1、DB61 1i f 1*DB61/T 413-2007 t I 2007-09-03 2007-09-03 DB611T 413-2007*tiFftJ:f:llJ tA*,H1HjL*ftJ:f:lAMmA.m*acQ*ftN:J:f:lAmA.Q I*ftA:*WWQ*tjf,ftJktt;fpQ I I I i I I I!i i !j I I!,J l;i I I DB61fT 413-2007 t f I t l 1;:$:tift/fUJI!7liffWlulE11A*4*D-:3jZ*mUfJiitlr1Jo*fiftttE m mTiX*4*D-1f:*itW!IJlEo 2
2、tJUiz31 iJ Imjc14 r:yux1LI=*iJjffiJi*tittEiJ iJ!).trnfi JjJ;:$:t;j;ttEiJ,o JLM:r:E BjtJjiJ iJ!J+1 ft:,;ltPiQfr JJi:tHi1.a.(iJ).rrmmT*ttE,W,.a.M*ttEiJ*MEJ1ffli3:X11:iJ1iifA&;:$:o fLfikff BWHfJtrmfll:,;ltiid#rA&;:$:JffJ:rFf;:$:flFttEo EN 12138:;J.*UJlirtcpD-1ji:Il1ttiJlifW!1E.D-MDH D1jZ*llil1i1i:lll?t
3、I.NAD:tj!JfiIi,*-A1rllil NADH f3-:li!Iw:-nut-:i%tfllil U mIJ lF Jfl-f.fr.,if 25C iJ fij It14:ftJ=5 1 U iJ mit.$tf ft PX;1 mol iJ0 A 11&7tf1tiEi:C r9tR*JJt hepes 4-f5:z.IJJ&z:.jliml I J 4 JliHf D-1jL (D-*tik)tD1jL*1i1i:JW(D-MDH)HiiJt5r.F,i!liJiNADf:,7lP=mz.tiko lltmz.lliliiL0:liP EE ffiJtf iJtft?Hff.
4、PX;jAIiil1i1i:*11.:ftfWt 0 D-MDH,dec arbJ)D-*1i1i:+NAD.p;jm11i1i:+CO:c+NADH+H NADH iJ,ffi D-1jL*!IiiJ,:!tit11:0 II NADHiJid:rn tE334,340!&365nm4!r iJ1l&7tJJEi:iJ-f:tJYJ3HL tI I5 lit1fIJ f 5.1 hepes 5.2 NAD 5.3 D-MDHI!5.4 NaClI!5.5 KCl I DB61!T 4132007 5.6 Na2HP04.2H2O 5.7 lim.5.8 HCl 5.9 NaOH 5.10 K
5、OH 5.11:m:*5.12 1Bj:ijitffl jij&litillj 6 iii6.1 nRl#t(5 mL,1 mL 50-200ILL 10-50IlU 6.2 t.nn.:7:;1.0em 6.3 91*:7:;Jlit,r:iJSiJt*,tE 340nm JIJ:AE 6.4 jJf:ll,11 10 11m 6.5 Ji IL,ll 6.6 ILi tf,jtIL n;*ftG;,J.&JIii 7 jmIJ-WiJJ 7.1 lit1f1J ililj 7.1.1 hepes;,*,j 190 mL:m:*r:p:5t!tlIJ:JJO/0.8 g NaCL 0.037
6、 g KCL 0.0135 g NazHPO,2H20,0.1 g 1i1rfJ,0.5 g hepes,3EJhfftJfi,J.j:j NaOH pH 7.05,100mlo 7.1.2 NAD I1-t:Hi ImL l1iJ*mflllf.52.5rng f1)&P:1J NAD T1t:,-15C-20C1:*tfo 7.1.3 D-MDH I1-t:Rl 0.6 mL.ffi:*?$M 8U Er-Jf;f)P;k)D1ji:lti:lHl;:Jmtl.!W.I1t,-15C-20C1*#o 7.1.4 HCl:2 mollL 7.1.5 NaOH:2mollL 7.1.6 KOH
7、:2mollL 7.2 litt$ililji7.2.1 1Ct;t J+j KOH llWl 25rnl m:H pH fll 78.:J.mm:l.JiJj.1im.1lJ:H 1.000 L 800 mL iJ11ft:i1I!uo 7.2.2;!ll=*5t*$&:i1*1!l:!.ffRn,!iH:,lfXLm 25ml.mKOH llWl pH 7-8,:JJQM!*1imff,1fJtJ L 000 1.800 mL;IJtiJ1lJt:W 0 7.2.3*CO2 f*=*jtf$&:t:ffr iJ CO1*,M:JJQ NaOH l KOH.i)aJ,i pH 7-8.Jtf
