凝乳酶活性的灵敏荧光测定法。
Sensitive fluorometric assay for the activity of chymosin.
作者信息
Yonezawa H, Uchikoba T, Kaneda M, Izumiya N
机构信息
Department of Chemistry, Faculty of Science, Kagoshima University, Japan.
出版信息
Int J Pept Protein Res. 1996 Jan-Feb;47(1-2):56-61. doi: 10.1111/j.1399-3011.1996.tb00810.x.
Fluorogenic substrates for chymosin [Dns-Leu-Ser-Phe-Trp-Ala-Leu-OCH2Py (I), Dns-Leu-Ser-Phe-Met-Trp-Leu-OCH2Py (II), Dns-Leu-Ser-Leu-Trp-Ala-Leu-OCH2Py (III), Dns-Leu-Ala-Phe-Trp-Ala-Leu-OCH2Py (IV), Dns-Leu-Ser-Phe-Leu-Ala-Leu-OCH2Py (V) and Dns-Leu-Ser-Phe-Phe-Ala-Leu-OCH2Py (VI)] were synthesized by a solution method. The obtained substrates I-VI were cleaved specifically (between the Phe and Trp residues for substrates I and IV, the Phe and Met residues for substrate II, the Leu and Trp residues for substrate III, the Phe and Leu residues for substrate V, and the Phe and Phe residues for substrate VI) by chymosin. The fluorescence of substrates I-IV (345 nm) increased with their hydrolysis, and hydrolysis rates were obtained by measuring the increase in fluorescence. The minimum detectable chymosin concentrations for substrates I and IV were about 1 nM; those for substrates II and III were about 4 and 2 nM. This assay method is very sensitive, and it is possible to determine the chymosin activity rapidly and easily. Substrates I and IV-VI were hydrolyzed by chymosin two times faster than substrates II and III. The effect of the amino-acid residues of the substrates on the hydrolysis rate is discussed.
通过溶液法合成了凝乳酶的荧光底物[丹磺酰-L-亮氨酸-L-丝氨酸-L-苯丙氨酸-L-色氨酸-L-丙氨酸-L-亮氨酸-邻甲氧基苄醇（I）、丹磺酰-L-亮氨酸-L-丝氨酸-L-苯丙氨酸-L-蛋氨酸-L-色氨酸-L-亮氨酸-邻甲氧基苄醇（II）、丹磺酰-L-亮氨酸-L-丝氨酸-L-亮氨酸-L-色氨酸-L-丙氨酸-L-亮氨酸-邻甲氧基苄醇（III）、丹磺酰-L-亮氨酸-L-丙氨酸-L-苯丙氨酸-L-色氨酸-L-丙氨酸-L-亮氨酸-邻甲氧基苄醇（IV）、丹磺酰-L-亮氨酸-L-丝氨酸-L-苯丙氨酸-L-亮氨酸-L-丙氨酸-L-亮氨酸-邻甲氧基苄醇（V）和丹磺酰-L-亮氨酸-L-丝氨酸-L-苯丙氨酸-L-苯丙氨酸-L-丙氨酸-L-亮氨酸-邻甲氧基苄醇（VI）]。所得到的底物I-VI被凝乳酶特异性切割（底物I和IV在苯丙氨酸和色氨酸残基之间、底物II在苯丙氨酸和蛋氨酸残基之间、底物III在亮氨酸和色氨酸残基之间、底物V在苯丙氨酸和亮氨酸残基之间、底物VI在苯丙氨酸和苯丙氨酸残基之间）。底物I-IV（345nm）的荧光随着它们的水解而增加，并且通过测量荧光的增加来获得水解速率。底物I和IV的最低可检测凝乳酶浓度约为1 nM；底物II和III的约为4 nM和2 nM。该测定方法非常灵敏，并且能够快速、容易地测定凝乳酶活性。底物I和IV-VI被凝乳酶水解的速度比底物II和III快两倍。讨论了底物氨基酸残基对水解速率的影响。
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