A Study of Several Bonds Hypersensitive to Proteases in a Complex Flavohemoenzyme, Yeast Cytochrome <em>b</em>2.

A detailed study of the polypeptide fragments formed, and of their evolution during the first stages of the trypsinolysis of two homologous active cytochrome b2 molecules, was carried out, These cytochromes are complex flavoheme-L-lactate dehydrogenases extracted from different yeasts and having different molar activities. Our purpose was to gather information concerning the bonds, which, in the native proteins, are the most sensitive to trypsin (termed here protease-hypersensitive bonds). The products resulting from the fist hydrolytic events were thus analysed using disc-electrophoresis in sodium dodecylsulfate according to the method of Shapiro et al. (1967). Electrophoretic patterns were related to the molecular weight and to the amount of each intermediate species as a function of the extent of reaction. Schemes of the series of events, (including several rate constants) were derived from this set of data. A tentative map of the homologous loci of protease-hypersensitive bonds along the chains of the two cytochrome b2 molecules is proposed. Considering that such bonds which are very reactive towards trypsin are certainly located on flexible and thus loosely structured regions at the surface of the molecule, this map gives a first insight into the overall secondary and tertiary structure of the chain which includes globules of high internal stability. important finding was that the relative reactivities of the various protease-hypersensitive bonds identified, depend on conditions such as ionic strength and the binding of a ligand which probably changes the conformation. The protein transitions are therefore possibly described in terms of modulation of the loci of these bonds and of their reactivity. [ABSTRACT FROM AUTHOR]