鎰忎箟锛氱嚎绮掍綋杞阿閰?2 (Mitochondrial hydrogenase-2, TH-2) 浣嶄簬绾跨矑浣撶殑鍐呰啘涓婏紝鍙堢О涓虹嚎绮掍綋澶嶅悎浣撳叚锛屽偓鍖 NADH+ NADP+ 鍜 NAD++ NADPH 鐩镐簰杞寲锛岃皟鑺傜嚎绮掍綋 NAD(H) 鍜 NADP(H) 骞宠　銆 鎶婇€嗗悜鍙嶅簲绉颁负 TH-2锛屽偓鍖 NADPH 鍜 NAD+ 鐢熸垚 NADP+ 鍜 NADH銆侟/p>

鍘熺悊锛歂ADH 鍜 NADPH 鍧囧湪 340nm 鏈夌壒寰佸惛鏀讹紝鍥犳 TH 鍌寲鐨勮浆姘㈠弽搴斾笉鑳藉鑷 340nm 鍚稿厜搴﹀彂鐢熷彉鍖栥€傜敤浜哄伐鍚堟垚搴曠墿 3-涔欓叞鍚″暥鑵哄槍鍛や簩鏍歌嫹閰?APAD+)鏇夸唬 NAD+锛孴H-2 鍌寲 APAD+ 杩樺師鐢熸垚 APADH锛孉PADH 鍦 375nm 鏈夌壒寰佸厜鍚告敹锛屾祴瀹 375nm 鍏夊惛鏀剁殑澧炲姞閫熺巼锛屾潵璁 绠 TH-2 娲绘€с€侟/p>

妫€娴嬫柟娉曪細鍒嗗厜鍏夊害娉旤/strong>

浜у搧缁勬垚鍙婁繚瀛樻潯浠讹細

缂栧彿	瑙勬牸	鍌ㄥ瓨鏉′欢
AK256-A	50 mL脳1 鐡迭/p>	-20鈩冧繚瀛橈紱
AK256-B	25 mL脳1 鐡迭/p>	-20鈩冧繚瀛橈紱
AK256-C	25 mL脳2 鐡迭/p>	4鈩冧繚瀛樸€侟/p>
AK256-D	绮夊墏脳2 鏀?/p>	-20鈩冧繚瀛橈紱
AK256-E	绮夊墏脳2 鏀?/p>	-20鈩冧繚瀛橈紱

鈥 姝ｅ紡娴嬪畾鍓嶅姟蹇呭彇 2-3 涓鏈熷樊寮傝緝澶х殑鏍锋湰鍋氶娴嬪畾銆侟/p>

鑷鐢ㄥ搧锛欬/strong>

鍙鍒嗗厜鍏夊害璁°€佸彴寮忕蹇冩満銆佹按娴撮攨銆佸彲璋冨紡绉绘恫鍣ㄣ€?mL 鐜荤拑姣旇壊鐨裤€佺爺閽点€佸啺鍜岃捀 棣忔按銆侟/p>

鏍峰搧鐨勫埗澶囷細

缁勭粐銆佺粏鑿屾垨缁嗚優涓優娴嗚泲鐧戒笌绾跨矑浣撹泲鐧界殑鍒嗙

1. 鍑嗙‘绉板彇 0.1g 缁勭粐鎴栨敹闆 500 涓囩粏鑳烇紝鍔犲叆 1mL AK256-A锛岀敤鍐版荡鍖€娴嗗櫒鎴栫爺閽靛寑娴嗐€侟/p>

2. 灏嗗寑娴嗘恫 600g锛?鈩冪蹇 5min銆侟/p>

3. 寮冩矇娣€锛屽皢涓婃竻娑茬Щ鑷冲彟涓€绂诲績绠′腑锛?1000g锛?鈩冪蹇 10min銆侟/p>

4. 涓婃竻娑插嵆涓洪櫎鍘荤嚎绮掍綋鐨勮優娴嗚泲鐧斤紝鍙敤浜庢祴瀹氫粠绾跨矑浣撴硠婕忕殑 TH-2(姝ゆ鍙€夊仛)銆侟/p>

5. 姝ラ 4 涓殑娌夋穩鍗充负绾跨矑浣擄紝鍔犲叆 500uL AK256-B锛岃秴澹版尝鐮寸(鍐版荡锛屽姛鐜 20% 鎴 200W锛 瓒呭０ 3s锛岄棿闅 10 绉掞紝閲嶅 30 娆?锛岀敤浜 TH-2 娲绘€ф祴瀹氥€侟/p>

