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The unfolding and refolding of ribonuclease in urea solutions

1964; Elsevier BV; Volume: 10; Issue: 2 Linguagem: Inglês

10.1016/s0022-2836(64)80044-9

1089-8638

Eric A. Barnard,

Enzyme Structure and Function

A study has been made of the unfolding of ribonuclease in urea solutions and of the refolding by molecules related to substrates. Unfolding is measured by the spectral blue shift, which arises from the change in environment of some tyrosine and phenylalanine residues as the protein unfolds, and is an index of a general conflgurational change. Unfolding occurs at readily measurable rates. An equilibrium situation of the protein is recognized, with the extent of unfolding varying with temperature, pH and urea concentration. The dependence of unfolding kinetics upon these variables has also been explored. A transition occurs to the unfolded form above 5 M-urea (at pH 7, 25°C), with midpoint at 6·7 M. About 12 urea molecules are apparently involved from a kinetic analysis, and 16 from an equilibrium viewpoint. Refolding by phosphates in 8 M-urea occurs, and is due to disturbance of the equilibrium by binding to the more folded form, and not to a process of direct interaction at the unfolded protein. The refolding is, therefore, very much slower than unfolding. Conversely, phosphates retard the unfolding by urea, to extents parallel to their affinities for the enzyme in water. Kinetic analysis shows one phosphate molecule is bound. Cytidine 2′-phosphate is the most effective of a series tested. Poly-metaphosphate is unusually effective as are-folder, on a molarity basis, but on a phosphate residue basis is of the same order of effectiveness as orthophosphate. As the ribonuclease molecule unfolds, the specific binding of cytidine 2′-phosphate, when followed independently, declines at the same rate. This, together with the pH dependences of refoldings and the behaviour of derivatives of ribonuclease, show that the unique binding site of ribonuclease is held non-covalently, and provide information on the components of this site.