Hardlei Tore Forsingdal, Morkbak Anne Louise, Nexo Ebba
Department of Clinical Biochemistry, AS, Aarhus University Hospital, Norrebrogade 44, DK 8000 Aarhus, Denmark.
Clin Biochem. 2007 Dec;40(18):1392-7. doi: 10.1016/j.clinbiochem.2007.09.008. Epub 2007 Sep 25.
Current extraction methods for cobalamins from serum influence the molecular characteristics of the vitamin. Therefore, an extraction procedure that leaves the cobalamins unchanged is needed.
Monoclonal antibodies towards transcobalamin (TC) and haptocorrin (HC) (in house) linked to magnetic microspheres were used for precipitation of the proteins. The cobalamins were subsequently released by degradation of TC or HC with subtilisin Carlsberg (EC.3.4.21.14).
Recovery for the extraction of (57)Co-cyanocobalamin ((57)Co-hydroxycobalamin) was higher than 99 (93)% and 92 (93)% from TC and HC respectively (n=10 (3)). No change in retention time and recovery was observed for (57)Co-hydroxycobalamin analyzed with HPLC before and after extraction with subtilisin Carlsberg. Endogenous cobalamin in serum was extracted from TC and HC and measured with routine methods. Enzymatic extracted cobalamin constituted 118-187% of that measured in serum with routine methods (n=16).
We present a method that allows extraction of cobalamins from their binding proteins without modifying the vitamin. The procedure may be useful especially for extractions aiming at characterizing the cobalamins bound to individual cobalamin binding proteins.
目前从血清中提取钴胺素的方法会影响维生素的分子特性。因此，需要一种能使钴胺素保持不变的提取方法。
使用与磁微球相连的抗转钴胺素蛋白（TC）和运钴胺素蛋白（HC）（自制）的单克隆抗体沉淀蛋白质。随后用嗜热栖热菌蛋白酶（EC.3.4.21.14）降解TC或HC来释放钴胺素。
从TC和HC中提取（57）Co-氰钴胺素（（57）Co-羟钴胺素）的回收率分别高于99%（93%）和92%（93%）（n = 10（3））。用嗜热栖热菌蛋白酶提取前后，通过高效液相色谱法分析（57）Co-羟钴胺素，其保留时间和回收率均未观察到变化。血清中的内源性钴胺素从TC和HC中提取出来，并用常规方法进行测量。酶促提取的钴胺素占常规方法测定血清中钴胺素的118 - 187%（n = 16）。
我们提出了一种从其结合蛋白中提取钴胺素而不改变维生素的方法。该方法可能特别适用于旨在表征与单个钴胺素结合蛋白结合的钴胺素的提取。
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