Chernyshenko V O
Protein Structure and Functions Department, Palladin Institute of Biochemistry, 9, Leontovych Str., Kiev, 01601, Ukraine,
Protein J. 2015 Apr;34(2):130-4. doi: 10.1007/s10930-015-9605-2.
Previously we purified fibrinogenase from venom of Echis multisquamatis and showed that the enzyme predominantly cleaves BβArg42-Ala43 peptide bond of fibrinogen. A much slower hydrolysis of its Aα-chain was also shown. To evaluate the accessibility of the hydrolysis sites to fibrinogenase's hydrolytic action, the pathway of cleavage of Aα- and Bβ-chains of fibrinogen, monomeric and polymeric fibrin desA and desAB has been investigated using western blot with monoclonal antibodies to Bβ 26-42 and Aα 20-78 of fibrinogen. The data indicated that the BβArg42-Ala43 peptide bond is available for cleavage in all forms of fibrin(ogen) with the exception of polymerized fibrin desAB. This is direct evidence of BβN-domain involvement in formation of protofibrils that makes it inaccessible to protease. The Aα-chain of fibrinogen remained intact after 3 min of incubation with fibrinogenase. Further incubation resulted in cleaving of the fibrin(ogen) αC-regions with the formation of two kinds of degradation products (~30 and ~60 kDa). In the case of monomeric fibrin desA or desAB we observed simultaneous hydrolysis of Aα and Bβ-chains and the cleavage of Aα-chain was more apparent for both forms of polymeric fibrin.
此前，我们从多鳞蝰蛇毒中纯化出纤维蛋白原酶，并表明该酶主要切割纤维蛋白原的Bβ链Arg42 - Ala43肽键。还发现其对Aα链的水解作用要慢得多。为了评估水解位点对纤维蛋白原酶水解作用的可及性，我们使用针对纤维蛋白原Bβ 26 - 42和Aα 20 - 78的单克隆抗体进行蛋白质印迹，研究了纤维蛋白原、单体和聚合纤维蛋白desA及desAB的Aα链和Bβ链的切割途径。数据表明，除了聚合纤维蛋白desAB外，Bβ链Arg42 - Ala43肽键在所有形式的纤维蛋白（原）中均可被切割。这直接证明了BβN结构域参与原纤维的形成，使得蛋白酶无法接近该位点。纤维蛋白原与纤维蛋白原酶孵育3分钟后，Aα链保持完整。进一步孵育导致纤维蛋白（原）αC区域被切割，形成两种降解产物（约30 kDa和约60 kDa）。在单体纤维蛋白desA或desAB的情况下，我们观察到Aα链和Bβ链同时被水解，并且对于两种形式的聚合纤维蛋白，Aα链的切割更为明显。
