Effect of self-degradation products on crystallization of protease thermolysin

Abstract

The effect of self-degradation products of protease thermolysin on the crystallization of thermolysin was investigated. Crystallizations were carried out at the concentration of the self-degradation products of 0 to 0.622 mg/ml, 5°C and pH 7.0. The initial concentration of thermolysin was constant (1.70 ± 0.01 mg/ml). Crystallizations were monitored by dynamic light scattering and photomicroscopy. The crystallization of thermolysin in the presence of the self-degradation products proceeded through two successive steps: the formation of primary particles and the formation of large crystals by the aggregation of the primary particles. Low concentration of the self-degradation products (0.212 mg/ml) accelerated the formation of the primary particles and also the formation of the large crystals. High concentration of the self-degradation products, however, inhibited the formation of the primary particles and their aggregation to the large crystals. As the result, a large number of small aggregates which had not grown to the large crystals were observed by photomicroscopy. An analysis of the crystals and the primary particles formed in the presence of the self-degradation products by gel filtration high performance liquid chromatography revealed that the self-degradation products are not incorporated in the primary particles, but are incorporated probably in the openings between the primary particles during the crystallization.