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Carboxypeptidase Digestion in the Presence of Detergents

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Carboxypeptidase Digestion in the Presence of Detergents COMMUNICATION J. Biochein. 102, 243-246 (1987) Carboxypeptidase Digestion in the Presence of Detergents Masaharu KAMO and Akira TSUGITA Department of Chemistry, Faculty of Science , Science University of Tokyo, Noda, Chiba 278 Received for publication, April 21, 1987 The detergents, sodium dodecyl sulfate (SDS), lauryl glutamate (LG), and octyl- (polydisperse)oligooxyethylene (Octyl-POE), were tested as to their effects on the activities of carboxypeptidases A, B, and P. In general, Octyl-POE showed little inhibition and SDS showed the strongest inhibition. Carboxypeptidase B was only slightly inhibited by SDS. The inhibitory effect of SDS on these enzyme activities depended not on its concentration but on its absolute amount. For a constant amount of SDS, the activities of carboxypeptidases A and B remained almost constant with increasing reaction volume. Commercial carboxypeptidase B is usually contaminated by carboxypeptidase A. With the addition of SDS, almost only carboxypeptidase B activity was detected. Carboxy (C)-terminal sequence analysis is still one SDS is one of the most widely used deter of the most important tasks in sequencing studies gents. It has a strong anionic dodecyl group and on proteins. Besides C-terminal stepwise chem a marked solubilizing effect on proteins. LG ical degradation (1), classical carboxypeptidase (Ajinomoto Co., Japan) is a newly developed mild digestion has also been widely used. However, anionic detergent. Octyl-POE is a mild but non many proteins and peptides are known to be ionic detergent, and was donated by Y. Rosenbush, insoluble under the usual enzymic digestion condi Biozentrum, Switzerland. Figure 1 shows the car tions and consequently are indigestible. One ap boxypeptidase activities with various concentra proach to overcome this difficulty could be the tions of detergents with a constant volume (100 addition of detergents for the carboxypeptidase ƒÊ1), and with a constant enzyme concentration (5 digestion (2, 3). In the present study, sodium ƒÊ g). Activity is expressed as relative activity i.e., dodecyl sulfate (SDS), lauryl glutamate (LG), and as the percentage of that in the absence of a octyl-(polydisperse)oligooxyethylene (Octyl-POE) detergent. were tested as to their practical use for carboxypep Octyl-POE was generally observed to have a tidases digestion. Small synthetic substrates were very mild effect on all the peptidases tested, LG used in order to prevent structural changes due to was next with a slightly higher inhibitory effect the addition of detergents. and SDS showed the most extensive inhibition. LG cannot dissociate and is insoluble at pH 2.5, therefore, carboxypeptidase P activity was not Abbreviations: SDS, sodium dodecyl sulfate; Octyl- tested with LG. Carboxypeptidase P has an opti POE, octyl-(polydisperse)oligooxyethylene; LG, lauryl mal pH of 5.5, but it is often used at pH 2.5 glutamate. Vol. 102, No. 2, 1987 243 244 COMMUNICATION most inhibitory effect, a more detailed study was carried out on its effect on adequate concentrations of the individual enzymes, as shown in Fig. 1. Figure 2 summarizes the effects of SDS on the enzyme activities with various enzyme concen trations with a constant volume (100ƒÊl), and with a constant detergent concentration, which differed with the individual enzyme. Except for in the case of carboxypeptidase B, an increase in enzyme concentration resulted in an increase in enzyme activity. This observation sug gests that the SDS concentration is not a crucial factor for the inhibition but rather that the rela tive amount of each of these enzymes compared to that of SDS is a more important factor for the inhibitory effect. In Fig. 2b, both the SDS concentration and Fig. 1. Inhibition of carboxypeptidase activities by the amount of enzyme were maintained at con three detergents. (a) Carboxypeptidase A (Sigma Bio stant levels but the volume was varied; i.e., the chem., U.S.A.) digestion: Leu-Trp-Met (3nmol; Serva amount of SDS increased with increasing reaction Fein Chem., B.R.D.) was dissolved in 100ƒÊl of 0.1M volume, and the relative SDS: enzyme ratio in pyridine-acetate-collidine buffer, pH8.2, containing creased with increasing volume. The data clearly various amounts of the detergents. Solid circles denote showed that as the relative ratio of SDS increased activities in the presence of SDS; open circles, those in the enzyme activity became more inhibited, con the presence of lauryl glutamate; and triangles, those firming the idea based on the data shown in Fig. in the presence of Octyl-POE. The reaction was ini tiated by the addition of 5ƒÊg/5ƒÊl of carboxypeptidase 2a. A, followed by incubation for 3 h at 37°C. To stop When the relative SDS: enzyme