Ribonuclease, transition temperature

Fig. 5.3. Transition temperature of ribonuclease (a) as a function of concentration of various added salts (b) as a function of concentration of various denaturants, urea and guanidium salts at pH 7.0, all solutions containing 0.15 M KCl and 0.013 Af sodium cacodylate protein concentration 0.385 mM (from von Hippel and Wong, 1965).

The preceding summary and Fig. 20 present a frame-by-frame account of the pathway for ribonuclease catalysis, based predominandy on knowledge of the structures of the various intermediates and transition states involved. The ability to carry out such a study is dependent on three critical features (1) crystals of the enzyme which diffract sufficiently well to permit structural resolution to at least 2 A (2) compatibility of the enzyme, its crystals, and its catalytic kinetic parameters with cryoenzymology so as to permit the accumulation and stabilization of enzyme-substrate complexes and intermediates at subzero temperatures in fluid cryosolvents with crystalline enzyme and (3) the availability of suitable transition state analogs to mimic the actual transition states which are, of course, inaccessible due to their very short lifetimes. The results from this investigation demonstrate that this approach is feasible and can provide unparalleled information about an enzyme at work. 

FIGURE 7.4 Transition of proteins from the native to the unfolded state or vice versa, (a) Ribonuclease at pH 3.15, as a function of temperature, (b) Lysozyme as a function of guanidinium chloride concentration, (c) Nuclease A as a function of pH. 

Privalov and Khechinashvili (1974) have investigated the thermal transition of five globular proteins ribonuclease, lysozyme, chymotrypsin, cytochrome c, and myoglobin. Figure 6.20 represents the AH jai/AHyn at various temperatures for all studied proteins. The average value is 1.05 0.03. This deviation from a two-state process (not exceeding 5%) was attributed to the existence of highly unstable intermediates. 

Thermal transitions of ribonuclease, chymotrypsinogen (with disulfide bonds intact) were reinvestigated by temperature jump in a time range of observation which could extend down to few microseconds allowing detection of rapid phases, under the same conditions (pH, concentration) used by Pohl (1968a). 

In and above the transition zone, fast and slow species exist at equilibrium they are forms of the heat-unfolded ribonuclease. The ratio of their concentrations UyUi) does not depend on temperature. 

---