title: Biochemical characterization of an L-asparaginase bioconjugate.
abstract: In this work is characterized a bioconjugate of L-asparaginase, obtained by linkage of palmitic acid chains to the native enzyme in the presence of substrate as a protein protective molecule. Comparisons between isoelectric points, hydrophobicity, pH, and temperature profiles for the bioconjugate and the native enzyme were performed. A shift of pI from 5.03 to 4.58 was observed after conjugation. The modified enzyme evidences a 10-fold increase of the hydrophobicity. A small shift from 7.5 to 7 of the pH for maximal catalytic activity and a 5 degree C increase of temperature for maximal activity were observed with conjugation. Stability studies in human serum and on storage evidence similar behaviors for both bioconjugate and native enzyme. The retention of catalytic activity of the bioconjugate is dependent on the presence of micelles. The bioconjugate evidenced 65% retention of activity when catalytic activity was assayed without a surfactant and 98-100% retention of activity when catalytic activity was assayed in the presence of surfactant micelles. The kinetic characteristics of the bioconjugate and of the native enzyme, in micelles of different hydrophobicities, were compared. The Michaelis constant of native enzyme is 0.030 mM, independent of the surfactant, and the Michaelis constant of the bioconjugate varies with the surfactant, from 0.036 to 0.046 mM.
authors.affiliation: Department of Biotechnology, Instituto Nacional de Engenharia e Tecnologia Industrial, Lisboa, Portugal. UBQ1@DB.INETI.PT
authors.forename: M B
authors.initials: MB
authors.lastname: Martins
authors.forename: A P
authors.initials: AP
authors.lastname: Gonçalves
authors.forename: M E
authors.initials: ME
authors.lastname: Cruz
mesh_terms: D001215:Asparaginase / Q000737:chemistry*; D004795:Enzyme Stability; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008823:Micelles; D019308:Palmitic Acid; D013696:Temperature
chemical_list: D008823:Micelles; D019308:Palmitic Acid; D001215:Asparaginase