Gladun D. Physico-chemical and catalytic properties of trypsin-like and fibrinogenolytic enzymes from Antarctic sea scallop Adamussium colbecki

The thesis is devoted to the study of structural and functional properties and catalytic constants of trypsin-like and fibrinogenolytic enzymes derived from the tissues of the Antarctic sea scallop A. сolbecki. Electrophoretic separation of the total A. colbecki extract allowed to detect protein bands with molecular weights of 157, 144, 130, 121, 110, 79, 61, 47, 44, 35 kDa. The method of two-dimensional electrophoresis revealed the presence of a certain number of protein stains in the plane of the 2D electrophoregram, the isoelectric points of which are located in two intervals of pH - from 3,5 to 4,0 and from 5,5 to 5,9. The range of molecular weights was from 30 to 235 kD. Enzyme-electrophoretic analysis showed a clear area of hydrolysis observed in the 25kDa region. The use of protease inhibitors EDTA and PMSF showed a dominant presence in the total extract of A. colbecki serine proteases and metal-dependent proteases. The trypsin-like enzyme was isolated by two steps using affinity chromatography on a SBTI-sepharose and Superdex 75 PG columns. Electrophoretic analysis of the obtained fraction showed the presence of only one protein band in the region of 23 kDa. An enzyme-electrophoretic study of the obtained fraction revealed only one clearly defined zone of hydrolysis indicating the presence of an active enzyme with a molecular weight of about 23 kDa. The trypsin-like enzyme effectively hydrolyzed the ligands formed by the carboxyl groups of Arg or Lys, especially those containing the Arg residue in the P1-position. The activity of this enzyme on substrates S2366 and S2238 was respectively in 4.36 and 1.68 times higher than that of S2251 substrate. Absence of activity on the substrate Ac-Phe-pNA is a confirmation of the trypsin-like nature of enzyme. The main kinetic constants of KM and Vmax were calculated from the Lineweaver-Burk graphs constructed for the respective substrates. The lowest value of KM and hence the highest affinity of the enzyme to the substrate was observed with the use of BApNA - in this case, the value of KM by an order of magnitude exceeded the value defined for substrates S2366 and S2238.