Studies on the irreversible thermal denaturation of Rnase A

Abstract

“STUDIES ON THE IRREVERSIBLE THERMAL DENATURATION OF RIBONUCLEASE A” SUBMITTED BY GIRISH SAHNI FOR THE Ph.D. DEGREE OF THE INDIAN INSTITUTE OF SCIENCE, BANGALORE–560012, INDIA** Processes whereby proteins and enzymes are irreversibly thermoinactivated are not clearly understood. Choosing RNase A, a protein whose structure and chemistry are known in great detail, as a model, a study has been made on the products formed during the irreversible thermal denaturation of the enzyme at neutral pH and 70?°C. In an earlier study from this laboratory, the isolation and partial characterization of the soluble intermediates formed during thermoinactivation of RNase A had been reported. The soluble protein fraction obtained after thermal treatment and fractionation by gel filtration on Sephadex G?75 was found to contain:

Oligomeric (RNase A_t?) Dimeric (RNase A_t?) Unfolded monomeric (RNase A_t?) Compactly folded monomeric (RNase A_t?)

RNase A_t? was generally considered to be unmodified RNase A. RNase A_t?, RNase A_t? and RNase A_t? were found to possess very low residual enzymatic activity (?5% of the native specific activity). The present study focuses on resolving the gel?filtration components—RNase A_t?, RNase A_t? and RNase A_t?—and examining their physicochemical and catalytic properties. It was also discovered that the presence of oxygen during thermoinactivation has a marked influence on the structure and properties of the initial soluble products. These oxygen?derived products have also been characterized. Influence of Experimental Variables Several variables were found to affect the thermoinactivation process. In the absence of oxygen, heating RNase A (20 mg/mL in 0.15 M NaCl, pH 7.0) at 70?°C led to:

Formation of turbidity after ~60 minutes Appearance of monomeric, dimeric, and oligomeric derivatives in solution

pH had a strong influence: even small increases in pH in the neutral range greatly decreased the incubation period preceding turbidity. However, the initial RNase A concentration did not affect the onset of turbidity, although it did significantly alter:

The specific activities of the soluble intermediates Their overall yields

These observations indicate that the gel?filtration components are heterogeneous mixtures. Nature of Structural Changes SDS?PAGE (±2?mercaptoethanol) showed:

Oligomeric and dimeric derivatives are linked by intermolecular disulfide bonds.

Amino?acid composition and tryptic?peptide maps (after performic acid oxidation) of RNase A_t? and RNase A_t? did not differ from native RNase A. However, diagonal peptide maps revealed non?native disulfide bonds, indicating that both derivatives consist of mixtures with different cystinyl pairings. Reversibility Studies All thermally denatured derivatives—RNase A_t?, RNase A_t? and RNase A_t?—could be reactivated to varying extents by:

Sulfhydryl–disulfide interchange (mixed GSH/GSSG) Reduction followed by re?oxidation

Gel?filtration of the reoxidized samples revealed regeneration of native?like RNase A, demonstrating that the primary cause of inactivation is disulfide?bond scission and mispairing. Heterogeneity of the Derivatives Detailed characterization showed:

RNase A_t? and RNase A_t? are heterogeneous. Urea?gradient electrophoresis revealed no unfolding transition, unlike native RNase A.

Activity?versus?urea experiments, however, did show transitions—indicating:

Only a small fraction is catalytically competent Most molecules have structures so disrupted that they cannot unfold further detectably

Trypsin and iodoacetate experiments confirmed the presence of:

A native?like, trypsin?resistant, catalytically active component A trypsin?labile, inactive majority

Interactions with S?Protein More definitive evidence of heterogeneity came from interaction studies with:

S?protein (des 1–20 RNase A) CM?His?119?S?protein (a chemically modified analog)

RNase A_t? regained nearly full activity with S?protein due to reassociation of its N?terminal S?peptide with the external partner. Detailed analysis showed:

Most constituents of RNase A_t? are inactive but readily bind S?protein A small intrinsically active component binds more weakly

Affinity Chromatography Resolution Using CTP–Sepharose, the active and inactive fractions of both RNase A_t? and RNase A_t? were resolved:

The active fraction of RNase A_t? (~15% native activity) was not reactivated by thiols The active fraction of RNase A_t? (~7% native activity) was greatly enhanced by thiol treatment, consistent with mispaired disulfide bonds

Fluorescence and near?UV CD spectra partially characterized these species. Effect of Oxygen In the presence of oxygen:

Polymerization at 70?°C, pH 7.0 was severely inhibited No turbidity formed The enzyme remained soluble even after long incubation (10 h)

Chemically modified, monomeric RNase A species accumulated. Though they co?eluted with native RNase A on gel filtration, ion?exchange chromatography resolved them into three non?native species. These oxygen?derived species:

Have conformations close to native RNase A Show reduced activity toward RNA (35–55%) Show high activity toward cyclic CMP Are more susceptible to urea and thermal destabilization