用紫外-可见光谱法测定酶动力学

酶活性经常在医学、生物化学和食品科学研究领域进行研究，以阐明反应发生的速率和酶-底物相互作用的亲和力。这些反应的速率可以用紫外-可见分光光度计准确地测量。Application Note Measurement of Enzyme Kinetics byUV-Visible Spectroscopy2/7Application Note Measurement of Enzyme Kinetics by UV-Visible Spectroscop y In tr od u c t ion Enzyme activ i ty i s frequently i nvest i gated in the med i c i nal， bioc h emistry， and food sc i ence researc h fields to eluc i date the r ate of wh i ch r eaction occurs and the affinity of the enzyme-substra t e i n teractions. The rates o f these reac ti ons can be accurately measured using a UV-Visib l e spect r ophotometer. When a n enzyme (E) binds wit h a subst r ate (S)， an intermediate or enzyme/substrate complex (ES) is prod u ced， wh i c h can f u r ther react and yie l d a by-product (P)， s h own i n Scheme 1. This biproduct can be quant i f i ed and observed by mo n itor i ng its maximum absorption wavelength. V-650 UV-Visi b le Spect r o p hoto m ete r The Micha e lis-Menten expression is commonly u sed to d e scribe the rate (v) of t h e enzyme r eaction. Wit h i ncreasi n g substrate concentrations [S]， the enzyme reac t ivity will asymptotically appro a ch its maximum velocity [V ]. T h e [K ] value is defi n ed b y t h e s ub strate co n cent r ation at hal f-maximum velocity Ⅴ/2. This [K ] value is referred to as the Michaelis-Menten constant. Based on th e eq u ation (1)， a saturat i on curve illus tr ated by a Michael i s-Me n ten plot i n Figu r e 2， describes the relation between the subst ra te conce n trat i on [S] a nd velocity [M . The[V]and[K ] v a lues ca n then b e deter mi ned by the plot of the s u bs tr ate concentrat i on and ve l oc i ty values derived from a series of exper i mental determina ti ons of the en z yme ac t ivity. Larger values of [V ] denote a higher maximum activity. T he [K ] value represents the af f inity of t he e n zyme f or the s u bst r a t e . Smal l er valu e s of [K ]i nd icate th a t th e enzyme and substrate are t ightly bound and f orm t h e ES complex mor e quick l y. On th e othe r h and， larger va lu es o f th e [K ] in dicate that t h e components are loos e ly bound a n d for m th e ES compl e x mor e slowly. Figure 1. Mi cha e l is -M e n t e n p lot. Determ i nation of the [Vo x ]and [K ]values by visual interpretation of the Michaelis-Men t en plot can result in errors for t h e kinet i cs values. Therefore， linearized plots that can be calcu l ated f rom variat i ons of equation (1) a r e prefer r ed to determine the va l ues. The L i neweave r -Burk， Hofstee， and Eadie plots are expressed as linear plots of t h e same data derived f r om the e n zyme kinetics react i ons . The [V]a n d [K ] va l ues can the n be determined by the slope and the i n tercepts of the vertical and h orizontal axes of these plots， offer i ng greater accu r acy. Taking t h e reciprocal o f the M i c h ael i s-Menten equat i on gives： Plotting 1/v as a fu n ction of 1/[S] is known as the Lineweaver-Burk plot (Fig u re 2). The slope (a) and intercept (b) of the li n ear curve is the n calcu l ated by the least-squares method and [V]and [Km ] values can be calculated by the fol l owing equa t ions ： Figure 2. L i n e w ea ver-B ur k p l o t . 28600 M a ry's Co u rt ， E a st o n ， M D 21601 US A USGO The Hofstee Plot is shown in Fig ur e 3 a n d is described b y the f ollowing equa ti on： where [S]/v is plotted as a function of [S]. The slope (a) a n d i n tercept (b) of the linear curve i s then calculated by the least-sq u ares method and [Vmax] and [Km] values ca n be ca l culated by the f ollowing equations： K=b.Vmax (7) Figure 3. Ho f stee p l ot. The Ead i e Plot i s shown in F i gu r e 4 and is described by t h e fol l owing equation： where [V] i s plotted as a f u n c t ion of v/[S]. The s l ope (a) and i n t ercept (b) of t h e line a r curve i s t h en ca l culated by t h e least -sq u ares method and [Vmax] and [Km] values ca n be ca l c u lated by the f ollowing equations： JASCO INC. 28600 M a ry's Co u rt ， E a st o n ， M D 21601 US A Figure 4. E a d i e p l o t . The Lineweaver-Burk plot is most useful whe n the enzymatic reaction h as a smaller su b strate concent