REFOLDING PROCESS OF OVALBUMIN FROM UREA-DENATURED STATE - EVIDENCE FOR THE INVOLVEMENT OF NONPRODUCTIVE SIDE-CHAIN INTERACTIONS IN AN EARLY INTERMEDIATE

Ovalbumin contains one cystine disulfide (Cys(73)-Cys(120)) and four c ysteine sulfhydryls (Cys(11), Cys(30), Cys(367), and Cys(382)) in a si ngle polypeptide chain of 385 amino acid residues, The refolding mecha nism of ovalbumin was investigated under disulfide-bonded and disulfid e-reduced conditions using the denatured protein state, D-A, as the st arting protein sample, For the preparation of D-A, the disulfide-intac t and disulfide-reduced forms of ovalbumin were denatured by protein i ncubation in 9 M urea at pH 2.2. When D-A was placed in a refolding bu ffer, PH 8.2, an intermediate state I-N was produced in either the dis ulfide-bonded or the disulfide-reduced condition; I-N showed about 60% of the native CD ellipticity at 222 nm and the intrinsic tryptophan f luorescence with the native spectrum peak but with decreased intensity , The formation of I-N as detected by far UV CD ellipticity was quite rapid and finished within a mixing dead time of 20 ms, When D-A was di luted with an acidic buffer, pH 2.2, a partially folded equilibrium in termediate I-A with the structural characteristics equivalent to those of I-N was formed, After the formations of I-N and I-A, the regains i n CD ellipticity and tryptophan fluorescence at pH 8.2 followed biphas ic kinetics in the disulfide-bonded condition but monophasic kinetics in the disulfide-reduced condition, As unexpected findings, the native disulfide in D-A and I-A underwent nonproductive disulfide rearrangem ents in the disulfide-bonded condition at an early refolding stage and then was recovered during the subsequent refolding. The integrity of overall refolding was confirmed by the observation that the proteins r efolded for 20 h in the disulfide-bonded and disulfide-reduced conditi ons showed, on differential scanning calorimetry analyses, almost exac tly the same denaturation temperatures as their native protein counter parts, These results were consistent with a refolding process for oval bumin which includes nonproductive side chain-side chain interactions in the early intermediate I-N, which requires subsequent reorganizatio n for the correct refolding.