Calculate the final concentrations of NAD+ in assay IIIA and the final concentrations of ethanol in assay IIIB. Add these values to the assay protocol tables. 2. Plot two substrate saturation curves (vo vs. [S] graphs): one for variable [NAD+] and another one for variable [ethanol]. Use the Logger Pro manual data entry files for Figures 6 and 7 to create these graphs (see the computer software instructions that follow). The files will convert the slope (?A/sec) of each Absorbance versus Time graph to initial velocity in Units (µmol/min) for each varying substrate concentrations. Use the hyperbolic curve fit function described below to draw the best smooth curve through the data points. See page 151 of this manual for instructions:

Hi I need help with Enzyme Kinetics for parts 3A and 3B pages 140-143

Enzyme kinetics is the study of the rates of enzyme catalyzed reactions. Kinetic studies have

several useful applications including the clinical diagnosis of disease, the design of new drugs,

and the elucidation of the catalytic mechanism of an enzyme. When one conducts a kinetics

experiment, the initial velocity (v0) of the reaction is measured in a series of reaction mixtures in

which the substrate concentration (S) is varied. All other parameters such as temperature, pH,

buffer composition, salt concentration, and enzyme concentration are held constant. When v0 is

plotted versus S, a hyperbolic relationship is usually observed (Figure 1). The graph is known as

a substrate saturation curve. The research team of Leonor Michaelis and Maud Menten first

proposed the theoretical basis for this relationship in 1913. The equation that they used to

describe this relationship is known as the Michaelis?Menten equation:

v0 = Vmax [S]

Km + [S] Vmax is defined as the maximum velocity of the enzyme, the fastest initial velocity that can be

observed for a particular set of reaction conditions (i.e. the parameters mentioned above). Km, the

Michaelis constant, is the substrate concentration that will produce an initial rate equal to onehalf Vmax. Km is a dissociation constant and indirectly measures the affinity of the enzyme for the

substrate. Lower values of Km indicate a higher the affinity of the enzyme for its substrate.

The Michaelis?Menten equation is based on

several assumptions. First, the enzyme has a

single substrate and catalyzes a two-step

reaction that can be described by the

following equation:

E+S ES E+P Where E is the enzyme, S is its substrate,

ES is the enzyme-substrate complex,

and P is the product of the reaction.

Figure 1. Dependence of v0 on substrate concentration. The second assumption is that the first step is the fast step of the reaction and the second step is

the slow, rate-limiting step. Third, rates are measured only after steady state is achieved. This

means that the rate of formation of the enzyme-substrate complex is equal to the rate of its

breakdown. Therefore, the concentration of the enzyme-substrate complex is constant while the

reaction is monitored. Michaelis?Menten kinetics is also known as steady-state kinetics for this

reason. To observe steady-state kinetics, rate measurements cannot be taken too early in the

course of the reaction. Fourth, initial rates are measured. This means that measurements are not

recorded too late in the course of the reaction. There are two important consequences to

measuring initial rates: (1) Very little substrate has been consumed and one may assume that the

substrate concentration does not change significantly during the course of the reaction. (2) Very

©Barbara T. Nash, Ph.D., Mercy College September 2012 130

little product has been formed so one may assume that the rate constant of the reverse of the

second step, k-2 is insignificant. This assumption is essential in the derivation of the Michaelis?

Menten equation, but that is beyond the scope of this discussion. Finally, because the second step

of the enzyme mechanism is the slow step, its rate constant, k2, is the rate constant for the overall

reaction. Therefore, the following equation applies:

vo = k2[ES]

The Km is an important property of an enzyme that characterizes it and make it possible to make

predictions about its behavior both in vivo and in vitro. When one sets up an enzyme assay,

using substrate concentrations that are greater than the Km, reaction rates will approach the Vmax.

Such conditions are desirable in order for reactions to proceed at reasonable rates. The activity of

an enzyme in vivo can change in response to the intracellular concentration of its substrate.

When the substrate concentration falls to a level significantly below the Km, the activity of the

enzyme will be insignificant. If two enzymes utilize the same substrate, the one with the lower

Km will be active at lower substrate concentrations than the one with the higher Km. Metabolic

pathways can be controlled in this manner. Although the Vmax of an enzyme constant for a

particular set of assay conditions, it varies with enzyme concentration and is therefore not a

fundamental property of an enzyme. The specific activity of an enzyme, the ratio of the Vmax to

the amount (mg) of enzyme is a constant that is used to characterize an enzyme.

