Hosmane R S, Peri S P, Bhadti V S, Macdonald V W
Department of Chemistry and Biochemistry, University of Maryland, Baltimore 21250, USA.
Bioorg Med Chem. 1998 Jun;6(6):767-83. doi: 10.1016/s0968-0896(98)00032-7.
The design, synthesis, and hemoglobin cross-linking studies of a novel organic reagent, bis[2-(4-carboxyphenoxy)carbonylethyl]phosphinic acid (BCCEP, 1) have been reported. The reagent was designed with the aid of molecular modeling, employing crystal coordinates of human hemoglobin A0. It was synthesized in three steps commencing from 4-t-butoxycarbonylphenol. The tri-sodium salt of 1 was employed to cross-link human oxyHb. While SDS-PAGE analyses of the modified hemoglobin product pointed to the molecular mass range of 32 kDa, the HPLC analyse suggested that the cross-link had formed between the beta 1-beta 2 subunits. The oxygen equilibrium measurements of the modified hemoglobin at 37 degrees C showed significantly reduced oxygen affinity (P50 = 31.3 Torr) as compared with that of cell-free hemoglobin (P50 = 6.6 Torr). The sigmoidal shape of O2 curves of the modified Hb pointed to reasonable retainment of oxygen-binding cooperativity after the cross-link formation. Molecular dynamics simulation studies on the reagent-HbA0 complex suggested that the most likely amino acid residues involved in the cross-linking are N-terminus Val-1 or Lys-82 on one of the-chains, and Lys-144 on the other. These predictions were consistent with the results of MALDI-MS analyses of the peptide fragments obtained from tryptic digestion of the cross-linked product.
已报道了一种新型有机试剂双[2-(4-羧苯氧基)羰基乙基]次膦酸（BCCEP，1）的设计、合成及血红蛋白交联研究。该试剂借助分子建模，采用人血红蛋白A0的晶体坐标进行设计。它从4-叔丁氧羰基苯酚开始分三步合成。1的三钠盐用于交联人氧合血红蛋白。虽然对修饰后血红蛋白产物的SDS-PAGE分析表明分子量范围为32 kDa，但HPLC分析表明交联是在β1-β2亚基之间形成的。与无细胞血红蛋白（P50 = 6.6 Torr）相比，在37℃下对修饰后血红蛋白进行的氧平衡测量显示氧亲和力显著降低（P50 = 31.3 Torr）。修饰后血红蛋白的O2曲线的S形表明交联形成后氧结合协同性得到了合理保留。对试剂-HbA0复合物的分子动力学模拟研究表明，交联中最可能涉及的氨基酸残基是一条链上的N端缬氨酸-1或赖氨酸-82，以及另一条链上的赖氨酸-144。这些预测与对交联产物胰蛋白酶消化得到的肽片段进行MALDI-MS分析的结果一致。
