Determination of lysozyme activity at low levels with emphasis on the milk enzyme

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.