S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins

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Abstract
Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a β‐sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the β‐sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined 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‐ovalbumin was more stable than ovalbumin by about 3 kcal mol−1. CD spectra, which indicated that S‐ovalbumin had less α‐helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native‐to‐S conformational change specifically affected the reactive center region. These data are consistent with a structure for S‐ovalbumin in which part of the reactive center loop has inserted into β‐sheet A to give a more stable structure, analogously to other serpins. However, the rate 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 compete effectively with the substrate pathway. This alone is sufficient to account for the absence of detectable inhibitory properties in ovalbumin.