hepes. pH 7.6; (0) Tris-HCI, pH 8.0: (m) Tris-HCI, pH 8.6.
Other results showed that 0.1 mM EDTA, 5 mM dithiothreitol, or I5 mM 2-mercaptoethanol in buffer (pH 7.5) did not interfere with inhibition of protease by PMSF. Neither ionic strength nor phosphate ion concentration had an effect on 20 PM PMSF, since the rate of inactivation of PMSF was the same in 10 and 150 mM sodium phosphate buffer (no sodium chloride or other salts). pH 7.0, and was identical to the pH 7.0 data in Fig. 2. Reactivation of PMS-chymotrypsin at 2X, pH 7.0, did not occur within 7 days. This was tested by comparing the proteolytic activity of PMSF-inhibited chymotrypsin to an identical sample prepared at the same time without PMSF. DISCUSSION The general protease assay based on azocoll was reliable and reproducible since duplicate samples generally differed by only several percentages in terms of absorbancies at 520 nm. Timed results of chymotryptic action on azocoll showed that under the described conditions, a single time point of 60 min would be valid for routine use in the comparison of paired samples with or without inclusion of PMSF. Stock solutions of PMSF in 2-propanol were fully stable at room temperature for at least 9 months after their preparation. In buffers. PMSF is often used at aconcentration of 100 PM for inhibition of proteases in various types of samples. To check the stability of aqueous 100 /.LM PMSF, an aliquot of 50 mg/ml of chymotrypsin was employed (see Materials and Methods) giving a final concentration of 20 pM enzyme.
Therefore the initial molar ratio of PMSF to chymotrypsin was 5 to 1. In order to do some experiments with a 1 to 1 molar ratio. PMSF was incubated in buffers at 20 PM. then assayed with 20 PM chymotrypsin. Other approaches to the I to 1 ratiogave results with too much scatter in the plotted results, e.g., aliquots of aqueous 100 pM PMSF did not provide reproducible enzyme inhibition, probably due to a degree of insolubility of the inhibitor in buffer or water.
578
GORDON
T. JAMES
In an aqueous environment free of inhibitable proteases PMSF becomes inactivated, presumably hydrolyzed by hydroxyl ion. Inactivation increases with the pH and is faster at 25 than at 4°C (Figs. 1 and 3). In the experiments with preincubation at 4”C, lag times or flat portions of the curves became more significant (Fig. 3) and were due to the routine use of excess PMSF relative to chymotrypsin. In experiments where the molar ratio of inhibitor to enzyme was 1 to 1, half-lives of 20 PM PMSF may be estimated for pH 7.0, 7.5, and 8.0 at 25°C (Fig. 2). Lifetimes of PMSF at different pH values (Figs. 1-3) should be taken into account where the inhibitor is included in buffers used for dialysis or for chromatographic elutions. For extraction of tissues or other operations where inhibition of proteolysis is desired, PMSF should ideally be added to the buffer system just before or during contact with the biological material. Since some biochemical systems benefit from the presence of metal chelators and/or reducing agents, the effect of EDTA, mercaptoethanol, or dithiothreitol on PMSF was checked although there was no a priori reason to expect that these chemicals would interfere with the action of PMSF on serine proteases. There was no such interference. Also ionic strength or phosphate ion concentration had no effect on PMSF in the range tested (see Results). Reactivation of PMSF-inhibited chymotrypsin did not occur for at least one week at pH 7.0 at 25°C. This result complements the data of Gold and Fahrney (13) who studied the desulfonylation of [14C]PMS-chymotrypsin. Between pH 4 and 8.5, the sulfonyl group was indefinitely stable (13). Significant desulfonylation did occur at lower or higher pH values (13) and at a wide range of pH values when urea was present (14). It was stated (14) without data that PMSF itself was stable for hours under the conditions employed (pH 7.0). Definitive data on PMSF stability under various conditions has not been presented in the literature. The present paper provides some of this information. ACKNOWLEDGMENTS This research was supported by NIH Grant Nos. NS 07701 and CA-5154.
REFERENCES I. Fahrney, D. E., and Gold. A. M. (1963) J. Amer. Chem. SW. 85, 997- 1000. Gold, A. M. (1965) Biochemistry 4, 897-901. 3. Prouty, W. F., and Goldberg, A. L. (1972) J. Biol. Chum. 247, 3341-3352. 4. Nooden, L. D., VanDenBroek. H.. and Sevall, J. S. (1973) FEBS Lerr. 29, 326-328. 5. Takeda, Y., Brewer, Jr., H. B., and Larner, J. (1975) J. Bid. Chum. 250, 8943-8950. 6. Ballal, N. R., Goldberg, D. A.. and Busch. H. (1975) Biochem. Biophys. Res. Commun. 2.
