Weidner M F, Grenier D, Mayrand D
Groupe de Recherche en Ecologie Buccale, Faculté de Médecine Dentaire et Faculté des Sciences et de Génie, Université Laval, Québec, Canada.
Oral Microbiol Immunol. 1996 Apr;11(2):103-8. doi: 10.1111/j.1399-302x.1996.tb00343.x.
The aim of the study was to examine whether proteolytic artifacts, which result in a loss and poor resolution of protein bands, occur during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis of cellular proteins from selected proteolytic (Porphyromonas gingivalis, Prevotella nigrescens and Treponema denticola) and non-proteolytic (Fusobacterium nucleatum) bacteria. Conditions to limit or prevent proteolysis were also investigated. Bacterial cells were incubated in solubilizing buffer (SDS+ beta mercaptoethanol) at room temperature for various periods of time before boiling. A control assay consisted of trichloroacetic acid-treated bacterial cells. Cellular proteins were separated by electrophoresis and stained with Coomassie blue. Proteolysis occurred very rapidly in the case of P. gingivalis (< 30 s), whereas a longer incubation time (> 1 h) was required to observe similar effects in P. nigrescens and T. denticola. No proteolysis was observed for F. nucleatum. In all cases, heat (100 degrees C) and low pH (< 4) treatments of bacterial cells could avoid production of proteolytic artifacts. Incorporation of specific protease inhibitors before solubilization of bacteria could also prevent proteolysis. More particularly, N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), iodoacetamide and diisopropylfluorophosphate (50 mM) were highly efficient for P. gingivalis, P. nigrescens and T. denticola, respectively. When outer membranes of P. gingivalis were prepared in the presence of TLCK, numerous additional protein brands, not seen in the absence of TLCK, were detected. The present study suggests that specific protease inhibitors, effective in preventing proteolysis, should be identified and added during cell fractionation and protein purification procedures.
本研究的目的是检测在对来自特定的蛋白水解菌（牙龈卟啉单胞菌、变黑普雷沃菌和具核梭杆菌）和非蛋白水解菌（具核梭杆菌）的细胞蛋白进行十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳分析期间，是否会出现导致蛋白条带丢失和分辨率降低的蛋白水解假象。同时还研究了限制或防止蛋白水解的条件。在煮沸之前，将细菌细胞于室温下在溶解缓冲液（SDS + β-巯基乙醇）中孵育不同时间。对照试验由经三氯乙酸处理的细菌细胞组成。通过电泳分离细胞蛋白并用考马斯亮蓝染色。牙龈卟啉单胞菌的蛋白水解发生得非常迅速（<30秒），而变黑普雷沃菌和具核梭杆菌则需要更长的孵育时间（>1小时）才能观察到类似效果。未观察到具核梭杆菌有蛋白水解现象。在所有情况下，对细菌细胞进行加热（100℃）和低pH（<4）处理可避免产生蛋白水解假象。在细菌溶解之前加入特定的蛋白酶抑制剂也可防止蛋白水解。更具体地说，N-α-对甲苯磺酰-L-赖氨酸氯甲基酮（TLCK）、碘乙酰胺和二异丙基氟磷酸（50 mM）分别对牙龈卟啉单胞菌、变黑普雷沃菌和具核梭杆菌非常有效。当在TLCK存在的情况下制备牙龈卟啉单胞菌的外膜时，检测到许多在没有TLCK时未见的额外蛋白条带。本研究表明，在细胞分级分离和蛋白质纯化过程中，应鉴定并添加有效防止蛋白水解的特定蛋白酶抑制剂。
