| Abstract: | 為應用一可準確分析蛋白質與GSH發生Mixed-disulfides(Glutathionation)的方法於動物實驗(In vivo)中,來分析當老鼠處於過氧化緊迫(Oxidative stress)時蛋白質的變化。本計畫利用等電點電泳法(Isoelectric focusing)可分離不同等電點蛋白質的特性,結合免疫西方墨點法(Immuno-Western blotting)的敏感性及專一性,提供一較傳統方法準確且省時的選擇。Carbonic anhydrase III(CA III)是一在公老鼠肝細胞中含量高且可發生Glutathionation的可溶性蛋白質,所以為本實驗研究對象,過氧化緊迫則分別以兩種廣效性殺草劑Paraquat(PQ)和Diquat(DQ)經腹腔注射誘發。首先,CA III利用兩步驟的DEAE-trisacryl M ion-exchange chromatography純化出,其純度最少95%。其次CA III抗血清則經由兔子來生成。經測試,此方法的靈敏度可達10ng。老鼠在經20或40mg/kg bw of PQ或85或170mg/kg bw of DQ腹腔注射5、15、40、120、240分後犧牲取樣。Thiobarbituric acid-reactive substances (TBARS)均於PQ或DQ注射後增加並顯現Time-dependent response。血漿Glutamate-pyruvate transaminase(GPT)活性,則僅於接受DQ處理的老鼠有增加,分別在低劑量注射後120分或高劑量注射後40分開始顯著上升(p<0.05)。肝細胞內GSSG濃度則不受PQ或DQ影響。至於GSH,在PQ高劑量處理40分鐘後,GSH濃度顯著較對照組為低(p<0.05),在DQ處理組,無論高或低劑量,則經120分鐘後,肝細胞內GSH濃度也顯著較低(p<0.05)。CA III Glutathionation在所有處理組中均無差異。由以上結果,顯示PQ和DQ確實能在肝細胞誘發氧化傷害,然而無法偵測到CA III Glutathionation的發生,可能是由於發生Dethiolation的緣故,就如同因GSH reductase作用使GSSG濃度無法因PQ或DQ的作用而增加。
A newly developed method which combining isoelectric focusing (IEF) and immunostaining is successful in analyzing protein-GSH mixed disulfides formation (glutathionation) under oxidative stress in an in vitro model. So far, several methods are widely used in determining protein mixed disulfides formation, such as radioisotope-based method and sodium borohydride reduction method, but they may limit in sensitivity, specificity, and/or be time consuming work. Therefore, this new method provides us an alternative and a powerful tool in this area. In present study, we try to apply this technique to measure carbonic anhydrase III (CA III)-GSH mixed disulfides formation in an in vivo model. Firstly, CA III was purified from rat liver by a two steps DEAE-trisacryl M ion-exchange chromatography and purity was over 95%. Then, CA III antiserum was produced by immunization of a rabbit with this purified CA III. As little as 10ng of CA III could be clearly identified. In animal experiment paraquat (PQ) and diquat (DQ) are served as oxidative stressors. Rats were given intraperitoneally either paraquat at 20 or 40mg/kg bw or diquat at 85 or 170mg/kg bw for 5, 15, 40, 120, and 240min., respectively, before sacrifice. PQ and DQ significantly increased the generation of thiobarbituric acid-reactive substances (TBARS) and showed a time-dependent response. The significant effect on plasma glutamate-pyruvate transaminase (GPT) was obtained in rats treated with DQ but not with PQ. Rats dosed with either 85 or 170mg/kg bw of DQ, GPT leakage was increased significantly (p<0.05) beginning at 120 and 40min respectively. Hepatic cellular GSSG contents were not affected of all treatments. However, hepatic reduced GSH contents was significantly decreased (p<0.05) in rats dosed with 40mg/kg bw of PQ beginning at 40 min and rats dosed with either dosage of DQ at 120 min. No change of CA III glutathionation was observed. Based on the effects of PQ or DQ on TBARS, results indicate that hepatic oxidative damage was actually generated. In this situation, a possible explanation of no detectable CA III S-glutathionation is probably due to the dethiolation, which similar to the role of GSH reductase on GSSG as seen in many reports. |