62, 972-982. 7.
Gaertner, F. H.. and Cole,
K. W. (1976) Arch.
Biochrm.
Biophys.
177, 566-573.
PHENYLMETHYLSULFONYL
FLUORIDE
579
8. James, G. T., Yeoman, L. C.. Matsui, S.. Goldberg. A.. and Busch, H. (1977) Biochemistry 16, 2384-2389. 9. Chong, M. T.. Garrard. W. T., and Bonner. J. (1974) Biochemisfry 13, 5128-5134. IO. Kumar. S. (1975) J. Bid. Chem. 250, 5150-5158. I I. James, G. T.. unpublished results. 12. Moore, G. L. (1969) And. Biochem. 32, 122-127. 13. Gold. A. M., and Fahrney, D. E. (1963) Biwhem. Biophys. Res. Commun. 10, 55-59. 14. Gold, A. M., and Fahrney, D. E. (1964) Biochemistry 3, 783-791.
BIOCHEMISTRY
Inactivation
86,
574-579 (1978)
of the Protease inhibitor Phenylmethylsulfonyl Fluoride in Buffers GORDON
Department
of Neurology.
University
T. JAMES
of Colorado
Medico1
Center,
Denver,
Colorado
80262
Received April 20, 1977; accepted December 12. 1977 Aqueous preparations ofphenylmethylsulfonyl fluoride (PMSF) become inactive toward proteases unless promptly brought into contact with protease. Inactivation of PMSF increases with increased pH and temperature. Half-lives ofthe inhibitor at 25°C are approximately I10,55, and 35 min at pH 7.0,7.5, and 8.0. respectively. At pH 8, 100 /.LM PMSF is almost completely inactivated within 1 hr at 25°C or within 22 hr at 4°C. Stock solutions of PMSF in 100% isopropanol are stable at 25°C for months if not longer. Reactivation of PMSF-inhibited chymotrypsin did not occur within 1 week at 25°C at pH 7.0.
Inhibition of proteases has become important not only for well-defined biochemical studies of pure proteases but also for prevention of proteolytic degradation of other proteins in biological preparations. The protease inhibitor phenylmethylsulfonyl fluoride (PMSF) is now widely used for these purposes. Fahrney and Gold (1) described the kinetics of inhibition of the reaction of PMSF and analogs with trypsin and chymotrypsin. It has been shown that the active site serine residue reacts with PMSF (2). A number of workers have used PMSF to prevent artifactual proteolytic modification of various proteins during purification or during other work (3-8). Chong et al. (9) found that a neutral chromatin protease was inhibited by PMSF. Besides proteases PMSF sometimes inhibits other types of enzymes, usually esterases, e.g., acetylcholinesterase (1). Kumar (IO) found that palmityl coenzyme A deacylase was selectively inhibited by PMSF within a pigeon liver fatty acid synthetase complex, and Gaertner and Cole (7) noted an inhibition of some of the biosynthetic enzymes in the aromatic complex of Neurospora, as well as an effective inhibition of a proteolytic contaminant. Human liver arylsulfatase A was partially inhibited by PMSF at or near the optimal pH for enzyme activity (11). The above reports show that PMSF is widely employed in protein chemistry and molecular biology. However little definitive data is available on the stability of this inhibitor under various conditions. This paper provides such data.
0003-?697178/0862-0574$02.00/O Copyright All rights
B 1978 by Academic Press, Inc. of reproduction in any form reserved.
