Studies on extracellular proteins from Staphylococcus aureus. III. Investigations on the heterogeneity of hyaluronate lyase using the method of isoelectric focusing

Abstract

Staphylococcus aureus, strain M18, was grown in liquid media. The proteins in the culture supernatants have been submitted to isoelectric focusing and separation in pH gradients. Two main components of hyaluronate lyase have been separated, with isoelectric points, p I, at 7.4 and 7.9, respectively. The reproducibility in the p I determinations was very good. The relative amount of activity in the peaks, however, varied in different culture batches. The component with p I 7.4 could be converted to get a p I of 7.9. The explanation for this is still obscure. The two components had the same elution volume on Sephadex gel filtration. If samples from the culture supernatant of a batch culture were taken after 6 and 14 h of cultivation, and then examined by isoelectric focusing, the main component was isoelectric at 7.4 in the first sample and at 7.9 in the second sample. The results indicate that the hyaluronate lyase molecule is susceptible to some modification. Fractions containing enzyme, and also sucrose and carrier ampholytes could be stored for several months at −20° with only small losses of activity. A protective effect of sucrose and/or ampholytes on the enzyme has been observed. Some other properties of the enzyme are also described, e.g. the activity of the enzyme when treated with cysteine, iodoacetic acid, N-ethylmaleimide and EDTA. In the assay system the enzyme was optimally active in acetate buffer with a broad maximum in the pH range 4.8 to 6.0. No difference between the components having different isoelectric points was observed.