Although the Vmax and Km of an enzyme can

be estimated from a substrate saturation curve,

this requires estimating the Vmax from an

asymptote. A more accurate way to determine

the Km and Vmax of an enzyme is by using the

linear transformation of the Michaelis?Menten

equation, the Lineweaver?Burk equation:

1

v0 = Km + Vmax [S] 1

Vmax A double-reciprocal plot (1/v0 versus 1/[S])

yields a straight line that can be evaluated by

linear regression. Exact values for Vmax

(y-intercept) and Km (the slope/y-intercept)

can be derived from this equation (Figure 2). Figure 2. A Lineweaver?Burk plot. Enzyme kinetics can be used to deduce the mechanism of an enzyme. For example, in

bisubstrate reactions (those involving two substrates), the enzyme can either bind both substrates

simultaneously during the course of the reaction, forming a ternary complex (Figure 3a), or the

first substrate can be converted to product and dissociates from the enzyme before the second

substrate binds (Figure 3b). The latter mechanism is known as a Ping-Pong, or doubledisplacement mechanism. The order of substrate binding to form a ternary complex may be

random (Figure 3a top) or ordered (Figure 3a bottom). Kinetic experiments can distinguish a

Ping-Pong mechanism (Figure 4b) from one involving a ternary complex (Figure 4a). ©Barbara T. Nash, Ph.D., Mercy College September 2012 131 Figure 3. Common mechanisms for enzyme-catalyzed bisubstrate reactions. Figure 4. Steady-state analysis of bisubstrate reactions. An enzyme inhibitor is a substance that binds to an enzyme in a specific manner and decreases

its activity without disrupting its structure. This definition distinguishes an inhibitor from a

protein denaturant. There are two main categories of inhibitors: (1) Reversible inhibitors, that

bind to enzymes via noncovalent interactions and (2) irreversible inhibitors that bind to enzymes

via a covalent bond. Three types of reversible inhibition can be distinguished by kinetic studies.

Competitive inhibitors are substrate analogs that bind to the active site of the enzyme (Figure 5

left). Inhibition is overcome at high substrate concentration. A series of kinetics experiments are

performed at several concentrations of inhibitor, a family of Lineweaver?Burk plots will be

generated that intersect on the y-axis (Figure 5 right). This pattern is characteristic of the

presence of a competitive inhibitor. Some examples of competitive inhibitors are sulfonamides

(Sulfa drugs), methotrecate, and 5-fluorouracil (cancer chemotherapy agents). Noncompetitive

inhibitors bind with equal affinity to both the free enzyme (E) and to the enzyme substrate

complex (ES) at a site other than the active site (Figure 6 left). Inhibition is not overcome at high

substrate concentrations. The characteristic family of Lineweaver?Burk plots for noncompetitive

inhibitors intersect on the x-axis (Figure 6 right). Some examples of noncompetitive inhibitors

are heavy metals, such as lead and mercury and chelating agents that bind essential metal

cofactors. Uncompetitive inhibitors bind reversibly to the enzyme-substrate complex, but not to

free enzyme (Figure 7 left). The Lineweaver?Burk plots that indicate the presence of an

uncompetitive inhibitor form a series of parallel lines (Figure 7 right). ©Barbara T. Nash, Ph.D., Mercy College September 2012 132 Figure 5. Competitive inhibition Figure 6. Noncompetitive inhibition.

If K1=K1', inhibition is noncompetitive. Figure 7. Uncompetitive inhibition Reference

Nelson, D.L.; Cox, M.M. Lehninger Principles of Biochemistry, 5th ed.; W.H. Freeman: NY,

2008; pp. 194?205. ©Barbara T. Nash, Ph.D., Mercy College September 2012 133 Biochemistry

