英语翻译请不用机译Measurement of protease activity by azocasein assay

英语翻译
请不用机译
Measurement of protease activity by azocasein assay
The assays were conducted as described previously (Scholze and Tannich 1994) with some minor modifications.Briefly,parasitic lysates were preincubated with 2 mM dithriothreitol (DTT) (Sigma) at 37°C for 10 min to activate protease activity.100 μl of 5 mg/ml azocasein (Sigma) solution was prepared in PBS (pH 7.4) and incubated with 100 μl of parasitic lysate for 1 h at 37°C.Reaction was stopped by adding 300 μl of 10% trichloroacetic acid and samples were incubated on ice for 30 min.Undigested azocasein was removed by centrifugation (5,000×g,5 min) and the resulting supernatant was transferred to a clean tube containing 500 μl of NaOH (525 mM).Absorbance was measured at 442 nM on a spectrophotometer (Tecan Magellan).For controls,lysates were boiled at 90°C for 15 min to inactivate proteases and 100 μl trypsin (2.5 mg/ml) was used as a positive control.
Analysis of proteases by nondenaturing gelatin-SDS-PAGE
This SDS-PAGE method requires the inclusion of a protein,usually gelatin,in an acrylamide gel.Gelatin is digested by proteases after electrophoresis and clear bands are seen after staining the gel with Coomassie Brilliant blue.Cell lysate (40 μg) of B isolate was analyzed by gelatin-SDS-PAGE for protease activity (Lockwood et al.1987) using the SDS-discontinuous buffer system of Laemmli (1970).The acrylamide concentration of resolving and stacking gel was 12.5 and 5%,respectively.Briefly,0.2% (w/v) gelatin (Sigma) was copolymerized into the resolving gel.Samples were not boiled so that enzymatic activity of the proteases upon renaturation would not be lost.After electrophoresis at a constant current of 30 mA/gel for 1.5 h,gels were immersed with continuous shaking for 1 h in 2.5% (v/v) Triton X-100 to remove the SDS and to allow the proteases to become active.The proteinases bands were developed by immersing the gels in incubation buffer containing 1 mM DTT for 3 h at 37°C.The bands were visualized by staining in 0.12% (w/v) Coomassie Brilliant blue R-250 for 30 min.Cell lysate was also analyzed by 12.5% gelatin-containing native gel (without SDS) to determine the number of proteases present and to investigate whether the proteases were single/multiple subunits.
Inhibition of proteases
To determine the type of proteases that are present in Blastocystis,effects of inhibitors on the activity of Blastocystis proteases were studied by incubating Triton-treated gels in incubation buffer with 1 mM DTT containing one of the following inhibitors:iodoacetamide (IA),EDTA,N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK),8-hydroxyquinoline,phenylmethylsulfonyl fluoride (PMSF) (all 1 mM); pepstatin A,leupeptin or antipain (all 100 μg/ml).All inhibitors were purchased from Sigma except pepstatin A (CHEMICON),IA (MP Biomedicals,LLC) and PMSF (BDH).Gels were stained with Coomassie Brilliant blue.
For azocasein assays,similar concentrations of protease inhibitors (IA,TLCK,pepstatin A,EDTA) were added during a 1-h incubation of parasitic lysate with azocasein.
Determination of pH optimum
The pH dependence of Blastocystis protease activity was investigated by incubating Triton X-100 treated gels in buffers of different pH ranging from 3.0 to 8.5.The following buffers were used:0.1 M glycine/HCl (pH 3.0),0.1 M sodium acetate/acetic acid (pH 4.0 and 5.5),0.1 M sodium phosphate (pH 7.0 and 7.6),and 0.1 M Tris–HCl (pH 8.5).All incubation buffers contained 1 mM DTT and gels were stained with Coomassie Brilliant blue.For azocasein assay,a 1-h incubation with parasitic lysate was performed in buffers of different pH containing 1 mM DTT.
本人仅在此先谢过上回黎明晴空所做的精彩回答!