r ation whi l e Hofstee plots are best used for l arger substrate co n cent r ations. Eadie plots ca n be used for a variety of substrate concen tr ations. When alkaline phosphatase (ALP) reacts with p-nitropheny l phosphate， i norgan i c phosphate and p-nitrophenol compounds are produced. The p-n i trophenol has a stable yellow color in an alkaline solution. This application note will describe how to determine t he substrate concent r ation， [S]， and velocity， v， using the absorption maximum of p-n itrophenol . Keyword s V-650，UV-Vi s i bl e/N I R， ca l ibra ti on c u rve ， quan ti t ative， Bio c h em i stry ， Kinet i cs ， Enz y me ， ST R -707 Water T her mo sta tt ed Cel l H o ld er wit h S t i rr er ， C S P-478 Sa m p l e h o l d e r Exper i mental The enzyme (0.0004 ug/mL of al k al in e phosphate i n 0.9 g/L of NaC I ) was mixed with a 0.1 M carbonate buffer solution (27.5 mg of Na2CO3 a n d 22.5 mg of NaHCO3) and 10 mM m agnesium c h loride to create t h e enzyme sol u tion. The initial p -nitrophenylphosphate substrate so l utions were 0.067，0.167， 0.333， 0.337， 0.833， 1.11， 1.67， 3.33 mmol /L and f inal concentrat i ons wer e 0.0067， 0.0167， 0.0333，0.0667， 0.0833， 0.111， 0.167， 0.333 mmol/L wh e n mixed wit h the enzyme solut i o n . To determine the absorpt i on maximum of p-nit r ophenol， 0.667 mM of the subst r ate solution was added to the enzyme solutio n . T h e absorption spectrum was measured after allowing t he reactants to react fo r 2000 seconds. The absorption maximum was observed at 402 nm (Figure 5). The ALP act i vity i n the subsequent experiments was determined by monitoring the 402 n m absorpt i on peak. JASCO INC. 28600 M a ry's Co u rt ， E a st o n ， M D 21601 US A A pp l i c a ti o n Lib r a r y： h t t p：//w ww .j a s co in c .c o m/ap pl i cat i o n s Figure 5. p-ni t roph e n ol ab so r pt i o n s p e c t r u m m e asu red a f ter 2000 se cond s . To exper i mentally determine the K and Vvalues， a b lank spectrum was measured with 1.5 mL of 0.1 M carbo n ate buffer solu t ion， 0.3 mL of 10 mM magnesium chloride sol u tion， and 0.9 mL of the enzyme solution in a 10 mm pathleng th cell. For the sample measurement， s h ortly after the sta r t of the measu r ement 0.067 mL of the subst r ate sol ut ion was i n jected into the sam p le cell . Both t he blank and sample measurements were repeated for di f f erent substra t e co n cent r ation s . Measurement Conditions Measurement Range 0-2000 seconds Data Interval 1 second Bandwidth 2.0nm Response Medium Wavelength 402 nm lemperature 37°C R esul t s Figure 6 shows the resul t s of the Time Cou r se measu r ements for eac h substrate concen tr ation. The gradient after t he sta r t of the react i on was calculated as the velocity， v， s h own i n Table 1. Figure 6. T i m e c o ur se m e as u re m e n t fo r v ary i n g s ub s t ra te co n ce n tr at ion s. 28600 M a ry's Co u rt ， E a st o n ， M D 21601 US A Table 1. Subs t ra te co n c e n tra t ions [S] and v e l o ci ty v. v abs/min 0.0067 0.004 0.0167 0.0333 0.0667 0.0833 0.111 0.039 0.167 0.048 0.333 0.061 The results were p lotted using the Michaelis-Mente n ， Li n eweaver-Burk， Hofstee， and Eadie algorit h ms， and the [Vmox]and [K] valu e s we r e calculated based on th e r e action k ine t ics. T h e se values are s h own in T able 2. For th e Micha el is -Men t e n val u es，[V]a n d[K ]values we r e i n itially ca l c u lat e d b y t he Ead i e p lot a n d the calculated valu e s were tak e n as t h e i n itial values. T he m eas u red v and calcul a ted v f rom equ a t i o n 1 we r e f itted by the least -squ a re method t o calculate the f ina l Vm ax and [K ] values. Table 2. R es u l ts o f t h e va r y i n g k i n et i c a n al y s es. Michaelis-Menten Lineweaver-Burk Hofstee Eadie max 0.0835 0.0815 0.0828 0.0818 K 0.1238 0.1179 0.1212 0.1187 Eqn. 0.0333 1/v=1.447×1/[S]+12.27 [S]/v=12.0721x[S]-1.4632 v=-0.01187xv/[S]+0.08185 28600 M a ry's Co u rt ， E a st o n ， M D 21601 US A Figure 7. The M i c h a e l i s -M en t en (A)， L in eweave r -B u r k (B )， H o f s t ee (C)， a nd Ead i e (D) p lo t s. 28600 M a ry's Co u rt ， E a st o n ， M D 21601 US A UASGO