pH dependence of the inhibition of chymotrypsin by a peptidyl trifluoromethyl ketone.
pH dependence of the inhibition of chymotrypsin by a peptidyl trifluoromethyl ketone.
复制标题
肽基三氟甲基酮抑制胰凝乳蛋白酶的 pH 依赖性。
DOI：
10.1021/bi00449a017
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Abeles,RH
中科院分区：
文献类型：
--
作者：
Brady,K;Liang,TC;Abeles,RH
材料和方法材料。[3H]乙酸酐（5 mCi，8.3 mCi/mmol）获自新英格兰核能公司。Sephadexes G-15和G-25购自Bio-Rad。N-BOC-L-亮氨酸、BTEE和N-琥珀酰基-L-丙氨酰基-L-丙氨酰基-L-脯氨酰基-L-苯丙氨酸对硝基苯胺（SAAPFpNA）来自Sigma Chemical Co.。所有其它试剂如前所述（Liang & Abeles，1987）。所有缓冲盐均购自Fisher Scientific。使用的缓冲液为50 mM甲酸钠（pH 4.0）、50 mM乙酸钠（pH 4.5-5.5）、50 mM磷酸钾（pH 6.0-7.5）、50 mM Tris（pH 7.5-9.0）和50 mM甘氨酸（pH 9.0-10.0）。通过添加KCl将所有缓冲液的离子强度调节至0.2M。N-[% H]乙酰基-L-亮氨酰基-L-苯丙氨酰基三氟甲烷的合成。N-（7V-BOC-L-亮氨酰基）-3-氨基-4-苯基-1.1。如先前所述制备1-三氟丁-2-醇（4种立体异构体的混合物）（Liang和Abeles，1987），不同之处在于使用N-BOC-L-亮氨酸代替N-乙酰基-L-亮氨酸。通过在二氯甲烷中用三氟乙酸在室温下处理肽2小时来完成BOC基团的去除。过量的三氟乙酸通过重复蒸发从碳酸氢钠中除去。然后将残余物在真空下干燥12小时。将1-三氟丁-2-醇（267 mg，0.6 mmol;四种异构体的混合物）和0.11 mL（1.0 mmol）N-甲基吗啉溶解于5 mL四氢呋喃中。然后将该溶液置于含有[3 H]乙酸酐（5 mCi，8.3mCi/mmol）的密封管的上部。在丙酮-干冰浴中冷却试管的同时，用玻璃棒敲击鹅颈密封。在破裂时，溶液被吸入管的下部。使该溶液在-78 ℃下反应30分钟，偶尔振摇.然后使其升温至室温并静置1 h。加入乙酸酐（0.01mL，0.1mmol），反应再进行2小时。然后将溶液转移到烧杯中，并在氮气流下除去溶剂。将残余物溶于5 mL二氧六环中，并如所述用碱性高锰酸钾氧化（Liang & Abeles，1987）。首先用乙酸乙酯和氯仿（1：1，Rf= 0.2），然后用氯仿和2-丙醇（9：1，Rf= 0.6）在制备型硅胶板上纯化放射性抑制剂。从乙醚/石油醚中重结晶后，得到55 mg（0.15 mmol）比活度为8.1 mCi/mmol的标记抑制剂。放射性与标准品化合物在三种不同的TLC系统（乙酸乙酯/氯仿，1：1，Rf= 0.2;氯仿/2-丙醇，9：1，Rf= 0.6;纯乙酸乙酯，Rf= 0.6）中合并。
Materials and Methods Materials.[3H] Acetic anhydride (5 mCi, 8.3 mCi/mmol) was obtained from New England Nuclear. Sephadexes G-15 and G-25 were purchased from Bio-Rad. iV-BOC-L-leucine, BTEE, and jV-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide (SAAPFpNA) were from Sigma Chemical Co. All other reagents were as previously described (Liang & Abeles, 1987). All buffer salts were purchased from Fisher Scientific. Buffers used were 50 mM sodium formate (pH 4.0), 50 mM sodium acetate (pH 4.5-5.5), 50 mM potassium phosphate (pH 6.0-7.5), 50 mM Tris (pH 7.5-9.0), and 50 mM glycine (pH 9.0-10.0). The ionic strength of all buffers was adjusted to 0.2 M by addition of KC1. Synthesis of N-[% H] Acetyl-L-Leucyl-L-Phenylalanyltrifluoromethane. iV-(7V-BOC-L-leucyl)-3-amino-4-phenyl-1.1. 1-trifluorobutan-2-ol (a mixture of 4 stereoisomers) was prepared as previously described (Liang& Abeles, 1987), except iV-BOC-L-leucine was used instead of TV-acetyl-L-leucine. Removal of the BOC group was accomplished by treating the peptide with trifluoroacetic acid in methylene chloride at room temperature for 2 h. Excess trifluoroacetic acid was removed by repetitive evaporationfrom carbontet-rachloride. The residue was then dried under vacuum for 12 h.The deprotected peptide/VL-leucyl-3-amino-4-phenyl-1.1. 1-trifluorobutan-2-ol (267 mg, 0.6 mmol; a mixture of four isomers) and 0.11 mL (1.0 mmol) of iV-methylmorpholine were dissolved in 5 mL of tetrahydrofuran. This solution was then put in the upper portion of a sealed tube containing [3H] acetic anhydride (5 mCi, 8.3 mCi/mmol). While the tube was cooled in an acetone-dry ice bath, the goose-neck seal was broken with strokes of a glass rod. The solution was sucked into the lower portion of the tube upon breakage. The solution was allowed to react at-78 C for 30 min with occasional shaking. It was then allowed to warm up to room temperature and stand for 1 h. Acetic anhydride (0.01 mL, 0.1 mmol) was added, and the reaction progressed for another 2 h. The solution was then transferred to a beaker and the solvent removed under a stream of nitrogen. The residue was taken up in 5 mL of dioxane and oxidized with alkaline potassium permaganate as described (Liang & Abeles, 1987). The radioactive inhibitor was purified on preparative silica plates first with ethyl acetate and chloroform (1: 1, Rf= 0.2) and then with chloroform and 2-propanol (9: 1, Rf= 0.6). This afforded 55 mg (0.15 mmol) of labeled inhibitor with a specific activity of 8.1 mCi/mmol after recrystallization from ether/petroleum ether. The radioactivity comigrated with the standard compound in three differentTLC systems (ethyl acetate/chloroform, 1: 1, Rf= 0.2; chloroform/2-propanol, 9: 1, Rf= 0.6; neat ethyl acetate, Rf=