Chemical modification of earthworm fibrinolytic enzyme with human serum albumin fragment and characterization of the protease as a therapeutic enzyme.
The strongest fibrinolytic protease (F-III-2) in the six enzyme proteins purified from earthworm, Lumbricus rubellus [N. Nakajima et al., Biosci. Biotech. Biochem., 57, 1726-1730 (1993)] has been modified chemically with fragmented human serum albumin (mol. wt., 10,000-30,000). The modified enzyme lost the antigenicity of the native enzyme and reacted with the antisera against human serum albumin, the human serum albumin fragments, and the conjugate with the native enzyme to form precipitation lines, which fused with each other. The conjugate was significantly more resistant to inactivation by protease inhibitors in rat plasma. The enzyme was a non-hemorrhagic protein and did not induce platelet aggregation. The enzyme kept potent proteolytic activity for fibrin and fibrinogen than that of human plasmin. The enzyme easily solubilized actual fibrin clots (thrombi) of whole blood induced by thrombin in a rat's vena cava. The continuous fibrinolysis for fibrin suspension in an enzyme reactor system using the modified enzyme immobilized to oxirane-activated acrylic beads has been achieved without any inactivation of the activity at least for more than 1 month. The N-terminal amino acid sequence of the protein was also investigated and the sequence showed local similarity to those of the serine proteases such as plasmin and chymotrypsin.
Nakajima
N
N
Department of Nutritional Science, Okayama Prefectural University, Japan.
Ishihara
K
K
Sugimoto
M
M
Sumi
H
H
Mikuni
K
K
Hamada
H
H
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004789:Enzyme Activation; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D005337:Fibrin / Q000187:drug effects / Q000378:metabolism; D005343:Fibrinolytic Agents / Q000737:chemistry* / Q000494:pharmacology*; D006470:Hemorrhage / Q000188:drug therapy; D006801:Humans; D008297:Male; D051379:Mice; D008969:Molecular Sequence Data; D009835:Oligochaeta / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry* / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects; D011480:Protease Inhibitors / Q000097:blood; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology*; D012709:Serum Albumin / Q000737:chemistry* / Q000494:pharmacology*
D004800:Enzymes, Immobilized; D005343:Fibrinolytic Agents; D010446:Peptide Fragments; D011480:Protease Inhibitors; D012709:Serum Albumin; D005337:Fibrin; D012697:Serine Endopeptidases