Synthesis, characterization and properties of sialylated catalase.
Colominic acid (CA), an alpha-(2-->8) N-acetylneuraminic acid (sialic acid) polymer (average molecular weight of 10 kDa) was activated by periodate oxidation of carbon 7 at the non-reducing end of the saccharide. The oxidized CA was then coupled to catalase by reductive amination in the presence of sodium cyanoborohydride. The extent of sialylation of catalase, estimated by ammonium sulfate precipitation as 3.8+/-0.4 (mean+/-S.D.) moles of CA per mole of catalase, did not improve significantly when depolymerized CA was used in the coupling reaction. At the end of the coupling reaction, sialylated catalase exhibited a two-fold (70%) retention of initial activity compared to enzyme controls (29-35%) subjected to the same conditions. Formation of sialylated catalase was confirmed by ammonium sulfate or trichloroacetic acid precipitation, molecular sieve chromatography and SDS-PAGE electrophoresis. Enzyme kinetics studies revealed an increase in the apparent Km of the enzyme from 70.0 (native) to 122.9 mmol l-1 H2O2 (sialylated catalase) indicating a reduction of enzyme affinity for the substrate (hydrogen peroxide) on sialylation. Compared to native enzyme, sialylated catalase was much more stable in the presence of specific proteinases, completely resisting degradation by chymotrypsin and losing only some of its activity in the presence of trypsin. The increased stability conferred to catalase by sialylation agrees with similar observations on enzymes modified by other hydrophilic molecules (e.g., monomethoxypoly(ethyleneglycol)) and suggests that steric stabilization with the biodegradable polysialic acid may prove an alternative means to render therapeutic proteins more effective in vivo.
Fernandes
A I
AI
Centre for Drug Delivery Research, School of Pharmacy, University of London, UK.
Gregoriadis
G
G
D000818:Animals; D002374:Catalase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D002850:Chromatography, Gel; D010450:Endopeptidases / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry; D006023:Glycoproteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007700:Kinetics; D015394:Molecular Structure; D008970:Molecular Weight; D010504:Periodic Acid / Q000378:metabolism / Q000494:pharmacology; D011134:Polysaccharides / Q000737:chemistry*; D011489:Protein Denaturation; D012794:Sialic Acids / Q000737:chemistry*
D006023:Glycoproteins; D011134:Polysaccharides; D012794:Sialic Acids; D010504:Periodic Acid; C014420:colominic acid; C009288:metaperiodate; D006861:Hydrogen Peroxide; D002374:Catalase; D010450:Endopeptidases