A Serine Protease Inhibitor from the Anemone Radianthus Macrodactylus: Isolation and Physicochemical Characteristics
I. N. Sokotun1, E. V. Leichenko1 #, T. I. Vakorina1, A. A. Eskov1, A. P. Ilina1, M. M. Monastyrnaya1 and E. P. Kozlovskaya1
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1Pacific Institute of Bioorganic Chemistry, Far East Division, Russian Academy of Sciences, pr. 100-letiya Vladivostoka 159, Vladivostok, 690022, Russia
Received: August 18, 2006; in final form: November 1, 2006
Abstract. A serine protease inhibitor with a molecular mass of 6106±2Da (designated as InhVJ) was isolated from the tropical anemone Radianthus macrodactylus by a combination of liquid chromatography methods. The molecule of InhVJ consists of 57 amino acid residues, has three disulfide bonds, and contains no Met or Trp residues. The N-terminal amino acid sequence of the inhibitor (19 aa residues) was established. It was shown that this fragment has a high degree of homology with the N-terminal amino acid sequences of serine protease inhibitors from other anemone species, reptiles, and mammals. The spatial organization of the inhibitor at the levels of tertiary and secondary structures was studied by the methods of UV and CD spectroscopy. The specific and molar absorption coefficients of InhVJ were determined. The percentage of canonical secondary structure elements in the polypeptide was calculated. The inhibitor has a highly ordered tertiary structure and belongs to mixed α/β or α+β polypeptides. It was established that InhVJ is highly specific toward trypsin (K i 2.49 × 10−9 M) and α-chymotrypsin (K i 2.17 × 10−8 M) and does not inhibit other proteases, such as thrombin, kallikrein, and papain. The inhibitor InhVJ was assigned to the family of the Kunitz inhibitor according to its physicochemical properties.
Key words: amino acid sequence, anemone, circular dichroism, inhibition constant, protease inhibitor, serine proteases, UV spectroscopy
Russian Journal of Bioorganic Chemistry 2007, 33 (4): 415-422