用固氮酪蛋白分析测定的蛋白酶活性
像之前所说的(Scholze and Tannich 1994)一样再通过一些小小的修饰就可以进行分析了.简单地讲,就是寄生虫溶菌液加入2mMDDT 在37℃保温10分钟,以激活蛋白酶的活性.准备100 μl （5 mg/ml）的固氮酪蛋白溶液,加入100 μl 的寄生虫溶菌液在37℃保温1个小时.加入300 μl 10%的三氯乙酰酸来终止反应,然后将样品在冰中保温30分钟.通过离心(5,000×g,5 min)除去未被消化的固氮酪蛋白,将剩下的上清液转移到已加入500μ(525 mM) NaOH的干净试管中.用分光光度计(Tecan Magellan)测定其在442 nM处的吸光度.为了便于控制,将溶菌液在90℃保温15分钟以灭活酶活性,然后
加入100 μl胰蛋白酶(2.5 mg/ml)用以正调节.
明胶—SDS—PAGE分析蛋白酶
这种SDS—PAGE需要蛋白溶液和明胶,而明胶通常用丙烯酰胺凝胶.电泳之后,蛋白质会侵入明胶,用Coomassie Brilliant蓝染色后会出现多条清晰的带.用—SDS—PAGE分析分离物B的细胞消化液(40 μg)的蛋白酶活性(Lockwood et al.1987)要用到Laemmli (1970).的SDS—非连续缓冲系统.凝胶分解和分层后的丙烯酰胺浓度分别是12.5 and 5%.简而言之,0.2% (w/v)的明胶能聚合成有分解作用的凝胶.这样样品就不用加热,蛋白酶也就不会失去酶活性了.电泳1.5小时(30mA 的直流电)后,加入2.5% (v/v) Triton X-100不断的轻摇凝胶1小时,这样做不但能除去SDS,还可以激活蛋白酶的活性.将凝胶浸入已加了1 mM DTT的缓冲液中在37℃保温3个小时,蛋白酶带将逐渐展开.用 0.12% (w/v) Coomassie Brilliant 蓝 R-250 染色 30分钟这些蛋白酶带就能显现出来了.还可以用12.5%的明胶--天然的凝胶（不含SDS）分析细胞溶菌液,确定蛋白酶的数目,同时研究它有几个亚基.
蛋白酶的抑制作用
可以通过将处理过Triton的凝胶放入含有1 mM DTT的保温的缓冲液保温来研究抑制剂对酵母菌蛋白酶活性的影响,确定酵母菌中蛋白酶的种类,DDT中含有下列抑制剂的一种：IA,EDTA,TLCK,8--羟喹啉 ,PMSF（都是1mM）；胃蛋白酶抑制剂A或者亮肽酶素,抗痛素（都是100 μg/ml）.除了胃蛋白酶抑制剂A,IA和PMSF外所有的抑制剂都可以从Sigma买到.凝胶可以用Coomassie Brilliant蓝染色.
问题补充：分析固氮酪蛋白时,在含有固氮酪蛋白的保温1小时寄生虫溶菌液加入相同浓度的蛋白酶抑制剂（IA,TLCK,胃蛋白酶抑制剂,EDTA）.
最适PH的测定
酵母菌蛋白酶活性与PH的关系可以通过在将处理过Triton X—100的凝胶放在PH（在PH3.0—8.5的范围内）不同的缓冲液中保温来测得.缓冲液的配制如下：0.1 M甘氨酸/HCl (pH 3.0),0.1 M乙酸钠/醋酸(pH 4.0 and 5.5),0.1 M磷酸钠(pH 7.0 and 7.6),and 0.1 M Tris–HCl (pH 8.5).所有的保温缓冲液都含有1 mM DTT,因此可用Coomassie Brilliant蓝染色凝胶.对于固氮酪蛋白的分析,可以用寄生虫溶菌液在含有1 mM DTT不同的PH保温1小时.