Rodén L, Yu H, Jin J, Ekborg G, Estock A, Krishna N R, Livant P
Department of Biochemistry and Molecular Genetics, School of Medicine, University of Alabama at Birmingham 35294, USA.
Anal Biochem. 1997 Dec 15;254(2):240-8. doi: 10.1006/abio.1997.2398.
The Morgan-Elson method for quantitative N-acetylhexosamine analysis is a two-step procedure comprising alkali treatment of the sugar and subsequent condensation of the resulting chromogens with p-dimethylaminobenzaldehyde (Ehrlich's reagent) to yield a colored product. In the present investigation, the products formed in the first step of the procedure were analyzed by high-performance liquid chromatography (HPLC) on a reversed-phase (C18) column, which was eluted with a water-methanol gradient; the absorbance of the effluent was monitored at 229 nm. The profile generated from alkali-treated N-acetylglucosamine exhibited two major peaks, in a ratio of approximately 2.5:1, which accounted for 94% of the total peak area. A third peak, accounting for 3% of the peak area, was eluted in an intermediate position, and several smaller peaks were also observed. The three predominant components, isolated by preparative HPLC, all gave a purple color on addition of Ehrlich's reagent, indicating that they were Morgan-Elson chromogens. The HPLC profile of alkali-treated N-acetylmannosamine was identical to that of the products generated from N-actylglucosamine, as was expected because of the elimination of the asymmetry at C-2 during formation of the chromogens. N-Acetylgalactosamine yielded two major peaks, which were eluted in the same positions as the two major products formed from N-acetylglucosamine, but the intermediate peak seen in the N-acetylglucosamine pattern was absent. The HPLC procedure allowed detection of as little as approximately 25 ng of N-acetylglucosamine and may therefore be of value as an alternative to the complete Morgan-Elson procedure when only small amounts of sample are available for quantitative analysis.
用于定量 N- 乙酰己糖胺分析的摩根 - 埃尔森方法是一个两步程序，包括对糖进行碱处理，以及随后将所得发色团与对二甲氨基苯甲醛（埃利希试剂）缩合以产生有色产物。在本研究中，该程序第一步形成的产物通过高效液相色谱（HPLC）在反相（C18）柱上进行分析，用甲醇 - 水梯度洗脱；在 229 nm 处监测流出物的吸光度。碱处理后的 N - 乙酰葡糖胺产生的图谱显示出两个主要峰，比例约为 2.5:1，占总峰面积的 94%。第三个峰占峰面积的 3%，在中间位置洗脱，还观察到几个较小的峰。通过制备型 HPLC 分离出的三种主要成分，加入埃利希试剂后均呈现紫色，表明它们是摩根 - 埃尔森发色团。碱处理后的 N - 乙酰甘露糖胺的 HPLC 图谱与 N - 乙酰葡糖胺产生的产物图谱相同，这是预期的，因为在发色团形成过程中 C - 2 处的不对称性被消除。N - 乙酰半乳糖胺产生两个主要峰，其洗脱位置与 N - 乙酰葡糖胺形成的两种主要产物相同，但 N - 乙酰葡糖胺图谱中出现的中间峰不存在。HPLC 程序能够检测低至约 25 ng 的 N - 乙酰葡糖胺，因此当只有少量样品可用于定量分析时，作为完整的摩根 - 埃尔森程序的替代方法可能具有价值。
