Jeffrey A. Hurlbut,' G. Reza Kavianian, Sun Y. Lee, Kern L. Nuttall, Samuel R. Gentry, and Terry L. Hassman Metrooolitan State Colleae " Denver. Colorado 80204
Enzyme Activity Experiments Using a Simple Spectrophotometer
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M a n y enzyme activity experiments involve t h e use of an expensive spectrophotometer (I, 2), a n d a s a result, these exoeriments a r e imoractical t o use i n either laree or lower division biochemistry classes. Since we frequently have 40 or more students i n our hiochemistrv sections. we decided t o develop a series of enzyme activity experiments using Spectronic 20 soedroohotometers. T h e experiments demonstrate t h e effect bf p ~ , - t e m p e r a t u r e ,a n d in.hihitors on enzyme activity, a n d t h e y allow t h e determination of K,, V,.,, a n d
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kcat.
The enzyme olpha-chymotrypsin (CT), is used, and N-glutarylL-phenylalsnine-p-nitroanilide(GPNA) is the substrate. CT reacts with GPNA to produce p-nitroaniline which absorbs strongly at 410 nm. The o-nitroaniline has a molar absomtivitv of 8200 M-'cm-' between p H of 7 and 8 111.This CT catalyredcleavage of GPNA obeys Michaelis-Menrun kinetics +JI and can easily be followed using a Spectronic 20. The GPNA and CT concentrations are such that the change in absorbance per minute is about 0.04 at a pH of 7.0 and at a temperature of 30% The GPNA and CT concentrations are also such that the change in absorbance per minute is linear up to an absorbance of 0.7. The rate is thus aceuratelv measured bv followine the increase in shaorhanre for four or more minutes. 'The rate can be expressed either asabsorbanre per minute or as moles per liter per mrnute since the path length and molar absorptiv~tyare known.
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Reagents The CT was purchased from Worthingtan Bioehemid Corporation (CDS 1475), and a stock solution was prepared by dissolving0.025 g of CT in 20 ml of 10W M HCI. If this solution is kept at O'C, it is stable for months. The GPNA was purchased from the Nutritional Biochemical Company, and 0.15 g of GPNA was dissolved in 25 ml of N.N-dimethylformamide (DMF). This gave a 0.015 M GPNA stock solution. The buffer was a 0.10 M K H I P O ~buffer and was prepared by dissolving 13.6 g of KHzP04in 1I of distilled water. The buffer was then brought to the proper p H (5.0,6.0,7.0,8.0,9.0, and 10) with50% aqueous KOH. The p H 10 buffer requires readjustment daily. The inhibitor, indole, was a J. T. Baker Chemical (N889) and wasprepared by dissolving the indole in DMF.
Experimental General Procedure. Warm up the Speetronic 20 and set the wavelength to 410 nm. Add 4.0 ml of buffer at the desired temperature to a Spectronic 20 tube (cuvet), and then pipet the desired aliquot of the stock GPNA solution into the same cuvet. Add the inhibitor for Procedure C or DMF for ProcedureD. Note that the GPNA and inhihitor are in DMF and that the total volume of DMF (0.50 ml) has t u remam runstant at 11% t V, V I Then add 0 50ml uf the stock C'I' sulutlon to the cwet. shake well, and allow the bubbler I n me. Set the abnorhanee to read 0 110w ~ t hthe ruvet msertedand s~multaneously start the time measurement. Finally, measure the time tosome convenient point after 4 min have elapsed. The rate, V ,can be expressed as absorbancelmin or converted to molefilmin using Beer's law since the total volume (5.0 ml), the cuvet path length (1.27 cm), and the molar absorotivitv (8200 M-'em-') (11 are known.
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442 1 Journal of Chemical Education
Amounts of GPNA and DMF Used in the Experiment. Procedure D
ml G P N A Stock
Solution
mi D M F
Final M G P N A in 5.0 mi of Array Solution ( X lo-')
Pmr~du. r o.~ A-Actiuitv and OH.Use the eeneral orocedure with . p H 5.0, 6.0,7.0, 8.0.9.0,'and 16 bufferr uhiFh have been heated to DOT. Exactly 0.50 ml uf the stock GPNAadution is used whlch gives a final GPNA concentration of 15 X 10-4.1f.The final rolutlm tern. perature may increase somewhat if the assay time is much longer than 4 mi". Also, t h e p H 10 buffer should be freshly made. Make a plotof rate versus pH. Procedure B-Aetiuity and Temperature. Again use the general procedure with the following changes. Place 4.0 ml of p H 7.0 buffer and 0.50 ml of the stock CT solution into a cuvet, and then place the cuvet into the appropriate water bath (0,10,20,30,40, and 50'C) for 10 min. Add 0.50 ml of the GPNA solution, shake, and set the absorhance as well as the time to 0.00. Return the cuvet to the water bath for 5 min and then measure the abrorhanre. Every 5 min measure the al,surbanceand return the cuvet 1,. the bath. Hepeat this procedure fur each iemperature. Determine the rate and make a plot of rate versus temperature. Procedure C-Aetiuity and Inhibitors. Three quick runs will demonstrate the effect of a competitive inhibitor (2) as well as provide the value for the inhibitor binding constant, Ki, if desired. For each run use the general procedure with the following exceptions. Use 4.0 ml a f p H 7.0 buffer at 30°C and0.40 ml of theGPNAstocksolution. For the run without inhibitor add 0.10 ml of DMF; for the first run with inhibitor add 0.10 ml of 0.20M indole in place of the 0.10 ml of DMF; and for the final run with inhibitor add 0.10 ml of 0.40 M indole. The general effect of an inhibitor will be demonstrated, and, if desired, the K; can be determined using a V d V i - I50 plot (2). Procedure D-Determination of, Km. . .. Vm.. .... .. and k,,,. Use the general procedure w ~ t hthe following modifications. Pipet 1.0 ml of p H 7.U buffer at 30°C into the cuvrt Then add the desired amount of GI'XA and DhlF ,see the rablr). Finally, add 0.50 mi of CT and determine the rate. Make either a Lineweaver-Burk plot (1) or a Woolf plot (1) and calculate the K, and V,., values. The k,,or turnover number can also be determined since k, = V,.JE, where V,., is the maximum rate expressed in molelllmin (not absorbaneelminute) and E , is the total molar enzyme concentration. The Etis 5.0 X 10V M since the molecular weight far CT is about 25,000. ~~~~
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Results and Discussion T h e increase i n absorbance was found t o h e linear with respect w time for any p H betweensand 10 up w a n absorbance of 0.7. Abovea p H of 10 hasecatalyzed cleavageof t h e C P N A occurred. Geneiallv. the runs were stormed shortlv after 4 min d u e to t h e temperature increase causeci b y t h e ~ p e c t r o n i c20. Since t h e change in ahsorbancelmin is constant, the Spec-
tronic 20 does not have to be set to 0.00 absorbance upon initial mixing. The cuvet can be thoroughly mixed and settled before setting the absorbance and
