TY - JOUR
T1 - Chemical modification of earthworm fibrinolytic enzyme with human serum albumin fragment and characterization of the protease as a therapeutic enzyme
AU - Nakajima, Nobuyoshi
AU - Ishihara, Kohji
AU - Sugimoto, Manabu
AU - Sumi, Hiroyuki
AU - Mikuni, Katsuhiko
AU - Hamada, Hiroki
N1 - Funding Information:
AcknOll'ledK1IIen!s. We are grateful to Mr. Y. Ishii. Eimei Co.. Ltd.. Miyazaki. Japan for supplying the earthworms. l.ul11hricliS ruhelllls. We also thank Professor Dr. K. ;\lakamura and Dr. S. Kondo. Institute for Chemical Research. Kyoto University for the amino acid sequence of the enzyme and the computer search of the homology from the protein sequence data bank. This research \vas financially supported in part by Ryobi Teien Memorial Foundation. Okayama. Japan in 1992 and by Yoshida Foundation for Science and Technology. Tokyo. Japan in 1993.
PY - 1996/1/1
Y1 - 1996/1/1
N2 - The strongest fibrinolytic protease (F-III-2) in the six enzyme proteins purified from earthworm, Lumhricus 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 chymotrvpsin.
AB - The strongest fibrinolytic protease (F-III-2) in the six enzyme proteins purified from earthworm, Lumhricus 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 chymotrvpsin.
KW - Chemical modification
KW - Fibrinolytic enzyme (of earthworm)
KW - Immobilized enzyme
KW - Protein sequence
KW - Serine protease (of earthworm)
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U2 - 10.1271/bbb.60.293
DO - 10.1271/bbb.60.293
M3 - Article
C2 - 9063978
AN - SCOPUS:0030087823
VL - 60
SP - 293
EP - 300
JO - Bioscience, Biotechnology and Biochemistry
JF - Bioscience, Biotechnology and Biochemistry
SN - 0916-8451
IS - 2
ER -