Baggenstoss Bruce A, Weigel Paul H
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, 73104, USA.
Anal Biochem. 2006 May 15;352(2):243-51. doi: 10.1016/j.ab.2006.01.019. Epub 2006 Jan 31.
Size exclusion chromatography-multiangle laser light scattering (SEC-MALLS) analyses of Escherichia coli membranes expressing Streptococcus equisimilis hyaluronan synthase (seHAS) demonstrated an inherent artifact (10-100 MDa) that coeluted with hyaluronan (HA) and skewed the apparent weight-average mass of HA to erroneously high values. Briefly heating samples to 65-75 degrees C eliminated this artifact and increased the yield of recovered HA due to the release of HA chains that were attached to membrane-bound HAS. Inclusion of alkaline phosphatase, which removed uridine 5'-diphosphate (UDP) produced during the reaction, improved the linearity of HA synthesis-even at high substrate use. Surprisingly, the addition of EDTA, to chelate Mg(2+) ions, did not completely stop the HAS reaction at 30 degrees C or at 4 degrees C. The best conditions for stopping the reaction without altering SEC-MALLS profiles of the product HA were to chill samples on ice in the presence of both EDTA and UDP. Even with excess substrate, the maximum size of product HA decreased as the enzyme concentration increased. Therefore, the maximum HA size made by HAS was determined by extrapolation to zero enzyme concentration. Using the above conditions, membrane-bound seHAS synthesized a cohort of HA products that steadily increased in weight-average molar mass, reaching a final maximal steady-state size of 4 to 6 MDa within 2-4 h.
对表达马链球菌透明质酸合酶（seHAS）的大肠杆菌膜进行尺寸排阻色谱 - 多角度激光光散射（SEC - MALLS）分析，结果表明存在一种固有假象（10 - 100 MDa），它与透明质酸（HA）共洗脱，并使HA的表观重均质量偏向错误的高值。将样品短暂加热至65 - 75摄氏度可消除此假象，并由于附着在膜结合HAS上的HA链的释放而提高回收HA的产量。加入碱性磷酸酶可去除反应过程中产生的尿苷5'-二磷酸（UDP），即使在高底物用量时也能改善HA合成的线性。令人惊讶的是，添加EDTA以螯合Mg(2+)离子，在30摄氏度或4摄氏度时并未完全停止HAS反应。在不改变产物HA的SEC - MALLS图谱的情况下停止反应的最佳条件是在EDTA和UDP存在下将样品在冰上冷却。即使底物过量，产物HA的最大尺寸也会随着酶浓度的增加而减小。因此，通过外推至零酶浓度来确定HAS产生的HA的最大尺寸。使用上述条件，膜结合的seHAS合成了一组HA产物，其重均摩尔质量稳步增加，在2 - 4小时内达到最终最大稳态尺寸4至6 MDa。
