Physicochemical studies on ovalbumin. 4. Characterization of an iodine-modified derivative by electrophoresis and sedimentation

Abstract

The 5:1 product of iodine oxidation of ovalbumin has been studied in electrophoresis over the pH range 4-8:5. A new procedure is described for obtaining more accurately the first moment positions of the boundaries, and has been used in the determination of mobilities. The mobility-pH curve is identical with that of ovalbumin between pH4 and 5.4, but is more negative at higher pH values. Above pH 6.7 the mobility difference is constant, and has the value 0.6 x 10 -5 cm.2sec.-1v-1. From electrophoresis experiments under modified conditions, the relation between the size of the [delta]-boundary and the excess buffer concentration in the supernatant solution has been established for both proteins: these relations have been used to calculate the valences of the proteins at pH 8.5. The values obtained were -10.6 and -11.6 for the native and modified materials respectively. The mobilility values were also used to calculate valences, giving corresponding values of -10.7 and -11.8. The sedimentation coefficient-concentration relationship was established for the modified product at pH 7.5. No difference from the behaviour of the native protein was detectable, both sets of data fitting the equation S20, w= 3.42-0.26c. With Fujita''s (1956) expressions, diffusion coefficients were calculated from the sedimentation patterns as a sensitive test for heterogeneity: identical behaviour indicating homogeneity within the limits of the test was found for both materials. A mechanism for the iodine-ovalbumin reaction is suggested to account for these observations and the previously determined sulphydryl and disulphide content It involves the initial formation of a mixed product, half containing sulphenic acid groups and half sulphinic acid groups, each half being substituted with a single sulphenyl iodide (SI) residue. Subsequent aerobic oxidation of the sulphenic acid portion to yield a single final product is thought to occur.