Glauner B
Max-Planck-Institut für Entwicklungsbiologie, Abteilung Biochemie, Tübingen, West Germany.
Anal Biochem. 1988 Aug 1;172(2):451-64. doi: 10.1016/0003-2697(88)90468-x.
About 80 different muropeptides, the subunits which comprise the polymer murein of Escherichia coli, were resolved by high-performance liquid chromatography. The muropeptides were released from isolated murein by complete digestion with muramidase from Chalaropsis spec. The separation method is based on reversed phase chromatography of the sodium borohydride-reduced compounds using ODS (C18) columns and a linear gradient elution with sodium phosphate buffer and methanol as organic modifier. The effect of temperature, pH, ionic strength, and the steepness of the gradient and of different support materials on the separation of the muropeptides was investigated. The new method represents a major improvement over previous methods with respect to resolution, sensitivity, and speed. Analytical as well as preparative separations can be realized. Quantitative analysis of murein composition is achieved by a linear gradient from 50 mM sodium phosphate, pH 4.31, to 75 mM sodium phosphate, pH 4.95, containing 15% methanol for 135 min on a 250 X 4.6 mm 3-micron Hypersil ODS column at 55 degrees C using a flow rate of 0.5 ml/min. With uv detection at 205 nm about 20 micrograms of murein per analysis is sufficient. The detection limit per compound is about 5 ng. A method for the evaluation of the analytical data allowing a convenient comparison of different muropeptide pattern is described.
通过高效液相色谱法分离出了约80种不同的微小肽，这些微小肽是构成大肠杆菌聚合物胞壁质的亚基。用来自Chalaropsis spec.的溶菌酶完全消化从分离出的胞壁质中释放出这些微小肽。该分离方法基于使用ODS（C18）柱对硼氢化钠还原的化合物进行反相色谱分离，并以磷酸钠缓冲液和甲醇作为有机改性剂进行线性梯度洗脱。研究了温度、pH值、离子强度、梯度陡度以及不同载体材料对微小肽分离的影响。与以前的方法相比，新方法在分离度、灵敏度和速度方面有了重大改进。可以实现分析分离和制备分离。通过在55℃下，以0.5毫升/分钟的流速，在250×4.6毫米3微米的Hypersil ODS柱上，使用从50毫摩尔/升pH 4.31的磷酸钠到75毫摩尔/升pH 4.95的磷酸钠，含15%甲醇的线性梯度洗脱135分钟，实现对胞壁质组成的定量分析。在205纳米处进行紫外检测时，每次分析约20微克胞壁质就足够了。每种化合物的检测限约为5纳克。描述了一种评估分析数据的方法，该方法便于比较不同的微小肽图谱。