娴嬪畾姝ラ锛欬/strong>

1. 鍒嗗厜鍏夊害璁?閰舵爣浠鐑 30 min 浠ヤ笂锛岃皟鑺傛尝闀垮埌 375 nm锛岃捀棣忔按璋冮浂銆侟/p>

2. 宸ヤ綔娑茬殑閰嶅埗锛氫复鐢ㄥ墠鍙 AK256-C銆丏銆丒 鍚勪竴鏀紝灏 AK256-D銆丒 杞Щ鍒 AK256-C 涓贩鍚堟憾 瑙ｏ紝缃簬 37鈩?鍝轰钩鍔ㄧ墿)鎴 25鈩?鍏跺畠鐗╃)姘存荡 5min锛涚幇閰嶇幇鐢紱

3. 鍦 1mL 鐜荤拑姣旇壊鐨夸腑鎸夐『搴忓姞鍏ヤ笅鍒楄瘯鍓侟/p>

璇曞墏鍚嶇О	娴嬪畾绠 (渭L)
鏍峰搧	100
宸ヤ綔娑秉/p>	1000
娣峰寑锛岀珛鍗宠褰 375 nm 澶勫垵濮嬪惛鍏夊€ A1 鍜 10min 鍚庣殑鍚稿厜鍊 A2锛岃绠桙/p> 螖A=A2-A1銆侟/p>

TH-2 鍚噺璁＄畻鍏紡锛欬/strong>

(1)鎸夋牱鏈矞閲嶈绠桙/p>

鍗曚綅鐨勫畾涔夛細姣廹 缁勭粐姣忓垎閽熶骇鐢? nmol APADH 瀹氫箟涓轰竴涓叾娲绘€у崟浣嶃€侟/p>

TH-2 娲绘€ (nmol/min/g 椴滈噸) = [螖A脳V 鍙嶆€幻?蔚脳d)脳10⁹]梅(W脳 V 鏍访稸 鏍锋€? 梅T = 82脳螖A梅W

(2)鎸夋牱鏈泲鐧芥祿搴﹁绠桙/p>

鍗曚綅鐨勫畾涔夛細姣 mg 缁勭粐铔嬬櫧姣忓垎閽熶骇鐢 1 nmol APADH 瀹氫箟涓轰竴涓叾娲绘€у崟浣嶃€侟/p>

TH-2 娲绘€ (nmol/min /mg prot) = [螖A脳V 鍙嶆€幻?蔚脳d)脳10⁹]梅(V 鏍访桟pr)梅T = 164脳螖A梅Cpr

鈥 铔嬬櫧瀹氶噺妫€娴嬪缓璁娇鐢ㄦ湰鍏徃锛欱CA Protein Assay Kit (C05-02001)

(3)鎸夌粏鑿屾垨缁嗚優瀵嗗害璁＄畻

鍗曚綅鐨勫畾涔夛細姣? 涓囦釜缁嗚弻鎴栫粏鑳炴瘡鍒嗛挓浜х敓1 nmol APADH 瀹氫箟涓轰竴涓叾娲绘€у崟浣嶃€侟/p>

TH-2 娲绘€ (nmol/min /10⁴cell) = [螖A脳V 鍙嶆€幻?蔚脳d)脳10⁹]梅(500脳V 鏍访稸 鏍锋€? 梅T = 0.164脳螖A

娉細 V 鍙嶆€伙細鍙嶅簲浣撶郴鎬讳綋绉紝1.1脳10^-3L锛浳碉細APADH 鎽╁皵娑堝厜绯绘暟锛?.7脳10³L / mol /cm锛沝锛 姣旇壊鐨垮厜寰勶紝1cm锛沄 鏍凤細鍔犲叆鏍锋湰浣撶Н锛?.1mL锛沄 鏍锋€伙細鍔犲叆鎻愬彇娑蹭綋绉紝0.5mL锛汿锛 鍙嶅簲鏃堕棿锛?0 min锛汣pr锛氭牱鏈泲鐧借川娴撳害锛宮g/mL锛沇锛氭牱鏈川閲忥紝g锛?00锛氱粏鑿屾垨缁嗚優 鎬绘暟锛?00 涓囥€侟/p>