Temperature dependence ribonuclease

A relative fluorescence intensity scale will be defined in part 1 of the experiment. The cuvette holder should be provided with a water jacket to maintain the temperature within 0.5°. It is recommended that you use the same cuvette for all measurements unless a pair is available that is very well matched. Fluorescence measurements are temperature dependent. It is necessary to maintain all reagents, except the ribonuclease solution, at 37°C, the same as the setting for the cuvette holder in the fluorimeter. 

Figure 16.12 The temperature-dependent behavior of the denaturation enthalpy and entropy of ribonuclease (RNase) and myoglobin (Mb) under the assumption that AjjjCp is constant (dashed line) or decreasing with increasing temperature (solid line). Reproduced with permission from P. L. Privalov, Ann. Rev. Biophys. Chem. 18, 47 (1989). 1989, by Annual Reviews http //www.AnnualReviews.org...

Fig. 2. Temperature dependence of the partial specific heat capacity for pancreatic ribonuclease A (RNase), hen egg-white lysozyme (Lys), sperm whale myoglobin (Mb), and catalase from Thermus thermophilus (CTT). The flattened curves are for RNase and Lys with disrupted disulfide cross-links and for apomyoglobin, when polypeptide chains have a random coil conformation without noticeable residual structure (Privalov et al., 1988).

Fig. 4. Temperature dependence of the specific enthalpy of denaturation of myoglobin and ribonuclease A (per mole of amino acid residues) in solutions with pH and buffer providing maximal stability of these proteins and compensation of heat effects of ionization (see Privalov and Khechinashvili, 1974). The broken extension of the solid lines represents a region that is less certain due to uncertainty in the A°CP function (see Fig. 2). The dot-and-dash lines represent the functions calculated with the assumption that the denaturation heat capacity increment is temperature independent.

Almog and hrier (1978) made a direct calorimetric measurement of the dependence of the heat of solution of ribonuclease A on water content (Fig. 2). The heat of solution drops strongly in the low hydration range 90% of the heat change is obtained at about half-hydration. The differential heat for transfer of water from the pure liquid to the protein is estimated from the data of Fig. 2 as 8 kcal/mol of water at the lowest hydration studied (the heat of condensation of water should be added for comparison with isosteric heats), and it decreases monotonically with increased hydration. There is no extremum at low hydration, unlike what has been reported based on the temperature dependence of the sorption isotherm. It is not clear whether this difference reflects inaccuracies in the data used in van t Hoff analyses of the sorption isotherms, or a complex hydration path that is not modeled properly in the van t Hoff analyses. 

Mandel AM, Akke M, Palmer AG 3. Dynamics of ribonuclease H temperature dependence of motions on multiple time scales. Biochemistry 1996 35 16009-16023. 

E.L. Kovrigin, R, Cole, J.P. Loria, Temperature dependence of the backbone dynamics of ribonuclease A in the ground state and bound to the inhibitor 5 -phosphothymidine (3 -5 )pyrophosphate adenosine 3 -phosphate, Biochemistry 42 (2003) 5279-5291. 

The mechanism of ribonuclease A (RNase A) activity has been widely studied by evaluating different aspects such as roles of the catalytic amino acids, different substrates, thio effects, organic solvents, and pH and temperature dependence [79]. The usually accepted mechanism is the general acid/base pathway where a... 

The temperature dependence of K can also be taken into account. However, in experiments with ribonuclease films (Nakajima and Scheraga, 1961), the temperature dependence of K did not have much effect on the computed values of and A(S e.. 

Fig. 101. Temperature dependence of specific rotation of ribonuclease at pH 6.5. The two curves correspond to increasing and decreasing temperature, respectively, during the experiment (Harrington and Schellman, 1956).

Figure 2.11. The dependence of the position of the fluorescence spectrum maximum on excitation wavelength for tryptophan in a model medium (glycerol) at different temperatures (a) and singletryptophan proteins (b). 1, Whiting parvalbumin, pH 6.S in the presence of Ca2+ ions 2, ribonuclease Th pH 6.5 3, ribonuclease C2, pH 6.5 4, human serum albumin, pH 7.0, +10"4 M sodium dodecyl sulfate 5, human serum albumin, pH 3.2 6, melittin, pH 7.5, +0.15 M NaCl 7, protease inhibitor IT-AJ from Actinomyces janthinus, pH 2.9 8, human serum albumin, pH 7.0 9, -casein, pH 7.5 10, protease inhibitor IT-AJ, pH 7.0 11, basic myelin protein, pH 7.0 12, melittin in water. The dashed line is the absorption spectrum of tryptophan.

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. 

In the absence of substrates, a relaxation process is observed in solutions of ribonuclease with the temperature jump method having a relaxation time of 0.1 to 1 msec. This relaxation time is independent of the enzyme concentration but is pH dependent. It can be attributed to an isomerization or conformational change of the enzyme, and a simple mechanism consistent with the data is... 

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