Blanche F, Thibaut D, Couder M, Muller J C
Département de Chimie Analytique, Centre de Recherche de Vitry, Rhône-Poulenc Santé, Vitry-sur-Seine, France.
Anal Biochem. 1990 Aug 15;189(1):24-9. doi: 10.1016/0003-2697(90)90038-b.
After initial pretreatment for removal of interfering substances, corrinoid precursors of cobalamin from cultures of Pseudomonas denitrificans were separated by HPLC with a gradient elution system. In this system, all the following compounds are separated in their dicyano form, and retention times are given: cobyrinic acid; cobyrinic acid a-amide; cobyrinic acid c-amide; cobyrinic acid g-amide; cobyrinic acid a,g-diamide; cobyrinic acid c,g-diamide; cobyrinic acid a,c-diamide; cobyrinic acid a,c,g-triamide; cobyrinic acid triamide, tetraamide, and pentaamide isolated from P. denitrificans; cobyric acid; cobinamide; cobinamide phosphate; GDP-cobinamide; cyanocobalamin 5'-phosphate; and cyanocobalamin. Application of this HPLC method to culture samples of P. denitrificans revealed that in this microorganism the level of cobyrinic acid and cobyrinic acid monoamide is far lower than that of all other corrinoid precursors of cobalamin and suggested that (i) the (R)-1-amino-2-propanol group is incorporated only after completion of all the other amidations and (ii) the amidations follow only one sequence. The usefulness of this HPLC method was further demonstrated by identifying the 57Co-labeled corrinoid precursors of cobalamin accumulated by cobalamin-deficient mutants of Agrobacterium tumefaciens. A TLC system that separates the different corrinoid intermediates (in their dicyano form) and cyanocobalamin is also described.
在进行去除干扰物质的初始预处理后，利用梯度洗脱系统通过高效液相色谱法（HPLC）分离反硝化假单胞菌培养物中钴胺素的类咕啉前体。在该系统中，所有以下化合物均以其二氰基形式分离，并给出了保留时间：钴胺酸；钴胺酸α-酰胺；钴胺酸γ-酰胺；钴胺酸α,γ-二酰胺；钴胺酸c,γ-二酰胺；钴胺酸α,c-二酰胺；钴胺酸α,c,γ-三酰胺；从反硝化假单胞菌中分离出的钴胺酸三酰胺、四酰胺和五酰胺；钴胺酸；辅酶酰胺；辅酶酰胺磷酸酯；GDP-辅酶酰胺；氰钴胺素5'-磷酸酯；以及氰钴胺素。将该HPLC方法应用于反硝化假单胞菌的培养样品，结果表明在这种微生物中，钴胺酸和钴胺酸单酰胺的水平远低于钴胺素的所有其他类咕啉前体，并表明（i）（R）-1-氨基-2-丙醇基团仅在所有其他酰胺化反应完成后才被掺入，且（ii）酰胺化反应仅遵循一个顺序。通过鉴定根癌土壤杆菌钴胺素缺陷型突变体积累的57Co标记的钴胺素类咕啉前体，进一步证明了该HPLC方法的实用性。还描述了一种薄层层析系统，该系统可分离不同的类咕啉中间体（以其二氰基形式）和氰钴胺素。
