Viglio S, Luisetti M, Zanaboni G, Döring G, Worlitzsch D, Cetta G, Iadarola P
Dipartimento di Biochimica A. Castellani, Università di Pavia, Italy.
J Chromatogr A. 1999 Jun 18;846(1-2):125-34. doi: 10.1016/s0021-9673(98)01056-5.
Micellar electrokinetic chromatography (MEKC) is a new method for analysing proteolytic activities simultaneously present in incubation mixtures. Here we demonstrate that MEKC differentiates between the enzymatic activities of Pseudomonas aeruginosa elastase (PsE) and human leukocyte elastase (HLE) or cathepsin G (Cat G) in assays using the chromogenic peptide substrates Suc-Ala-Ala-Ala-NA or Suc-Ala-Ala-Pro-Phe-NA, respectively (where Suc = succinyl and NA = 4-nitroaniline/u-nitroanilide). When PsE and Cat G were incubated at equimolar ratio with Suc-Ala-Ala-Pro-Phe-NA, the PsE-specific cleavage products PheNA and Suc-Ala-Ala-Pro were detected whereas inhibition of the metalloproteinase PsE with EDTA resulted in detection of NA and Suc-Ala-Ala-Pro-Phe only. Similarly, when PsE and HLE were incubated at equimolar ratio with Suc-Ala-Ala-Ala-NA, the PsE-specific cleavage products Suc-Ala and Ala-Ala-NA were detected whereas at an PsE-HLE ratio 1:50, both the PsE-specific and the HLE-specific cleavage products NA and Suc-Ala-Ala-Ala were separated. MEKC also allowed determination of the kinetic constants for the interactions of PsE, Cat G and HLE with the substrates considered.
胶束电动色谱法（MEKC）是一种用于分析孵育混合物中同时存在的蛋白水解活性的新方法。在此，我们证明了在分别使用生色肽底物Suc-Ala-Ala-Ala-NA或Suc-Ala-Ala-Pro-Phe-NA（其中Suc =琥珀酰基，NA = 4-硝基苯胺/对硝基苯胺）的测定中，MEKC能够区分铜绿假单胞菌弹性蛋白酶（PsE）与人白细胞弹性蛋白酶（HLE）或组织蛋白酶G（Cat G）的酶活性。当PsE和Cat G与Suc-Ala-Ala-Pro-Phe-NA以等摩尔比孵育时，检测到了PsE特异性裂解产物PheNA和Suc-Ala-Ala-Pro，而用EDTA抑制金属蛋白酶PsE则仅检测到NA和Suc-Ala-Ala-Pro-Phe。同样，当PsE和HLE与Suc-Ala-Ala-Ala-NA以等摩尔比孵育时，检测到了PsE特异性裂解产物Suc-Ala和Ala-Ala-NA，而在PsE-HLE比例为1:50时，PsE特异性裂解产物和HLE特异性裂解产物NA和Suc-Ala-Ala-Ala都被分离出来。MEKC还能够测定PsE、Cat G和HLE与所考虑底物相互作用的动力学常数。
