Synthesis, characterization and immunogenicity of silk fibroin-L-asparaginase bioconjugates.
L-asparaginase (ASNase) is one basic drug in the treatment of acute lymphoblastic leukemia (ALL). Because its half-life time is too short and it is easy to arouse allergic reaction, use in practical clinic is considerably limited. Silk fibroin (SF) with different molecular mass from 40 to 120 kDa is a natural biocompatible protein and could be used as a novel bioconjugate for enzyme modification to overcome its usual shortcomings mentioned above. When the enzyme was bioconjugated covalently with the water-soluble fibroin by glutaraldehyde, the enzyme kinetic properties and immune characteristics in vivo of the resulting silk fibroin-L-asparaginase (SF-ASNase) bioconjugates were investigated in detail. The results show that the modified ASNase was characterized by its higher residual activity (nearly 80%), increased heat and storage stability and resistance to trypsin digestion, and its longer half-life (63 h) than that of intact ASNase (33 h). The abilities of intact and modified ASNases to arouse allergic reaction are 2(4) and 2(1) antibody titers, respectively. Bioconjugation of silk fibroin significantly helps to reduce the immunogenicity and antigenicity of the enzyme. The apparent Michaelis constants of the modified ASNase (K(m(app))=0.844 x 10(-3)mol L(-1)) was approximately six times lower than that of enzyme alone, which suggests that the affinity of the enzyme to substrate l-asparagine elevated when bioconjugated covalently with silk fibroin. SF-ASNase bioconjugates could overcome the common shortcomings of the native form. Therefore, the modified ASNase coupled with silk fibroin has the potential values of being studied and developed as a new bioconjugate drug.
Zhang
Yu-Qing
YQ
Silk Biotechnology Laboratory, School of Life Science, Soochow University, No. 1 Shizi Street, Suzhou, Jiangsu 215006, PR China.
Zhou
Wei-Lin
WL
Shen
Wei-De
WD
Chen
Yu-Hua
YH
Zha
Xin-Min
XM
Shirai
Koji
K
Kiguchi
Kenji
K
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D000937:Antigen-Antibody Reactions; D001215:Asparaginase / Q000008:administration & dosage / Q000096:biosynthesis* / Q000097:blood / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000493:pharmacokinetics; D001672:Biocompatible Materials; D003378:Counterimmunoelectrophoresis; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004655:Emulsions; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D005349:Fibroins / Q000378:metabolism*; D005620:Freund's Adjuvant / Q000494:pharmacology; D005976:Glutaral / Q000494:pharmacology; D006207:Half-Life; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007274:Injections, Intraperitoneal; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D011233:Precipitin Tests; D011489:Protein Denaturation; D011817:Rabbits; D047011:Silk; D012995:Solubility; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology
D000276:Adjuvants, Immunologic; D001672:Biocompatible Materials; D003432:Cross-Linking Reagents; D004655:Emulsions; D047011:Silk; D005349:Fibroins; D005620:Freund's Adjuvant; D014357:Trypsin; D001215:Asparaginase; D005976:Glutaral