Synthesis of the vinyl sulfone and chloroethyl sulfone derivatives of poly(ethylene glycol) (PEG) is described. The chloroethyl sulfone (CES-PEG) is rapidly converted to the vinyl sulfone (VS-PEG) in the presence of base but is stable in water at neutral pH. Reactions with small molecules such as beta-mercaptoethanol and N alpha-acetyllysine show that the vinyl sulfone derivative is highly selective for reaction with sulfhydryl groups relative to reaction with amino groups. Also, VS-PEG is stable in water. These properties indicate that VS-PEG should be useful for selective attachment of PEG to protein cysteine groups. This hypothesis was verified by reacting VS-PEG with cysteine groups of reduced ribonuclease (RNase); the reaction is rapid and selective at pH 7-9. Reaction at lysine sites of unreduced RNase occurs slowly at pH 9.3 and is essentially complete after 100 h. Amino acid residues other than lysine and cysteine are not reactive toward VS-PEG. The covalent linkage between VS-PEG and lysine or cysteine groups is shown to be stable.

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The efficacy of preventing ischemia-reperfusion damage by employing native or modified (mPEG-SOD) superoxide dismutase in an experimental animal model of acute ischemia of the left hindlimb was tested. Four hours and thirty minutes complete warm ischemia was induced in the left hindlimb of 43 Wistar rats, by clamping the femoral artery and monitoring its efficacy with Laser Doppler flowmetry. After ten days, a significative difference (p = 0.004) of the survival leg rate was found in the group treated with mPEG-SOD (86.6%) compared with the control group (30%). Histomorphological and ultrastructural analysis were performed at different time intervals confirming what the clinical course had already pointed out. These results show that SOD in its modified form, despite the lower dosage, can provide good protection against ischemia/reperfusion injury of muscles.

Enolase from Bacillus stearothermophilus was homogeneous in the ultracentrifuge and had the hydrodynamic properties of a globular protein with a molecular weight of 3.51 times 105 (sedimentation‐diffusion) and 3.60 times 105 (Archibald approach to equilibrium). Electrophoretic and gel filtration measurements in presence of dissociating agents indicated that the enzyme consists of eight apparently identical polypeptide chains of molecular weight of 47 000. The denaturation of enolase has been studied as a function of urea concentration. A broad transition curve was observed, the enzyme in 8 M urea being inactive and dissociated into monomers. The far‐ultraviolet circular dichroism spectrum of enolase in 8 M urea solution indicates extensive unfolding of the globular structure. Accordingly, fluorescence emission measurements indicate that the tryptophanyl residues of the denatured enolase are largely exposed to the aqueous solvent medium. The enzyme can be reversibly denatured by 8 M urea and the enzymatic activity then recovered by dilution 1:100 of the denatured enzyme into buffer or by removing urea by dialysis. Under suitable conditions and in the presence of Mg2+‐ions, 90–100% of original activity was recovered and the renatured enzyme was indistinguishable from the native one, as judged from several enzymological and physicochemical criteria, including molecular weight. The far‐ultraviolet circular dichroism spectrum and the fluorescence emission properties of the native enzyme were regained very rapidly (less than 1 min), while enzymatic activity was recovered much more slowly. Octameric enzyme species were obtained on refolding the denatured enzyme at temperatures between 4 and 55.

Twenty Wistar rats were divided into two groups. Both underwent acute ischaemia followed by reperfusion of the left hind limb. The first group was a control group while the second was treated with PAcM-SOD. The survival percentage of the limb after 10 days was 30% for the first group and 70% for the second. Neither linear regression nor correlation were found between groups as far as the survival percentage of the limb after 10 days and reperfusion pmO2 data were concerned. After ten days the histomorphological analysis was significant regarding the fibre diameter and the percentage of central located nuclei in the specimens of PAcM-SOD treated limbs compared to normal limbs, but not when compared to the muscular fibres of the control group. Comparing these results with others obtained with native SOD and monomethoxypoly(ethylene glycol) modified SOD (mPEG-SOD) used in the same experimental model, we can conclude that the clinical and morphological results were better using mPEG-SOD, and that PAcM-SOD does have a protective effect on ischaemic muscle damage, although it is not as effective as mPEG-SOD in preventing ischaemia/reperfusion injury.

The octameric enolase from Bacillus stearothermophilus was immobilized onto Sepharose 4B activated by the cyanogen bromide reaction under conditions for achieving essentially a single‐point attachment. The immobilized enzyme was dissociated with guanidine hydrochloride to yield bound monomeric enolase. The Sepharose‐bound subunit regained activity upon removal of the denaturant. It was also possible to rehybridize immobilized monomers to native octamers. Of note, the thermal stability of the immobilized enolase subunit does not appreciably differ from that of the parent soluble octameric enzyme. Thus, these results indicate that single subunits of thermophilic enolase are active and that oligomerization is not a prerequisite for the enzymic activity as well as for thermal stability.