The heat sensitivity of cytokine-inducing effect of lipopolysaccharide
The heat sensitivity of cytokine-inducing effect of lipopolysaccharide
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DOI：
10.1189/jlb.1205738
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发表时间：
2006-08-01
影响因子：
5.5
通讯作者：
Tsan, Min-Fu
中科院分区：
文献类型：
--
作者：
Gao, Baochong;Wang, Yun;Tsan, Min-Fu
通过煮沸进行热灭活已被广泛用作确定所观察到的蛋白质制剂的影响是否是脂多糖（LPS）污染的结果的标准。然而，LPS烟碱诱导活性的热敏感性尚未被表征。在目前的研究中，我们证明了内毒素活性，即，鲎变形细胞裂解物凝胶活性和LPS（大肠杆菌K-12 JM 83、K-12 LCD 25和F583）的肿瘤坏死因子α（TNF-α）诱导活性对煮沸敏感。通过煮沸15分钟的热处理足以抑制类似于90%的LPS TNF-α诱导活性。热诱导的LPS活性失活不是煮沸的LPS粘附到容器壁上的结果，即，聚丙烯管，或煮沸的LPS的聚集。此外，煮沸的LPS保留了其结合多粘菌素B的能力。LPS中蛋白质（卵清蛋白）的存在不影响LPS的热敏性。相反，煮沸减少了LPS聚集体的大小，如通过使用天然聚丙烯酰胺凝胶电泳所确定的。同样地，二磷酰脂质A（DPLA）的TNF-α诱导活性也对煮沸敏感。煮沸的DPLA的薄层色谱分析显示，DPLA TNF-α诱导活性的热诱导失活不是其转化为单磷酰脂质A的结果。我们的结论是，TNF-α诱导活性的LPS和DPLA是敏感的沸腾，并建议热敏感性作为一个指标，是否观察到的影响的蛋白质制剂是LPS污染的结果应谨慎使用。J. Leukoc. 80：359-366;2006.
Heat inactivation by boiling has been widely used as a criterion to determine whether the observed effects of a protein preparation are a result of lipopolysaccharide (LPS) contamination. However, the heat sensitivity of LPS cytokine-inducing activity has not been characterized. In the current study, we demonstrated that the endotoxin activity, i.e., Limulus amebocyte lysate-gelating activity, and the tumor necrosis factor a (TNF-alpha)-inducing activity of LPS (Escherichia coli K-12 JM83, K-12 LCD25, and F583) were sensitive to boiling. Heat treatment by boiling for 15 min was sufficient to inactivate similar to 90% of the LPS TNF-alpha-inducing activity. The heat-induced inactivation of LPS activities was not a result of adherence of boiled LPS to the wall of the container, i.e., polypropylene tubes, or aggregation of boiled LPS. In addition, boiled LPS retained its ability to bind polymyxin B. The presence of protein (ovalbumin) in LPS did not affect the heat sensitivity of LPS. Conversely, boiling reduced the size of LPS aggregates as determined by electrophoresis using native polyacrylamide gel. Likewise, the TNF-a-inducing activity of diphosphoryl lipid A (DPLA) was also sensitive to boiling. Thin-layer chromatographic analysis of boiled DPLA revealed that the heat-induced inactivation of DPLA TNF-a-inducing activity was not a result of its conversion to monophosphoryl lipid A. We conclude that the TNF-alpha-inducing activity of LPS and DPLA is sensitive to boiling and suggest that heat sensitivity as an indicator of whether the observed effects of a protein preparation are a result of LPS contamination should be used with caution. J. Leukoc. Biol. 80: 359-366;2006.