Kinetic correlations between the disulfide bond reduction and the induced conformational change of proteins.
Kinetic correlations between the disulfide bond reduction in excess dithioerythritol and the induced conformational change were studied on two proteins, bovine alpha-lactalbumin and soybean trypsin inhibitor, at 25 degrees C and pH 8.0-8.5 by measuring the absorbance of oxidized dithioerythritol at 310 nm and the ellipticity at 270 nm, respectively. With alpha-lactalbumin, in the absence of guanidine hydrochloride (Gdn x HCl) or in dilute Gdn x HCl, the kinetics for the bond reduction and the conformational change were both of a biphasic type. The fast phase was complete within a few seconds and was associated with the reduction of some of the disulfide bonds and with almost complete loss of the tertiary structure. The slow phase was associated with the reduction of other disulfide bonds. In concentrated Gdn x HCl, the kinetics of both processes were observed as a single phase, the rate of which was similar to that of the slow phase in the absence of Gdn x HCl or in dilute Gdn x HCl. In all cases studied, the rate of the bond reduction was similar to that of the conformational change induced. By correcting the change in absorbance at 310 nm to a contribution from the protein due to the conformational change, the number of bonds which are reduced in the fast phase in the absence of Gdn x HCl was determined to be 1.0-1.1. It was shown, taking observations of others and theoretical results into account, that the bond reduced in the fast phase might be the one between Cys 6 and Cys 120. On the other hand, on of two bonds of soybean trypsin inhibitor in the native form was reduced in the fast phase without any loss of the tertiary structure, and the other was reduced in the slow phase. Considering the results of other researchers, it was concluded that the bond reduced in the fast phase of the inhibitor is a 136-145 bond.
D000818:Animals; D002417:Cattle; D004220:Disulfides; D004226:Dithioerythritol; D007700:Kinetics; D007768:Lactalbumin; D010084:Oxidation-Reduction; D011487:Protein Conformation; D014361:Trypsin Inhibitors
D004220:Disulfides; D014361:Trypsin Inhibitors; D004226:Dithioerythritol; D007768:Lactalbumin