574
PHENYLMETHYLSULFONYL MATERIALS
FLUORIDE
575
AND METHODS
Bovine trypsin, a-chymotrypsin, and phenylmethylsulfonyl fluoride (PMSF) were purchased from Sigma Co. Pronase, azocoll, and N-2hydroxyethylpiperazine-N’-2-ethanesulfonic acid (Hepes) were from Calbiochem. Stock solutions of 10 and 100 mM PMSF in 100% 2-propanol were made and stored at room temperature. Proteases were dissolved in cold 10e3 N HCl at 5 mg/lOO ~1 and kept in an ice bath. PMSF stability at 100 PM in buffers was studied by adding 1~1 of 100 mM PMSF stock solution to 1.0 ml of the buffer under study. Then 10 ~1 of protease solution (0.5 mg) was added immediately for zero-time samples or optimum inhibition samples. Otherwise 10 ~1 of protease was added at a timed interval per sample so that preincubation of PMSF in buffer could occur before coming in contact with protease. Each timed sample also consisted of a control tube, i.e., 1 ~1 of 2-propanol was added rather than the PMSF stock solution; protease was added when appropriate. Some experiments were also performed with 20 pM PMSF in buffer, with an appropriate aliquot of 10 mM PMSF stock solution (see Discussion). After addition of protease to samples with or without PMSF, incubation was carried out for 5 min to allow reaction with PMSF. Then 4.0 ml of the buffer under study was added and mixed, and 1.O ml of the resulting 5-ml solution was transferred to a tube containing 5 mg of solid azocoll. The optical density of solubilized peptides containing the azo dye was measured at 520 nm after centrifugation of remaining insoluble azocoll(12). Blank controls consisted of above components minus proteases. The calculated molar ratio of PMSF to chymotrypsin under the above routine conditions was either 5 to I or 1 to 1, as noted in the Results. These would be the actual ratios for zero-time samples and for PMSFpreincubated samples where no hydrolysis or inactivation of PMSF had occurred. Stability studies of PMSF in 2-propanol were performed by taking aliquots of the stock solutions at monthly intervals into buffer followed immediately by addition of chymotrypsin as described above. RESULTS
Production of soluble azo-peptides by chymotryptic cleavage of azocoll was nearly linear for 2 hr. Blanks with no protease with or without PMSF or carrier solvent gave zero absorbancies at 520 nm. Therefore the method of assay was valid. Assays with pronase or trypsin gave similar results except that when PMSF was used it did not inhibit trypsin and especially pronase as efficiently as chymotrypsin. Stock solutions of either 10 or 100 mM PMSF in 2-propanol at 25°C were fully active for many months after their preparation. At a 1 to 1 molar ratio
576
GORDON
T. JAMES
30 60 PREINCUBATION
FIG. I. Inactivation of PMSF preincubated at 25°C addition of chymotrypsin. Buffers used were 10 mM in chloride: (0) sodium phosphate, pH 7.0; (A) Hepes, PMSF concentration: 100 PM. Fully active PMSF (I chymotrypsin.
90 TIME
120 (min)
in buffers as a function of time prior to buffering species and 150 mM in sodium pH 7.5: (W) Tris-HCI. pH 8.0. Initial .O) is defined by complete inhibition of
of inhibitor to enzyme, complete inhibition of chymotrypsin was found at all intervals tested: 1, 2, 3, 5, and 9 months after preparation of PMSF in 100% 2-propanol. Approximate lifetimes of 100 PM PMSF in various buffers at 25°C are shown in Fig. I, relative to inhibition of 20 PM chymotrypsin. At pH 7.0 the inhibitor is still in excess of 20 PM concentration after 2 hr of incubation in buffer, since the enzyme was fully inhibited. However, almost complete inactivation of PMSF occurred after 45-60 min of preincubation at pH 8.0. When PMSF was preincubated at 20 p.~ in buffers at 25°C and then checked with 20 PM chymotrypsin, it was shown that the inhibitor is inactivated at pH 7.0 (Fig. 2). This also occurred at pH 6.0 (data not shown) but was considerably slower than at pH 7.0. As seen in Fig. 2, half-lives of 20 PM PMSF are about 110, 55, and 35 min at pH 7.0, 7.5, and 8.0, respectively. When 100 PM PMSF was preincubated in buffers at 4°C rather than 25°C and then tested with chymotrypsin, complete loss of inhibitory activity occurred within 30, 22, and 10 hr at pH 7.6, 8.0, and 8.6, respectively (Fig. 3).
PREINCUBATION
TIME
(mid
FIG. 2. Same as Fig. 1 except that in the assay with chymotrypsin, the molar ratio of PMSF to enzyme was 1 to 1 rather than 5 to 1. using 20 pM inhibitor preincubated in the buffer (see Discussion).
PHENYLMETHYLSULFONYL
FLUORIDE
577
FIG. 3. Decrease in protease inhibition as in Fig. 1 except that PMSF was preincubated at 4°C. Buffers employed had the same compositions as for Fig. I. The pH values given are those measured at 4°C: (A) Hepes. pH 7.6; (0) Tris-HCI, pH 8.0: (m) Tris-HCI, pH 8.6.
Other results showed that 0.1 mM EDTA, 5 mM dithiothreitol, or I5 mM 2-mercaptoethanol in buffer (pH 7.5) did not interfere with inhibition of protease by PMSF. Neither ionic strength nor phosphate ion concentration had an effect on 20 PM PMSF, since the rate of inactivation of PMSF was the same in 10 and 150 mM sodium phosphate buffer (no sodium chloride or other salts). pH 7.0, and was identical to the pH 7.0 data in Fig. 2. Reactivation of PMS-chymotrypsin at 2X, pH 7.0, did not occur within 7 days. This was tested by comparing the proteolytic activity of PMSF-inhibited chymotrypsin to an identical sample prepared at the same time without PMSF. DISCUSSION The general protease assay based on azocoll was reliable and reproducible since duplicate samples generally differed by only several percentages in terms of absorbancies at 520 nm. Timed results of chymotryptic action on azocoll showed that under the described conditions, a single time point of 60 min would be valid for routine use in the comparison of paired samples with or without inclusion of PMSF. Stock solutions of PMSF in 2-propanol were fully stable at room temperature for at least 9 months after their preparation. In buffers. PMSF is often used at aconcentration of 100 PM for inhibition of proteases in various types of samples. To check the stability of aqueous 100 /.LM PMSF, an aliquot of 50 mg/ml of chymotrypsin was employed (see Materials and Methods) giving a final concentration of 20 pM enzyme.
