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檸檬酸合酶(CS)活性檢測試劑盒

CS（EC 2.3.3.1）廣泛(fan)存在于動物、植物、微(wei)生物和(he)(he)培養細胞的線粒體(ti)基(ji)質中，是三羧酸循(xun)環個限(xian)速酶，是三羧酸循(xun)環主要調(diao)控位(wei)點(dian)之一。 CS 催化(hua)乙酰 CoA 和(he)(he)草酰乙酸產(chan)生檸檬(meng)酰輔酶 A，進(jin)一步水解產(chan)生檸檬(meng)酸；該(gai)反應(ying)促使無(wu)色的 DTNB 轉變成黃色的 TNB，在 412nm 處有特征(zheng)吸(xi)光值。

檸檬酸合酶(CS)活性檢測試劑盒

組織、細菌(jun)或細胞中胞漿蛋白與線粒體(ti)蛋白的分(fen)離： ① 稱取(qu)約 0.1g 組織或收集 500 萬細胞，加(jia)入 1mL 提(ti)取(qu)液和 10μL 試劑(ji)(ji)一，用冰浴勻漿器或研缽(bo)勻漿。 ② 將勻漿液 600g，4℃離心 5min。 ③ 將上清(qing)液移另一離心管中，11000g，4℃離心 10min。 ④ 上清(qing)液即(ji)胞漿提(ti)取(qu)物，可用于測定從線粒體(ti)泄漏的 CS。 ⑤ 在沉(chen)淀中加(jia)入 200μL 試劑(ji)(ji)一和 2μL 試劑(ji)(ji)二，反復吹打(da)充分(fen)混勻，用于 CS 測定。 ⑥ 檸檬酸(suan)合酶酶活即(ji)沉(chen)淀和上清(qing)液中的酶活之和。