Chemistry 354

Laboratory Exercises 7 & 8

Kinetics of ADH

Purpose

1. To follow the yeast alcohol dehydrogenase reaction to estimate Vmax

2. To determine the relationship between initial rate and enzyme concentration

3. To determine the Km, Vmax, and specific activity of alcohol dehydrogenase

Introduction

Yeast alcohol dehydrogenase (ADH) assay

Enzyme assays are widely used in biochemistry for a variety of purposes. Specific enzymes are

assayed to assist in their purification, to identify expression patterns in various tissues, or to

understand enzyme mechanisms and the influence of altered conditions. Some enzymes are used

as reporters in laboratory procedures. Examples of this application include the following: (1) The

increase in specific activity of a mitochondrial enzyme can be used to demonstrate the

enrichment of mitochondria in subcellular fractions during purification steps. (2) The presence of

an organ-specific isozyme activity in the serum can be used to diagnose damage to a specific

organ. (3) Alkaline phosphatase conjugated to an antibody can be used to monitor the location of

the corresponding antigen in microscopy or to quantitate the antigen in ELISA. (4) The presence

of ??galactosidase activity can be used to demonstrate the presence of bacterial colonies

containing the gene for this enzyme.

ADH is an enzyme used by yeast in the reversible fermentation reaction that converts

acetaldehyde to ethanol. For our purposes in assaying the enzyme activity, we will measure the

reverse reaction, i.e. ethanol to acetaldehyde. Because NADH absorbs light at 340 nm and the

absorbance of NAD+ at this wavelength is close to zero, the production of NADH can be

followed in a spectrophotometer as an increase in absorbance at 340 nm. The rate of the reaction

will be directly proportional to the change in absorbance per unit time.

+ CH3-CH2-OH + NAD + CH3-C=O + NADH + H |

H Important notes about the design of the assay:

1. Since a proton is produced during the reaction, the equilibrium will be dependent upon pH.

Optimum pH is known to be 8.6. The enzyme is increasingly unstable at pH values below 6.0

or above 8.5. The enzyme is stored in a phosphate buffer at pH 7.5 for maximum stability

and the assay is performed in a sodium pyrophosphate buffer at pH 8.8 for optimum activity.

Sodium pyrrophosphate has four titrateable groups and hence, four pKas: 0.9, 2.0, 6.6, and

9.4. ©Barbara T. Nash, Ph.D., Mercy College September 2012 134

2. ADH is a zinc metalloenzyme. Each subunit binds two atoms of zinc. One zinc atom is

bound to the active site and participates in the catalytic mechanism; the other stabilizes the

quaternary structure of the enzyme. (See the SDS-PAGE lab for the determination of the

quaternary structure of ADH). The enzyme has a sensitive sulfhydryl group. Although some

+3

+2

assay systems include the chelator, EDTA, to complex trace metals such as Fe and Cu

that may interact with this group, high levels of EDTA inhibit the enzyme by chelating zinc

and causing subunit dissociation. Our assay system does not include a chelator.

3. The rate of change in absorbance at 340 nm can be converted to the rate of change in

concentration of NADH using Beer's Law. The extinction coefficient for NADH at 340 nm is

3

-1

-1

6.22 x 10 M cm . See the sample calculation in the appendix to this lab on page 211.

4. The kinetic constants, Km values for ethanol and NAD+, have been determined under several

different assay conditions. Because these values vary with assay conditions (57-400 µM for

+

NAD and 13-21 mM for ethanol), it is not strictly accurate to use Km values from one assay

protocol to another. We are using the protocol from the Sigma Chemical Company. The

kinetic constants have not been published for this system. You will compare your

experimental values to the Km values reported by the biochemistry students from the Fall

2005 semester (see step 7 in the section entitled, ?Data Analysis?).

Efficient assays will use at least 10 x Km values for both substrates to give 91% saturation;

i.e., 91% of the enzyme is found in ES complexes. You should remember that Km gives 50%

saturation and be able to prove that 10 x Km gives 91% saturation and 100 x Km gives 99%

saturation. To conserve substrate, but give a workable assay, we will use approximately 10

times the greatest value of Km that has been reported. The concentrations of NAD+ and

ethanol in this assay are 4.0 mM and 200 mM respectively.

5. Commercial yeast ADH usually has a specific activity around 300 units/mg protein. One unit

of activity is defined as the amount of enzyme that converts 1.0 µmol of ethanol to

acetaldehyde per minute at pH 8.8 and 25?C. Therefore, 1 mg of ADH protein would be

expected to produce 300 µmoles of acetaldehyde and NADH per minute under the

appropriate conditions. Since 300 µmoles of NADH in a 1-mL assay would be expected to

yield an absorbance of 1866, it is clear that we need to measure the activity of much less than

1 mg enzyme. About 0.025 - 0.125 µg would be a better range to assay over a 1-minute

period.

