The effect of NaCl and CaC12 on the aggregation and denaturation temperature of ovalbumin was examined in the pH interval 3-11. It was found that ovalbumin had maximal thermal stability between pH values of 6-10. NaCl did not affect the denaturation temperature, while a small decrease was observed with CaClz even at the low concentration range investigated. Both salts extended the pH interval of thermal aggregation, but at the alkaline side of the isoelectric point CaClz suppressed the aggregation temperature more than did NaCI. The aggregation properties of S-ovalbumin were also investigated and found to be similar to those of ovalbumin, although aggregation occurred at a higher temperature level. The quality of the aggregates was described by their dry matter content. Aggregates appeared as transparent gels, opaque gels, gel-like precipitates and precipitates. The addition of NaCl gave predominantly gel aggregates at the alkaline side of the isoelectric point, while precipitates dominated when CaC12 was added. Gelling is an intermediate phenomenon which occurs between the two extremes of precipitating and nonaggregating conditions. Special conditions, in terms of electrostatic repulsion between the protein molecules, are required for gelling, which could be achieved by manipulating with pH, type of salt, salt concentration or added detergent. The difference between aggregation and denaturation temperature correlated with the dry matter content of the aggregates formed. When aggregation temperature was well below denaturation temperature, the resulting aggregates had a high dry matter content (precipitates). Gelling, on the other hand, was observed at an aggregation temperature equal to or above the denaturation temperature.
The thermal precipitation and denaturation of conalbumin was studied separately at different pH values, low concentrations of an anionic detergent and salt concentrations relevant to food products. Sodium dodecylsulphate (SDS) was used as a model detergent and was found to lower the limit for thermal precipitation from pH 10-1 1 to pH 6-8 leaving a neutral pH region open for heat processing without any decrease in solubility. This protection against precipitation took place at the expense of a reduced thermal stability of conalbumin. Analysis at linearly increasing temperatures was shown to be a productive method to study the coupling between the thermal precipitation and denaturation processes. This coupling was examined at two heating rates, 10 and 1.25"C/min, respectively. Two types of precipitation behaviour were identified, a rapidly and a slowly sedimenting one. At the lower heating rate and under conditions where rapidly sedimenting precipitates were formed there was a close correlation between precipitation and denaturation. The precipitation was, however, always completed prior to denaturation. When approaching conditions where no precipitation took place, which also implied a successive transition towards slowly sedimenting precipitates, the precipitation process became delayed compared to denaturation. Precipitation could thus be registered at temperatures far above those of complete denaturation. The precipitates formed showed a considerable variation in particle size and water holding capacity.
The kinetics of the Streptomyces griseus protease-3-catalyzed hydrolysis of p-nitrophenylacetate have been studied by stopped-flow techniques with widely varied initial concentrations of enzyme and substrate. The results obtained are consistent with a three-step mechanism in which there is a rapid equilibration between enzyme and substrate to form a Michaelis complex with a dissociation constant in the order of 10 mM, followed by a moderately rapid formation of an acyl-enzyme and a rate-limiting deacylation of this intermediate. Estimated rate constants for the latter two reaction steps are 15 s-l and 0.09 s-l, respectively.The presence of a nucleophile such as methanol results in a partitioning of the deacylation process, reflected by a linear dependence of the maximum steady-state reaction velocity on the concentration of methanol. This observation provides confirmatory evidence for a rate-limiting deacylation step in the catalytic mechanism.Pronase, the extracellular proteolytic enzyme preparation from Streptomyces griseus, contains several components with protease activity [l, 21, some of which have been shown to be serine proteases [3-51. One of these serine enzymes (herein referred to as Streptomyces griseus protease 3) has received particular attention since it, despite its microbiological origin, exhibits striking sequential similarities to pancreatic serine enzymes [6] and has a broad substrate specificity showing similarities to both chymotrypsin and elastase [4,7].Acyltransfer reactions to water or other nucleophilic acceptors, catalyzed by serine or thiol hydrolases, have often been interpreted in view of the minimal kinetic scheme s P1
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