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首页> 外文期刊>The protein journal >Striking Effects of Storage Buffers on Apparent Half-Lives of the Activity of Pseudomonas aeruginosa Arylsulfatase
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Striking Effects of Storage Buffers on Apparent Half-Lives of the Activity of Pseudomonas aeruginosa Arylsulfatase

机译：储存缓冲液对铜绿假单胞菌芳基硫酸酯酶活性的表观半衰期的显着影响

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To obtain the label enzyme for enzyme-linked-immunoabsorbent-assay of two components each time in one well with conventional microplate readers, molecular engineering of Pseudomonas aeruginosa arylsulfatase (PAAS) is needed. To compare thermostability of PAAS/mutants of limited purity, effects of buffers on the half-activity time (t (0.5)) at 37 A degrees C were tested. At pH 7.4, PAAS showed non-exponential decreases of activity, with the apparent t (0.5) of similar to 6.0 days in 50 mM HEPES, but similar to 42 days in 10 mM sodium borate with > 85 % activity after 15 days; protein concentrations in both buffers decreased at slower rates after there were significant decreases of activities. Additionally, the apparent t (0.5) of PAAS was similar to 14 days in 50 mM Tris-HCl, and similar to 21 days in 10 mM sodium phosphate. By sodium dodecyl-polyacrylamide gel electrophoresis, the purified PAAS gave single polypeptide; after storage for 14 days at 37 A degrees C, there were many soluble and insoluble fragmented polypeptides in the HEPES buffer, but just one principal insoluble while negligible soluble fragmented polypeptides in the borate buffer. Of tested mutants in the neutral borate buffer, rates for activity decreases and polypeptide degradation were slower than in the HEPES buffer. Hence, dilute neutral borate buffers were favorable for examining thermostability of PAAS/mutants.
机译：为了使用常规酶标仪在一个孔中每次获得两种成分的酶联免疫吸附测定的标记酶，需要铜绿假单胞菌芳基硫酸酯酶（PAAS）的分子工程设计。为了比较纯度有限的PAAS /突变体的热稳定性，测试了缓冲液对37 A时半衰期（t（0.5））的影响。在pH 7.4时，PAAS显示出活性的非指数下降，在50 mM HEPES中的表观t（0.5）近似为6.0天，但在10 mM硼酸钠中的表观t（0.5）在15天后具有> 85％的活性;活性显着降低后，两种缓冲液中的蛋白质浓度均以较慢的速率降低。此外，PAAS的表观t（0.5）类似于在50 mM Tris-HCl中的14天，和类似于在10 mM磷酸钠中的21天。通过十二烷基聚丙烯酰胺钠凝胶电泳，纯化的PAAS得到单个多肽。在37 A的温度下保存14天后，HEPES缓冲液中有许多可溶性和不溶性片段化多肽，但硼酸盐缓冲液中只有一种主要的不溶性而可忽略的可溶性片段化多肽。与HEPES缓冲液相比，在中性硼酸盐缓冲液中测试的突变体的活性降低速率和多肽降解速度较慢。因此，稀释的中性硼酸盐缓冲液有利于检查PAAS /突变体的热稳定性。

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