S-OVALBUMIN, AN OVALBUMIN CONFORMER WITH PROPERTIES ANALOGOUS TO THOSE OF LOOP-INSERTED SERPINS

Most serpins are inhibitors of serine proteinases and are thought to u ndergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a bet a-sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhi bitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critica l point (P14) in the reactive center region, which prevents loop inser tion into the beta-sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have exami ned the properties of two forms of ovalbumin: the native protein and S -ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S-ovalb umin was more stable than ovalbumin by about 3 kcal mol(-1). CD spectr a, which indicated that S-ovalbumin had less alpha-helix than native o valbumin, and H-1 NMR spectra, which indicated very similar overall st ructures, suggest limited conformational differences between the two f orms. From comparison of the susceptibility of the reactive center reg ion of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native-to-S con formational change specifically affected the reactive center region. T hese data are consistent with a structure for S-ovalbumin in which par t of the reactive center loop has inserted into beta-sheet A to give a more stable structure, analogously to other serpins. However, the rat e of loop insertion appears to be very much lower than for inhibitory serpins. Thus, although loop insertion does not appear to be prevented by the presence of arginine at P14, it occurs at too slow a rate to c ompete effectively with the substrate pathway. This alone is sufficien t to account for the absence of detectable inhibitory properties in ov albumin.