ratio was kept the enzymatic reaction, a drop of formic acid was added constant, the enzyme activity remained constant to the reaction mixture. The mixture was dried up regardless of variation in the reaction volume; and the residue dissolved in 0.01M HCl, and then the when the volume was increased, the SDS concen amino acids liberated from the C-terminus were directly tration decreased because a constant amount of analyzed with an amino acid analyzer (Irika A5500). SDS was present in the reaction mixture. Except (b) Carboxypeptidase B (Sigma Biochem.) digestion: for carboxypeptidase P at pH 2.5, the above pre Leu-Trp-Met-Arg (Serva) was used as the substrate in diction was experimentally proved, as shown in 0.1M pyridine-acetate-collidine buffer, pH8.2, and the Fig. 2c. It is known that carboxypeptidase P is liberated Arg was determined with the amino acid inactive at pHs lower than 2.0. Carboxypeptidase analyzer. (c) and (d) Carboxypeptidase P (Takara, P activity at pH2.5 was peculiarly observed to Japan) digestion: Leu-Trp-Met was used as the sub strate in both 0.1M pyridine-acetate buffer, pH5.5 increase with decreasing SDS concentration. This (c), and 0.1M pyridine-formate buffer, pH2.5 (d), may be due to the enzyme being unstable at lower respectively. pHs. A typically insoluble protein, cucumber green mottle mosaic virus coat protein, was subjected to because peptide bonds containing acidic amino carboxypeptidase digestion. Its C-terminal se acids are only digestible at this lower pH. Also, quence is Ser-Lys-Ala (4). One microgram of the carboxypeptidase P is free from endoprotease ac protein was digested with 5ƒÊg carboxypeptidase tivity occasionally found at pH 5.5-6.5. It is A in 100ƒÊl of 0.1M pyridine-acetate-collidine noticeable that even in the presence of SDS, car buffer, pH8.2, for 3 h at 37°C, with and without boxypeptidase B is almost fully active. 0.2% SDS. Without SDS, the liberation of Ala Since SDS is one of the most useful deter- amounted to 18% but with SDS, 82% Ala was gents known, but at the same time exhibits the liberated. J. Biochem. CARBOXYPEPTIDASE DIGESTION IN DETERGENTS 245 Fig. 2. Effects of SDS on carboxypeptidase activities. The experimental conditions were the same as given in Fig. 1 except for the following: (a) The enzyme concentration was changed from 0 to 25ƒÊg/100ƒÊl; (1) carboxypep tidase A, (2) carboxypeptidase B, (3) carboxypeptidase P (pH 5.5), and (4) carboxypeptidase P (pH 2.5). The concentration of SDS was kept constant for each of the enzymes throughout each set of experiments; 1% for car boxypeptidases A and B, 0.1% for carboxypeptidase P (pH5.5) and 0.005% for carboxypeptidase P (pH2.5). The final volume of the reaction mixture was 100ƒÊl. (b) The volume of the reaction mixture was changed from 25 to 500ƒÊl. The concentration of SDS was kept constant for each set of experiments: 1% for carboxypep tidases A and B, 0.1° o for carboxypeptidase P (pH5.5) and 0.005% for carboxypeptidase P (pH2.5). The amount of enzyme was kept constant for each set of experiments; (1) 50ƒÊg carboxypeptidase A, (2) 5ƒÊg carboxypeptidase B, (3) 10ƒÊg carboxypeptidase P (pH5.5), and (4) 25ƒÊg carboxypeptidase P (pH2.5), respectively. (c) The volume of the reaction mixture was changed from 25 to 500ƒÊl. The amount of SDS was constant (but the concentration was varied) for each set of experiments; 1mg SDS for carboxypeptidases A and B, 0.1mg SDS for carboxypeptidase P (pH5.5), and 5ƒÊg SDS for carboxypeptidase P (pH2.5). The amount of enzyme was kept constant for each set of experiments; (1) 50ƒÊg carboxypeptidase A, (2) 5 pg carboxypeptidase B, (3) 10ƒÊg carboxypeptidase P (pH 5.5), and (4) 25ƒÊg carboxypeptidase P (pH2.5). As shown in Fig. 1, SDS inhibits carboxypep the liberation of Met dramatically decreased but tidase A but hardly affects carboxypeptidase B the liberation of Arg was maintained. Arg libera activity. In practice, commercial preparations of tion was not even inhibited with the addition of carboxypeptidase B are almost always contami up to 1% SDS. Thermolysin, which is known to nated by carboxypeptidase A. Thus it appeared have the sequence, Phe-Asp-Ala-Val-Gly-Val-Lys, we could use this SDS inhibition to obtain pure at its C-terminus (5), was digested with carboxypep carboxypeptidase B activity. As shown in Fig. tidase B. Denatured thermolysin was prepared 3, the substrate, Leu-Trp-Met-Arg, was used to by boiling at 100°C for 10min in the presence of test this proposition. In the absence of SDS, the 100mM EDTA, followed by dialysis. Digestion carboxypeptidase B preparation liberated not only with carboxypeptidase B (5ƒÊg, pH8.2, 100ƒÊl) the theoretical amount of Arg but also 75% of without SDS at 37°C for 8 h liberated many amino Met. When SDS (more than 0.1%) was added, acids including all those along the C-terminal se- Vol. 102, No. 2, 1987 246 COMMUNICATION t o be used.
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