SPECIFICITY OF ENZYMATIC PROTEIN CLEAVAGE IN GLYCEROL VS WATER

最近，我们发现枯草杆菌蛋白酶嘉士伯可以 裂解蛋白(BSA和核糖核酸酶A)作为模型 基质)在整齐的甘油中。我们现在正在解决的问题是 质谱学方法有：(I)是蛋白质水解物的位置 在甘油中的裂解作用(即酶的专一性)与在水中的相同？ (Ii)是不含甘油的多肽中蛋白质分解的产物(与 未封闭的羧基末端)或相应的甘油酯(即， 水或甘油在酶中起亲核剂的作用吗 乳沟？)为了解决这些问题，含有牛血清白蛋白和 用MS检测水和甘油中的枯草杆菌菌素 并且在各自的溶剂中孵化几个小时后， 根据FPLC分析，已经发生了显著的酶切割。 比较了在水和甘油中形成的蛋白质纤维。 回答问题(I)及(Ii)。将确定是否(I) 一种简单的方法就可以改变酶蛋白切割的特异性 改变反应介质，以及(Ii)新材料(蛋白质 甘油三酯)可以用我们的方法制备。要测试 概括我们的发现，研究将扩展到其他蛋白酶 (热裂解酶、胰凝乳酶)。

Recently, we discovered that the protease subtilisin Carlsberg can cleave proteins (BSA and ribonuclease A have served as model substrates) in neat glycerol. Questions that we are addressing now by means of mass spectrometry are: (i) Are the positions of proteolytic cleavage (i.e., enzyme specificity) in glycerol the same as in water? (ii) Are the products of proteolysis in glycerol free peptides (with unblocked carboxyl termini) or the corresponding glycerides (i.e., does water or glycerol act as a nucleophile in the enzymatic cleavage?). To address these questions, samples containing BSA and subtilisin, both in water and in glycerol, are examined by MS at to and after several hours of incubation in the respective solvents when, according FPLC analysis, significant enzymatic cleavage has occurred. Protein fi7agments formed in water and those in glycerol are compared to answer questions (i) and (ii). It wiH be ascertained whether (i) onecan alter the specificity of enzymatic protein cleavage simply by altering the reaction medium, and(ii) new materials (protein glycerides) can be prepared using our approach. To test the generality of our findings, studies will expand to other proteases (thermolysin, chymotrypsin).