1、組(zu)織(zhi)中(zhong) CS 活(huo)力(li)的(de)(de)計算(suan): （1）按(an)(an)樣(yang)本(ben)(ben)蛋(dan)(dan)(dan)白(bai)濃(nong)(nong)度計算(suan)：單(dan)位(wei)(wei)的(de)(de)定(ding)義：37℃或(huo) 25℃下(xia)(xia)每(mei) mg 組(zu)織(zhi)蛋(dan)(dan)(dan)白(bai)在反(fan)應(ying)體(ti)系(xi)中(zhong)每(mei)分(fen)(fen)鐘催化產(chan)(chan)生 1 nmol TNB 定(ding)義為(wei)一(yi)個(ge)(ge)酶活(huo)力(li)單(dan)位(wei)(wei)。 CS 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)（U/mg prot）=ΔA 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)÷ε÷d×V 反(fan)總(zong)(zong)÷（Cpr 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)×V 樣(yang)本(ben)(ben)）÷T=1050×ΔA 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)÷Cpr 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing) CS 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)（U/mg prot）=ΔA 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)÷ε÷d×V 反(fan)總(zong)(zong)÷（Cpr 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)×V 樣(yang)本(ben)(ben)）÷T =1050×ΔA 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)÷Cpr 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian) CS（U/mg prot）=CS 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)+CS 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)=1050×ΔA 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)÷Cpr 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)+1050×ΔA 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)÷Cpr 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)。（2）按(an)(an)樣(yang)本(ben)(ben)鮮重(zhong)計算(suan)：單(dan)位(wei)(wei)的(de)(de)定(ding)義：37℃或(huo) 25℃下(xia)(xia)每(mei) g 組(zu)織(zhi)在反(fan)應(ying)體(ti)系(xi)中(zhong)每(mei)分(fen)(fen)鐘催化產(chan)(chan)生 1 nmol TNB 定(ding)義為(wei)一(yi)個(ge)(ge)酶活(huo)力(li)單(dan)位(wei)(wei)。 CS 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)（U/g 鮮重(zhong)）=ΔA 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)÷ε÷d×V 反(fan)總(zong)(zong)÷（W×V 樣(yang)本(ben)(ben)÷V 提(ti)取(qu)）÷T =1050×ΔA 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)÷W CS 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)（U/g 鮮重(zhong)）=ΔA 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)÷ε÷d×V 反(fan)總(zong)(zong)÷（W×V 樣(yang)本(ben)(ben)÷V 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)）÷T =212×ΔA 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)÷W CS（U/g 鮮重(zhong)）=CS 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)+CS 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)=1050×ΔA 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)÷W+212×ΔA 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)÷W。 2、細(xi)菌或(huo)培養(yang)細(xi)胞(bao)中(zhong) CS 活(huo)力(li)的(de)(de)計算(suan): （1）按(an)(an)樣(yang)本(ben)(ben)蛋(dan)(dan)(dan)白(bai)濃(nong)(nong)度計算(suan)：單(dan)位(wei)(wei)的(de)(de)定(ding)義：37℃或(huo) 25℃下(xia)(xia)每(mei) mg 蛋(dan)(dan)(dan)白(bai)在反(fan)應(ying)體(ti)系(xi)中(zhong)每(mei)分(fen)(fen)鐘催化產(chan)(chan)生 1 nmol TNB 定(ding)義為(wei)一(yi)個(ge)(ge)酶活(huo)力(li)單(dan)位(wei)(wei)。 CS 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)（U/mg prot）=ΔA 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)÷ε÷d×V 反(fan)總(zong)(zong)÷（Cpr 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)×V 樣(yang)本(ben)(ben)）÷T=1050×ΔA 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)÷Cpr 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing) CS 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)（U/mg prot）=ΔA 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)÷ε÷d×V 反(fan)總(zong)(zong)÷（Cpr 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)×V 樣(yang)本(ben)(ben)）÷T=1050×ΔA 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)÷Cpr 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian) CS（U/mg prot）=CS 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)+CS 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)=1050×ΔA 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)÷Cpr 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)+1050×ΔA 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)÷Cpr 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)。（2）按(an)(an)細(xi)菌或(huo)細(xi)胞(bao)密度計算(suan)：單(dan)位(wei)(wei)的(de)(de)定(ding)義：37℃或(huo) 25℃下(xia)(xia)每(mei) 1 萬個(ge)(ge)細(xi)菌或(huo)細(xi)胞(bao)在反(fan)應(ying)體(ti)系(xi)中(zhong)每(mei)分(fen)(fen)鐘催化產(chan)(chan)生 1 nmol TNB 定(ding)義為(wei)一(yi)個(ge)(ge)酶活(huo)力(li)單(dan)位(wei)(wei)。 CS 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)（U/104 cell）=ΔA 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)÷ε÷d×V 反(fan)總(zong)(zong)÷（500×V 樣(yang)本(ben)(ben)÷V 提(ti)取(qu)）÷T=2.1×ΔA 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing) CS 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)（U/104 cell）=ΔA 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)÷ε÷d×V 反(fan)總(zong)(zong)÷（500×V 樣(yang)本(ben)(ben)÷V 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)）÷T =0.42×ΔA 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian) CS（U/104 cell）=CS 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)+CS 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)=2.1×ΔA 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)+0.42×ΔA 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)。第(di) 3 頁 共 3 頁 ε：TNB 的(de)(de)消光(guang)(guang)系(xi)數，13.6×10-3mL/(nmol·cm)；V 反(fan)總(zong)(zong)：反(fan)應(ying)體(ti)系(xi)總(zong)(zong)體(ti)積(ji)，0.2 mL；d：比色皿(min)光(guang)(guang)徑，1cm；V 樣(yang)本(ben)(ben)：加入的(de)(de)樣(yang)本(ben)(ben)體(ti)積(ji)，0.007mL；V 提(ti)取(qu)：提(ti)取(qu)液體(ti)積(ji)，1mL；V 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)：溶解沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)的(de)(de)總(zong)(zong)體(ti)積(ji)，0.202mL；T：反(fan)應(ying)時間，2min；Cpr 上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)：上(shang)(shang)(shang)(shang)(shang)(shang)(shang)清(qing)(qing)(qing)液的(de)(de)蛋(dan)(dan)(dan)白(bai)濃(nong)(nong)度，mg/mL；Cpr 沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)：沉(chen)(chen)(chen)(chen)(chen)(chen)淀(dian)(dian)(dian)(dian)(dian)溶解后的(de)(de)蛋(dan)(dan)(dan)白(bai)濃(nong)(nong)度, mg/mL；W：樣(yang)本(ben)(ben)鮮重(zhong)，g。