The thermodynamic stability of an enzymatically active derivative (sample LHO of hen egg lysozyme containing three, presumably, native disulfide bonds and carboxymethylated cysteines at positions 6 and 127 and exhibiting characteristics of the intact enzyme has been studied with respect to the formation of disulfide bonds. The procedure is to measure the equilibrium between the native and nonnative forms, after reequilibrating the disulfide bonds in the presence of β-mercaptoethanol, by gel filtration on Bio-Gel P-30. The native form has a lower hydrodynamic volume than the nonnative forms. To test whether the equilibrium has been reached, three different disulfide bonded forms of sample LH1 (native, reduced, and "scrambled") are exposed to the same concen-tration of β-mercaptoethanol (1.5 mM) at pH 8.0 and 37°C, which permits the disulfide interchange. All three samples show similar ratios (3:2) of native to nonnative forms after 16 h. This ratio is reached within 5 h in the case of native sample LH1. Consequently the apparent equilibrium appears to be reached. In these same conditions intact lysozyme shows only the native form. Thus, sample LH1 is less stable than the intact protein with respect to disulfide bond formation, but more stable than the nonnative isomers of sample LH1 obtained by reshuffling the disulfide bonds. This latter point has been tested by measuring the proportions of all the partially reduced forms of sample LH1 by trapping the free sulfhydryl groups present in the equilibrium mixture with iodoacetic acid.
|Original language||English (US)|
|Number of pages||7|
|State||Published - 1978|
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