Effects of monomethoxypoly(ethylene glycol) modification of ribonuclease on antibody recognition, substrate accessibility and conformational stability.
The effects of modification of bovine pancreatic ribonuclease A by monomethoxypoly(ethylene glycol) (MPEG) were examined for changes in recognition by antiRNase antibodies, enzymatic activity against low and high molecular weight substrates and conformational stability to temperature elevation. Modified forms of RNase were prepared containing an average of 4, 9, and 11 mol of MPEG/mol protein, by amino group modification. These were analysed by binding to RNase antibodies crosslinked to solid phase-immobilized protein A. The affinity column was incorporated into a high performance liquid chromatograph and the RNase species were studied by both zonal and frontal analytical affinity chromatography. An antibody dissociation constant of 7.6 x 10(-8) M was found for unmodified RNase, as compared to values of 1.3 x 10(-7) and 1.2 x 10(-6) M for RNase with 4 and 9 covalently bound MPEG chains, respectively. Modification also led to progressive loss of enzymatic activity against RNA, down to 3% for the most highly modified enzyme. In contrast, enzymatic activity against cytidine-2',3'-cyclic monophosphate was suppressed to a maximum of only 33% at the highest modification level, and the stability to temperature, as followed by circular dichroism, was reduced only partially, from 67 degrees C for native protein to 57 degrees C for RNase with 11 mol equivalents MPEG incorporated. The above differential effects on enzymatic activity, antibody binding and temperature effects are consistent with the view that MPEG modification has relatively small effects on conformational stability and small molecule accessibility, but more dramatic effects on large molecule (substrate as well as antibody) accessibility.(ABSTRACT TRUNCATED AT 250 WORDS)
Caliceti
P
P
Department of Pharmaceutical Sciences, University of Padua, Italy.
Schiavon
O
O
Veronese
F M
FM
Chaiken
I M
IM
D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D002417:Cattle; D007120:Immunochemistry; D066298:In Vitro Techniques; D011092:Polyethylene Glycols; D011487:Protein Conformation; D012259:Ribonuclease, Pancreatic / Q000276:immunology / Q000378:metabolism; D013379:Substrate Specificity
D000906:Antibodies; D011092:Polyethylene Glycols; C028210:monomethoxypolyethylene glycol; D012259:Ribonuclease, Pancreatic