Laser Raman spectroscopic studies of the thermal unfolding of ribonuclease A.

The reversible thermal denaturation of bovine pancreatic ribonuclease A at pH 5 in 0.1 M NaCl over the range 32-70 degrees C as studied by Raman spectroscopy proceeds in a gradual manner consistent with a stepwise unfolding process rather than as a transition between two states. Conversion of residues from helical or pleated-sheet geometry to some intermediate geometry, as followed by means of the amide I and III lines, reveals that substantial amounts of the helical and pleated-sheet conformations remain at 70 degrees C. Changes in the strength of hydrogen bonding by the tyrosyl residues are indicated by the intensity ratio of the doublet at 830-850 cm(-1) and changes in the geometry of the disulfide bridges by the frequency and half-width of the Raman line near 510 cm(-1) due to the S-S vibration. Vibrations of C-S bonds in the methionines and cystines are used to monitor conformational changes in these residues. While there are small quantitative differences in temperature dependence among these probes, all agree in placing the malting temperature at or near 62 degrees C. The Raman data are quantitatively consistent with the six-stage scheme of unfolding of A.W. Burgess and H.A. Scheraga [(1975), J. Theor, Biol. 53, 403], except that no change in the environment of the tyrosines is seen until 45 degrees C.