Refolding process of ovalbumin from urea-denatured state. Evidence for the involvement of nonproductive side chain interactions in an early intermediate.

摘要: Abstract Ovalbumin contains one cystine disulfide (Cys73-Cys120) and four cysteine sulfhydryls (Cys11, Cys30, Cys367, Cys382) in a single polypeptide chain of 385 amino acid residues. The refolding mechanism ovalbumin was investigated under disulfide-bonded disulfide-reduced conditions using the denatured protein state, DA, as starting sample. For preparation disulfide-intact forms were by incubation 9 M urea at pH 2.2. When DA placed buffer, 8.2, an intermediate state IN produced either or condition; showed about 60% native CD ellipticity 222 nm intrinsic tryptophan fluorescence with spectrum peak but decreased intensity. formation detected far UV quite rapid finished within mixing dead time 20 ms. diluted acidic 2.2, partially folded equilibrium IA structural characteristics equivalent to those formed. After formations IA, regains 8.2 followed biphasic kinetics condition monophasic condition. As unexpected findings, underwent nonproductive rearrangements early stage then recovered during subsequent refolding. integrity overall confirmed observation that proteins refolded for h showed, on differential scanning calorimetry analyses, almost exactly same denaturation temperatures their counterparts. These results consistent process which includes side chain-side interactions IN, requires reorganization correct