Disulfide bonds in egg-white riboflavin-binding protein. Chemical reduction studies.

All eight disulfide bonds in the apo-form of egg white riboflavin-binding protein were easily reduced by 2-mercaptoethanol and dithiothreitol. These bonds exhibited nearly the same reactivity, thus they appeared to be exposed in the native structure, or 'superficial'. The cleavage of protein disulfides resulted in a loss of riboflavin-binding capacity. A correlation between these two related processes, analysed by kinetic and statistical methods, suggested a single bond to be essential for binding of riboflavin by the apoprotein. In the riboflavin-apoprotein complex the disulfides were rather poorly reducible but they still constituted a single reactivity class. The essential bond was not protected against modification, suggesting it was located out of the riboflavin-binding site. A postulated subunit structure of riboflavin-binding protein was not confirmed. The cleavage of disulfides caused some aggregation of the protein molecules. Only dimers and high polymers were formed, the former being relatively stable. Hydrophobic forces were probably involved in the formation of dimers.