Dang C V, Ebert R F, Bell W R
J Biol Chem. 1985 Aug 15;260(17):9713-9.
Calcium is required for effective fibrin polymerization. The high affinity Ca2+ binding capacity of fibrinogen was directly localized to the gamma-chain by autoradiography of nitrocellulose membrane blots of fibrinogen subunits incubated with 45Ca2+. Terbium (Tb3+) competitively inhibited 45Ca2+ binding to fibrinogen during equilibrium dialysis, accelerated fibrin polymerization, and limited fibrinogen fragment D digestion by plasmin. The intrinsic fluorescence of Ca2+-depleted fibrinogen was maximally enhanced by Ca2+ and Tb3+, but not by Mg2+, at about 3 mol of cation/mol of fibrinogen. Protein-bound Tb3+ fluorescence at 545 nm was maximally enhanced by resonance energy transfer from tryptophan (excitation at 290 nm) at about 2 mol of Tb3+mol of fibrinogen and about 1 mol of Tb3+/mol of plasmic fragment D94 (Mr 94,000). Fibrinogen fragments D78 (Mr 78,000) and E did not show effective enhancement of Tb3+ fluorescence, suggesting that the Ca2+ site is located within gamma 303 to gamma 411, the peptide which is absent in fragment D78 but present in D94. When CNBr fragments of the carboxyamidated gamma-subunit were assayed for enhancement of Tb3+ fluorescence, peptide CBi (gamma 311-336) bound 1 mol of Tb3+/mol of CBi. Thus, the Ca2+ site is located within this peptide. The sequence between gamma 315 and gamma 329 is homologous to the calmodulin and parvalbumin Ca2+ binding sites.
有效的纤维蛋白聚合需要钙。通过用45Ca2+孵育的纤维蛋白原亚基的硝酸纤维素膜印迹放射自显影，将纤维蛋白原的高亲和力Ca2+结合能力直接定位到γ链上。在平衡透析过程中，铽（Tb3+）竞争性抑制45Ca2+与纤维蛋白原的结合，加速纤维蛋白聚合，并限制纤溶酶对纤维蛋白原片段D的消化。在约3摩尔阳离子/摩尔纤维蛋白原时，Ca2+耗尽的纤维蛋白原的固有荧光通过Ca2+和Tb3+最大程度增强，但不被Mg2+增强。在约2摩尔Tb3+/摩尔纤维蛋白原和约1摩尔Tb3+/摩尔血浆片段D94（分子量94,000）时，色氨酸（290nm激发）的共振能量转移使545nm处的蛋白质结合Tb3+荧光最大程度增强。纤维蛋白原片段D78（分子量78,000）和E未显示Tb3+荧光的有效增强，表明Ca2+位点位于γ303至γ411内，该肽段在片段D78中不存在但在D94中存在。当测定羧酰胺化γ亚基的CNBr片段对Tb3+荧光的增强作用时，肽CBi（γ311 - 336）结合1摩尔Tb3+/摩尔CBi。因此，Ca2+位点位于该肽段内。γ315和γ329之间的序列与钙调蛋白和小清蛋白的Ca2+结合位点同源。
