RNase radicals

The reaction of H atoms with bovine pancreatic ribonuclease A (RNAse A) has been studied by steady-state /-radiolysis of lipid vesicle suspensions containing RNAse A. The inactivation of RNAse A caused by interaction of H atoms with protein involved selective attack on methionine residues and was connected with release of diffusible thiyl radicals [reaction (36)] ... 

RS radicals enter the bilayer, then reach and isomerize the lipid double bonds from mto trans vide supra, Scheme 1). The conclusion that Met residues in RNAse A are the major source was further confirmed by monitoring of vesicle /-irradiation containing RNAse T1 (two S-S bridges, no Met residues) where the formation of the trans isomer was found only to 0.6% after 1 kGy. 

RNase A Representative Enzyme. Both H and OH radicals in fluid, aqueous solutions react with RNase in ways that lead to inactivation. Mee and coworkers (30) have found that H leads to aggregation, and that cystine, methionine, and tyrosine are mainly affected. Adams and coworkers (31), investigating the specificity of free radical reaction with RNase, found that OH and Br2 are effective inactivators because they rapidly react with histidine, which is associated with the active site. 

The change in the rate constants with temperature for the reactions of ribonuclease (RNase) with hydrated electrons and OH radicals was measured. The RNase molecule unfolds reversibly at elevated temperatures exposing sites particularly reactive towards hydrated electrons. The theoretical treatment leads to an estimate of the encounter frequencies for differently shaped macromolecules with small radiolytically produced solvent radicals. The derived encounter frequencies are compared with experimentally determined rate constants. Values as high as 1013 M"1 secr1 are understandable. 

Experiments to establish the rate constant of the OH radical with the RNase molecule at different temperatures were also carried out. 32/aM RNase and 100KCNS were allowed to compete for the OH radicals. Within the experimental error no sudden increase of the rate constant for the reaction of OH with RNase was observed at the transition temperature. The rate constant increased from 2.6 X 1010 M 1 sec.-1 at 20°C. to 5.2 X 1010 M hecr1 at 60°C. 

It appears that almost all of the HS radicals formed in the labeling reaction react with the protein and do not recombine in the gas phase. In native RNase about 70% of the HS- radicals react with disulfide bridges (Reaction 8), while 30% add to carbon radicals (Reaction 6). The latter process could, under conditions previously discussed, cause a distortion of the tritium distribution. The reactions of HS- radicals in reduced proteins remain to be investigated. 

STIMULATION OF RNAse ACTIVITY BY THE FREE RADICAL OF AA An vitvo experiment for demonstrating the Inductive effect of MDHA on the enzyme RNAse was carried out using pure AA, RNA and RNA e (Chlnoy et al, 1973 a). In the reaction medium containing AA, RNA and RNAse, the enzymic degradation of RNA was slow as seen In Fig, 11,2, In another medium which contained only AA and RNA together with a few drops of 2 2 RNAse), the velocity of RNA... 

From the foregoing evidence it appears that AA stimulates RNAse activity only In the presence of H2O2. This is brought about by the formation of the free radical of AA, viz, MDHA, Further studies supported the marked stimulation of AA - FR special peroxidase activity by exogenous application of AA + H2O2 (Chlnoy et al, 1973). Similarly, Chlnoy, N,J, (1978) and Chlnoy, N.J, et al, (1982) have also... 

FIGURE 11.2, Stimulation of RNAse activity by the free radical of AA CChinoy et al., 1973 a). 

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