Yonezawa H, Uchikoba T, Kaneda M
Department of Chemistry, Faculty of Science, Kagoshima University, Korimoto.
J Biochem. 1997 Aug;122(2):294-9. doi: 10.1093/oxfordjournals.jbchem.a021752.
N-Pepstatinyl-N'-dansyldiaminopropane (dansyl-pepstatin) was prepared by the coupling of pepstatin A and N-dansyl-diaminopropane. The dansyl-pepstatin obtained strongly inhibited pepsin activity by forming a 1:1 complex. The fluorescence of the dansyl group (excitation at 320 nm, and emission near 520 nm) increased with the formation of the complex. The increase in fluorescence of dansyl-pepstatin solution was proportional to the amount of added pepsin, chymosin and cathepsin D until dansyl-pepstatin was saturated by these enzymes and at higher protease concentrations the fluorescence did not increase further. Therefore, the net amounts of active pepstatin-sensitive carboxyl proteases could be determined by detecting the inflection point of increased fluorescence upon addition of the protease to a dansyl-pepstatin solution of known concentration. Moreover, the protease concentrations of many samples were obtained easily by measurements of increased fluorescence compared with that caused by authentic protease solution. The minimum detectable amount of pepsin was about 20 pmol. On the other hand, the fluorescence did not increase upon mixing with inactivated pepsin, chymotrypsin, or trypsin. The K(i) value of dansyl-pepstatin for pepsin was similar to that of pepstatin A. It was possible to determine the amount of chymosin contained in rennet by this method. The inactivation curve of pepsin in pH 6.5 buffer was also determined quickly and easily by the use of this method. This assay method for pepstatin-sensitive carboxyl proteases is very simple and easy, and it is possible to determine the net amounts of active pepstatin-sensitive carboxyl proteases even in crude mixtures.
N-胃蛋白酶抑制素基-N'-丹磺酰二氨基丙烷（丹磺酰-胃蛋白酶抑制素）是通过胃蛋白酶抑制素A与N-丹磺酰二氨基丙烷偶联制备而成。所得到的丹磺酰-胃蛋白酶抑制素通过形成1:1复合物强烈抑制胃蛋白酶活性。丹磺酰基团的荧光（激发波长为320 nm，发射波长在520 nm附近）随着复合物的形成而增强。丹磺酰-胃蛋白酶抑制素溶液荧光的增强与添加的胃蛋白酶、凝乳酶和组织蛋白酶D的量成正比，直到丹磺酰-胃蛋白酶抑制素被这些酶饱和，在更高的蛋白酶浓度下荧光不再进一步增强。因此，通过检测向已知浓度的丹磺酰-胃蛋白酶抑制素溶液中添加蛋白酶后荧光增强的拐点，可以确定活性胃蛋白酶敏感羧基蛋白酶的净含量。此外，与由纯蛋白酶溶液引起的荧光增强相比，通过测量荧光增强可以轻松获得许多样品的蛋白酶浓度。胃蛋白酶的最低可检测量约为20 pmol。另一方面，与失活的胃蛋白酶、胰凝乳蛋白酶或胰蛋白酶混合时荧光不增强。丹磺酰-胃蛋白酶抑制素对胃蛋白酶的K(i)值与胃蛋白酶抑制素A的相似。通过这种方法可以测定凝乳酶中凝乳酶的含量。使用这种方法还可以快速简便地测定胃蛋白酶在pH 6.5缓冲液中的失活曲线。这种针对胃蛋白酶敏感羧基蛋白酶的检测方法非常简单易行，甚至在粗混合物中也可以确定活性胃蛋白酶敏感羧基蛋白酶的净含量。