6. It is important to measure the initial velocity of the reaction (vo). In order to accomplish this,

all reagents except enzyme should be added to the cuvette, mixed well, and used to zero the

spectrophotometer. The appropriate amount of enzyme is then added and mixed quickly, and

placed in the spectrophotometer. Absorbance readings are then taken at 10-second intervals.

The change in absorbance per minute (?A/min) is then calculated from the slope of a graph

of absorbance versus time. ©Barbara T. Nash, Ph.D., Mercy College September 2012 135

Procedure

Materials:

Spectrophotometer interfaced to a computer

Cuvettes (1 mL)

Vortex mixer

Marking pen and tape

Test tube rack

16 mm x 150 mm test tubes

microfuge tube

Parafilm

Digital micropipets (L-10, P-20, P-200, P-1000) and tips

Waste beaker

Distilled water

Reagents :

A. 44.0 mM sodium pyrophosphate (PPi) buffer pH 8.8

sodium pyrophosphate decahydrate (molecular weight = 446.1 g/mol)

9.81 g/500 mL, pH adjusted to 8.8 with 8% phosphoric acid

B. 1.00 M Ethanol

Absolute ethanol (molecular weight 46 g/mol, density 0.7893 g/mL)

29 mL diluted to 500 mL with distilled water

+ C. 20.0 mM NAD

(molecular weight = 663.4 g/mol)

The molecular weight is adjusted for the water and acetone content present in each lot in

order to prepare the solution.

D. 10.0 mM sodium phosphate

monobasic anhydrous sodium phosphate (molecular weight = 120.0 g/mol)

0.60 g/500 mL

E. 10.0 mM sodium phosphate buffer, pH 7.5

0.71 g/500 mL dibasic anhydrous sodium phosphate, adjusted to pH 7.5 with reagent D.

F. 10.0 mM sodium phosphate buffer, pH 7.5 containing 0.1% (w/v) bovine serum albumin

G. Alcohol Dehydrogenase Enzyme Solution (concentrate)

Yeast ADH is provided commercially as a lyophilized solid. Stock concentrated enzyme is

1.00 mg/mL in ice-cold Reagent E. It is kept on ice during the lab. ©Barbara T. Nash, Ph.D., Mercy College September 2012 136

H. Alcohol Dehydrogenase (diluted solution)

Each group will need to prepare a diluted stock enzyme (1/200). This dilution is made in cold

Reagent F. ADH is unstable in a dilute solution and the BSA functions to keep the enzyme

sufficiently stable for kinetic studies to be performed. In a microfuge tube, add 2 µL of the

concentrated ADH solution to sufficient ice-cold phosphate buffer to dilute the enzyme

1/200. This diluted enzyme solution should be kept on ice during the experimental period and

discarded at the end of the day.

Note: The BSA concentration is kept at a constant value of 0.003% in the assay system by

keeping the total of the volumes of Reagents F and H at 33 µL in a 1 mL assay solution.

Thus, the amount of enzyme added the assay mix can be varied while keeping the BSA

concentration constant.

Getting Started

1. Turn to page 147 for instructions on

a. Setting up the spectrophotometer-computer system

b. Calibrating and blanking the spectrophotometer on the assay mixture

c. Finding the absorbance peak for NADH

d. Setting up the data collection parameters.

2. Follow the instructions on Table A on the next page to prepare assay mix in two cuvettes.

3. Use one cuvette to blank the spectrophotometer and allow it to find the absorbance peak for

NADH as described in IC on page 147 of these instructions.

4. Use the second cuvette for the first kinetics run as described on the following page. ©Barbara T. Nash, Ph.D., Mercy College September 2012 137

Methods Part I: Determination of the Initial Rate of the ADH Reaction

Table A: Assay Mix for the First Kinetics Run

The following reagents are used to prepare a mixture for a single enzyme assay:

Stock solution

Volume added

Final concentration in 1 mL

44.0 mM PPi, pH 8.8

22.0 mM

500. µL

1.00 M Ethanol

200. mM

200. µL

+

4.00 mM

200. µL

20.0 mM NAD

H2O

----------------67.0 µL

Reagent F

----------------23.0 µL

Total volume

----------------990. µL

ADH Enzyme Assay

1. Prepare a 990 µL assay solution directly in a cuvette, cover with Parafilm, mix well.

2. Add 10.0 µL diluted enzyme to the tube, cover with Parafilm, and mix well by inverting the

cuvette twice.

