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首页> 外文期刊>Biochemistry >BINDING OF COBALAMIN AND COBINAMIDE TO TRANSCOBALAMIN FROM BOVINE MILK
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BINDING OF COBALAMIN AND COBINAMIDE TO TRANSCOBALAMIN FROM BOVINE MILK

机译：牛乳中氨基甲酸酯和氨基甲酸酯与反式氨基甲酸酯的结合

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We have studied the interaction between transcobalamin (TC) and the ligands cobalamin (Cbl) and cobinamide (Cbi). Partially purified TC from bovine milk was depleted of endogenous Cbl by 8 M urea treatment. Unsaturated TC was adsorbed on CM-Sepharose in order to ensure fast separation of the matrix-bound protein from the reaction medium. The forward reaction TC + Cbl --> TC-Cbl (rate constant k(+)(Chl) and the backward reaction and the backward reaction TC-Cbl-->TC+Cbl (k(-)(Cbl)) were followed in time. A single-step binding model (with no intermediate protein-ligand complex) was sufficient to fit the data. The calculated rate constant were k(+)(Cbl)=-0.6 nM(-1) min(-1) and k(-)(Cbl)=1.3 X 10(-4) min(-1), which corresponded to the TC-Cbl dissociation constant K-D(Cbl)=0.2 pM. Reaction between TC and Cbl developed against electrostatic forces, and the effective charges of the interacting species were estimated as both +1 or both -1. The competition between Cbl and Cbi for TC was studied, which resulted in determination of the relevant rate constants for Cbi: k(+)(Cbi)=0.03 nM(-1), and K-D(Cbi)=1 nM. Slow dissociation of TC-Cbl guarantees its stability in plasma for 5-10 h, while Cbi bound to TC would be transferred to haptocorrin in less than 1 h.
机译：我们研究了反钴胺素（TC）与配体钴胺素（Cbl）和钴胺（Cbi）之间的相互作用。通过8 M尿素处理，从牛乳中部分纯化的TC去除了内源性Cbl。不饱和TC吸附在CM-Sepharose上，以确保从反应介质中快速分离出基质结合蛋白。跟踪正向反应TC + Cbl-> TC-Cbl（速率常数k（+）（Chl）和反向反应和反向反应TC-Cbl-> TC + Cbl（k（-）（Cbl））一步结合模型（没有中间蛋白-配体复合物）足以拟合数据，计算出的速率常数为k（+）（Cbl）=-0.6 nM（-1）min（-1）和k（-）（Cbl）= 1.3 X 10（-4）min（-1），对应于TC-Cbl解离常数KD（Cbl）= 0.2 pM。TC和Cbl之间的反应因静电力而产生，并且相互作用物种的有效电荷估计为+1或均为-1，研究了Cbl和Cbi之间对TC的竞争，从而确定了Cbi的相关速率常数：k（+）（Cbi）= 0.03 nM（-1）和KD（Cbi）= 1 nM。TC-Cbl的缓慢解离保证了其在血浆中的稳定性5-10小时，而与TC结合的Cbi将在不到1小时的时间内转移到结合蛋白中。

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