McKenzie H A, White F H
Anal Biochem. 1986 Sep;157(2):367-74. doi: 10.1016/0003-2697(86)90639-1.
A method is described for the determination of lysozyme (muramidase) activity, whereby sensitivity is maximized by incubation of the reaction mixture (sample, buffer, and substrate (Micrococcus luteus] over an extended period. This approach is made feasible by exploiting our observation that the lytic reaction follows simple kinetic order during this time (e.g., 700 min for bovine lysozyme and 960 min for the eggwhite enzyme at low concentrations). After this period, the reaction rates diminish, indicating biphasic behavior, and eventually become negligible. The kinetic order may vary with both the type of lysozyme and the buffer system used. The limit of detection for bovine milk lysozyme is 100 pg/ml reaction mixture, equivalent to 6 ng/ml milk, for a 50-microliters sample (with reference to hen eggwhite lysozyme). With these limits, the method has proven valuable in our comparative studies, particularly for low levels of activity in bovine milk, but also in secretions and tissue extracts from various other eutherian, metatherian, and prototherian mammals. The method may also be applied to investigation of structure and function in modified forms of the enzyme.
本文描述了一种测定溶菌酶（胞壁质酶）活性的方法，通过延长反应混合物（样品、缓冲液和底物[藤黄微球菌]）的孵育时间，使灵敏度最大化。我们观察到在此期间裂解反应遵循简单的动力学顺序（例如，低浓度下牛溶菌酶为700分钟，蛋清酶为960分钟），这使得该方法可行。在此时间段之后，反应速率降低，表明存在双相行为，最终变得可以忽略不计。动力学顺序可能因溶菌酶类型和所用缓冲系统而异。对于50微升样品（以鸡蛋白溶菌酶为参照），牛乳溶菌酶的检测限为100皮克/毫升反应混合物，相当于6纳克/毫升牛奶。基于这些检测限，该方法在我们的比较研究中已证明具有价值，特别是对于牛乳中低水平的活性，而且对于各种其他真兽类、后兽类和原兽类哺乳动物的分泌物和组织提取物也有价值。该方法还可应用于对该酶修饰形式的结构和功能的研究。