Therefore the initial molar ratio of PMSF to chymotrypsin was 5 to 1. In order to do some experiments with a 1 to 1 molar ratio. PMSF was incubated in buffers at 20 PM. then assayed with 20 PM chymotrypsin. Other approaches to the I to 1 ratiogave results with too much scatter in the plotted results, e.g., aliquots of aqueous 100 pM PMSF did not provide reproducible enzyme inhibition, probably due to a degree of insolubility of the inhibitor in buffer or water.
578
GORDON
T. JAMES
In an aqueous environment free of inhibitable proteases PMSF becomes inactivated, presumably hydrolyzed by hydroxyl ion. Inactivation increases with the pH and is faster at 25 than at 4°C (Figs. 1 and 3). In the experiments with preincubation at 4”C, lag times or flat portions of the curves became more significant (Fig. 3) and were due to the routine use of excess PMSF relative to chymotrypsin. In experiments where the molar ratio of inhibitor to enzyme was 1 to 1, half-lives of 20 PM PMSF may be estimated for pH 7.0, 7.5, and 8.0 at 25°C (Fig. 2). Lifetimes of PMSF at different pH values (Figs. 1-3) should be taken into account where the inhibitor is included in buffers used for dialysis or for chromatographic elutions. For extraction of tissues or other operations where inhibition of proteolysis is desired, PMSF should ideally be added to the buffer system just before or during contact with the biological material. Since some biochemical systems benefit from the presence of metal chelators and/or reducing agents, the effect of EDTA, mercaptoethanol, or dithiothreitol on PMSF was checked although there was no a priori reason to expect that these chemicals would interfere with the action of PMSF on serine proteases. There was no such interference. Also ionic strength or phosphate ion concentration had no effect on PMSF in the range tested (see Results). Reactivation of PMSF-inhibited chymotrypsin did not occur for at least one week at pH 7.0 at 25°C. This result complements the data of Gold and Fahrney (13) who studied the desulfonylation of [14C]PMS-chymotrypsin. Between pH 4 and 8.5, the sulfonyl group was indefinitely stable (13). Significant desulfonylation did occur at lower or higher pH values (13) and at a wide range of pH values when urea was present (14). It was stated (14) without data that PMSF itself was stable for hours under the conditions employed (pH 7.0). Definitive data on PMSF stability under various conditions has not been presented in the literature. The present paper provides some of this information. ACKNOWLEDGMENTS This research was supported by NIH Grant Nos. NS 07701 and CA-5154.
REFERENCES I. Fahrney, D. E., and Gold. A. M. (1963) J. Amer. Chem. SW. 85, 997- 1000. Gold, A. M. (1965) Biochemistry 4, 897-901. 3. Prouty, W. F., and Goldberg, A. L. (1972) J. Biol. Chum. 247, 3341-3352. 4. Nooden, L. D., VanDenBroek. H.. and Sevall, J. S. (1973) FEBS Lerr. 29, 326-328. 5. Takeda, Y., Brewer, Jr., H. B., and Larner, J. (1975) J. Bid. Chum. 250, 8943-8950. 6. Ballal, N. R., Goldberg, D. A.. and Busch. H. (1975) Biochem. Biophys. Res. Commun. 2.
62, 972-982. 7.
Gaertner, F. H.. and Cole,
K. W. (1976) Arch.
Biochrm.
Biophys.
177, 566-573.
PHENYLMETHYLSULFONYL
FLUORIDE
579
8. James, G. T., Yeoman, L. C.. Matsui, S.. Goldberg. A.. and Busch, H. (1977) Biochemistry 16, 2384-2389. 9. Chong, M. T.. Garrard. W. T., and Bonner. J. (1974) Biochemisfry 13, 5128-5134. IO. Kumar. S. (1975) J. Bid. Chem. 250, 5150-5158. I I. James, G. T.. unpublished results. 12. Moore, G. L. (1969) And. Biochem. 32, 122-127. 13. Gold. A. M., and Fahrney, D. E. (1963) Biwhem. Biophys. Res. Commun. 10, 55-59. 14. Gold, A. M., and Fahrney, D. E. (1964) Biochemistry 3, 783-791.