A. N. SMOLELIS" AND S. E. HARTSELL Laboratories of Bacteriology, Department of Biological Sciences, Purdue Univer8ity, Lafayette, Indiana
Received for publication October 28, 1951
Since the initial discovery of lysozyme by Fleming (1922), nuimerous attempts have been made to describe the properties of this enzyme. The absence of a reliable method for the determination of enzymatic activity, however, has contributed to the incompleteness and to the inconsistency of the facts found in the literature. The availability of crystalline, egg white lysozyme has made it possible to study quantitatively some of the factors which affect its lytic activity. The assay method reported by Smolelis and Hartsell (1949) has been found to be accurate and highly adaptable to the study of this enzyme. This report concenms its application to the effect of pH, salts, ions, temperature, and manner of preparation of the cell suspension, on the activity of lysozyme. MATERIALS AND METHODS
Preparation of the cell suspension. To expedite the testing of lytic activity by lysozyme'and to reduce the possibility of daily variations associated'with the use of live cells of Micrococcus lysodeikticus, as noted by Meyer and Hahnel (1946), an effort was made to use killed cells. Lysozyme is capable of 'causing lysis of both live and dead cells of this species. Boasson (1938) used phenolized cells; 4 therefore,tis method was tried first. This technique provided an extremely variable cell preparation especially with pure lysozyme and the spectrophotometric method of assay. After storage of these cells at 4 C reproducible results in replicate tests were not obtained. Desiccation of the phenol-killed cells with'acetone did not provide a satisfactory cell preparation. Such cells gave variable results and were difficult to resuspend properly. It was assumed'that' acetone influenced the'sensitivity of M. lysodeikticus by reason of its fat solvent action on 'certain lipoidal layers near the cell wall, thus'changing the physiochemical factors essential to lysozyme action or to cell solubility. Lyophilization of a distilled' water suspension of untreated cells provided a satisfactory preparation which 'could be easily rehydrated, easily stored at 4 C, and would give reproducible results, even though autolytic enzymes might still be active when such cells were employed. M. lysodeikticu4s, strain ATCC 4698, was used in these studies. Table 1 presents the effect on the sensitivity to lysis obtained when other agents were used to kill M. lysodeikticus. "These data constitute, in part, a thesis submitted to the Graduate School of Purdue University in partial fulfillmenl of the requirements for the Doctor of Philosophy degree. Grateful appreciation is expressed to the Purdue Research Foundation for a research grant while completing these studies. 'Present address: Armour and Company, Chicago, Illinois. 665
A. N. SMOLELIS AND S. E. HARTSELL
An examination of the data shows that treatment of cells by any method prior to exposure to lysozyme invariably caused a decreased lytic response. This is contrary to the findings of Feiner et al. (1946). These authors reported that little or no change in cell sensitivity to lysis was observed after the organism was subjected to repeated washings with saline, lyophilization, precipitation with ice cold acetone, one per cent phenol, or exposure to ultraviolet light. Our data are in agreement with Epstein and Chain (1940) who state that, "Even M. lysodeikticus will not be lysed, or incompletely so, if its proteins are denatured by heat, organic solvents (alcohol, acetone, etc.) or iodine." TABLE 1 A comparison of the bacteriolytic responses of killed cells* AVERAGE PER CENT TRANSMISSION
Live cells Live QF
1:200,000 1:400,000 1:800,000 1:1,600,000 1:3,200,000...