8、f.kg 15?t.1fJH 1.000 1.800 mL);iJ11Tt:!0 7.3 f&JIJ(t IltHT)7.3.1 ii:S)!1 2 1 DB61ff 413-2007 liUlITI.,*iXJJQA 1.OOOmL hepes tjftP,0.100mL NAD 11F 1.800 mL:m:7l,$15-i:6 7t)IttJllUtJ3tfll(AI),:PJJIlA O.050 mL D-MDH I.ff,M$15-&)SZ5EJ&;C:k11 20:7t),IttIJ&Ytlim(A.),aIJ&Ytlim&=(A2-AI)280 7.3.2 JIJ Itt!tlI
9、TI.,*iXJJIlA 1.OOOmL hepes tjl1l,O.100mL NAD I.fF,0.100 mL ff,1.700 mL 1t7l$ili:6:7t)iiIttIJ&Ytltm(AI),:pJJJIlA 0.050 mL D-MDH I.fF,M$1r&)SZ5EPX:(*11 20:7t).IttIJ&Ytltm(A.),ffIJ&Ytlim&=(A.-AI)I,lbo 1 i9!IJJ JJIlA!tellIla9m hepes tjH NADI.fF ffm:X7l aX1 ff;fig 1.000mL 1.000mL 0.100mL 0.100mL-0.100mL
10、1.800mL 1.700mL$iI.*1 6:7tJw;IttIJ&YtJ.tm(AI)o JJIlAfmJ7f&)sz 0.050mL 0.05OmLD-MDH I.ff:7tJJlJ;Jldffaa9IJ&Ytltm&,fflffIJ&Ytltm&aIJ&Ytlim.&Iij1tJJIj0():l:.A=(A2-AI)11iI.-(ArAI)213.(1)lI)a9IJ&Ytltf.a&)SZ*-f-0.100.1fg1tJJUl1i1iJrla9*o 8 it.:ftHt!l0(2)it_m&:V/XMW c=-X L1 A X f.(2)eXdXV2 X1000*:1 C;fiWoJ
11、9ta9mffl:giLI;fimlHJ9tIJ*fFJ9t:l:o D-1fl*D1f:fFJ9t:l:134.09 g/mo1,D1fl*D.!f:1f:fF MW 132.07g/mol VI!tlITI.*.m.IJ,.V2 fflJffm.IJA,.a f ffm.a9ffr d!tlITI.IJYt;f.!i!,J(!ffLa em e NADPHlJmYt:.:tE 340 nm:=6.3 1 x mmor1 x eml 3 DB61rr 4132007 9 D-:!jfUmIJP-ttr.Jlitfi 9.1 DMm.-*tr.JD*m.9.2 tr.JJ2i:m 9.2.1;
12、tfi;fjQ)HfiIJ&J2i:f*r:p 9.2.2:!:$JftHEiilHY;E:=:&J2i:ffrfnH&JlZAA*,A2ffiBIl,tm O.OSOmL R1mtf:ljnIJ:f.&nBirrp,&Iltf;ltJ(Omin),.OO(Ar)*W*&,JlZ.fim.&m.&m*:!Ju.9.2.3 m,.M*li.,ffMMrpNW.tto*2 iiti1 R1m hepes J1fl A 1.000mL ff:rfn 1.000mL fjf(ff.-1.000mL t+Mt+*$f1 1.000mL NADTft=;1&O.lOOmL O.lOOmL O.lOOmL
13、O.lOOmL nu-nn.O.lOOmL O.050mL)JfHH&O.lOOmL O.050mL 1.800mL 1.700mL 1.700mL 1.700mL itU3ft.-x 100%.(3)Ll A/,iff 4 I t DB61tr 413-2007 fIJfj:iF;A t(*4t11Jfj:iF;);iJ:*J!q f)2,ff1litlS9fltitP,!WJtU:%TJ5IlffT 2C-SC.i1!ic1li1jpxS9 NAD IfttE 2C-8CPJ)E fr I=ffd,eX;tE-15C-25C*fttrPJ1*ff.=:1-Ji Q i1!iC1IitlnZS9 D-MDH ItFtE 2C8CPJ)E 5:R.i)(.I I I f j A:ffS9,m:*,:ffSmS9.1 I I 5,tLj!j9JUJtU7tJJtflIt!HAi8/J(tLOO.100),)JlIJ:ffmJ5Ilm:mr5ltJl,!PtE 7.3.2 J)EJ:f:lilJ;lJo I 1!I I I I I I J t I,I I II i I f,r,t tI!i I!,!5 I I