= Ribonuclease A refolding experiment
Anfinsen, Haber, Sela and White's 1961 experiments began by treating bovine pancreatic <ribonuclease A> with <mercaptoethanol> in $8\,\mathrm{mol\,L^{-1}}$ <urea>. <Urea> promotes unfolding; <mercaptoethanol> breaks <protein disulfide bonds>. The <peptide bonds> along the chain remain intact. After separating the chain from those reagents, the authors allowed its reactive <sulfur> groups to form <protein disulfide bonds> in the presence of <molecular oxygen>, at <pH> $8.2$ and <temperature> $23$–$24\,{}^{\circ}\mathrm C$. They examined <protein> <concentrations> of $0.1$ and $2.0\,\mathrm{mg\,mL^{-1}}$. Measurements showed recovery of native properties under these conditions; the isolated major component had normal activity toward both <RNA> and a synthetic <substrate>. See https://williams.chemistry.gatech.edu/course_Information/6572_fall_2021/papers/ANFINSEN_1961.pdf[Anfinsen, Haber, Sela and White, The Kinetics of Formation of Native Ribonuclease during Oxidation of the Reduced Polypeptide Chain, PNAS 47, 1309–1314 (1961), pp. 1309–1311].
The observation supports the sufficiency of that intact sequence to recover native <protein structure> in the tested surroundings without a folded template. It does not establish that every <protein> refolds, that the original living <biological cell> needed no assistance, or that activity always returns immediately. <Protein folding>, forming <protein disulfide bonds> and recovering an <active site> are related processes rather than interchangeable measurements.
Anfinsen's lecture also discusses forming <protein disulfide bonds> while the chain remains in concentrated <urea>. This gave mostly inactive material; after removing <urea>, a small amount of <mercaptoethanol> permitted rearrangement toward the native form. As an exact combinatorial check, eight labeled reactive <sulfur> groups have $7\times5\times3\times1=105$ ways to be paired into four <protein disulfide bonds>: choose the partner of one group, then repeat with the unpaired groups. Only one pairing is the native one. Equal counting does not mean that all pairings are equally likely experimentally, nor that actual <protein folding> searches them uniformly. See https://web.iitd.ac.in/~amittal/1973_Anfinsen_Science.pdf[Anfinsen's Nobel lecture, 1973 publication: scrambled ribonuclease and the 105 pairings, p. 223].
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