3. Start the computer-interfaced data collection by clicking on Collect as soon as you mix the

reaction mixture. (See instructions for the use of the spectrophotometer software in the

Appendix to the Kinetics Lab.

4. Insert the cuvette in the spectrophotometer and begin reading absorbance at specific times

after the enzyme addition. Take readings every 10 seconds for 10 min (6 readings per

minute). Note whether or not the reaction rate levels off.

5. Data will be recorded in Table 1 and Figure 1 (Absorbance versus Time for Initial Rate

Calculation). See the appendix to these instructions for an example.

Data Analysis

1. Calculate the final concentration of each reagent in the 1-mL assay solution. Record these

values in the table above.

2. If the amount of NADH produced did not provide a range of absorbances that was easy to

read during the first minute, add more or less enzyme to improve the readability and data

analysis of the assay

3. Calculate the initial velocity (vo) of the reaction (µmol/min) from the slope of the absorbance

versus time graph by using Beer's Law and the extinction coefficient (See the sample

calculation in the Appendix to the Kinetics Lab)

4. Estimate the specific activity of the enzyme (Units/mg) from the initial velocity you

calculated in step 3 of this section and the total amount of enzyme (mg) you used in the

assay. Use the sample calculation in the appendix as a guide for this calculation. ©Barbara T. Nash, Ph.D., Mercy College September 2012 138

Methods Part II: Determination of the Relationship between Enzyme Activity (vo) and

Enzyme Concentration.

All cuvettes will contain 1-mL final volume of assay solution.

Table B: Assay Mix for Absorbance versus Time for Varying Enzyme Concentration

Stock solution

44.0 mM PPi, pH 8.8

1.00 M Ethanol

+ 20.0 mM NAD

H2O

Total volume Volume for 1x Mix

500. µL

200. µL

200. µL

67.0 µL

967 µL Volume for 10x Mix

5.0 mL

2.0 mL

2.0 mL

0.67 mL

9.67 mL Additions for Varying Amounts of ADH

cuvette #

Reagent F

ADH (1/200) 1

28.0 µL

5.00 µL 2

23.0 µL

10.00 µL 3

18.0 µL

15.00 µL 4

13.0 µL

20.00 µL 5

8.00 µL

25.0 µL Enzyme Assay

1.

2.

3.

4.

5.

6. Prepare 9.67 mL of 10x assay mix in a test tube according to the protocol in the table above.

Mix the assay mix well and transfer 967 µL to each of 5 cuvettes.

To one cuvette, add 28 µL of Reagent F, cover with Parafilm and mix well.

Add 5 µL of enzyme to this cuvette, cover with Parafilm, and mix well.

Read absorbance at 10-second intervals for 90 seconds. Record all values

Repeat steps 3, 5, and 6 using 10 µL, 15 µL, 20 µL, and 25 µL of enzyme. Vary the amount

of Reagent F as shown in the table above. It is not necessary to re-blank the cuvette for each

of these assays.

9. Record your data will be in Table 2 and Figure 2 (Absorbance versus Time for Varying

Enzyme Concentrations). ©Barbara T. Nash, Ph.D., Mercy College September 2012 139

Data Analysis

1. Calculate the final enzyme concentration (µg/mL) in each assay in Part II.

2. Were all the reactions fast enough to measure a significant amount of NADH produced

during the reaction time? Were all the reactions slow enough so that the absorbance versus

time curves are linear for a sufficient period of time to enable you to calculate initial

velocities? If not, you may need to repeat some of the assays using a different amount of

enzyme.

3. Record the following data into the Logger Pro manual data entry file for Figure 3. See page

150 of this lab manual for instructions):

(1) The volume of ADH used for each run.

(2) The corresponding concentration of ADH.

(3) The slope (?A/sec) of the plot of Absorbance versus Time for each run.

The file will convert the slope of the Absorbance versus Time graph to initial velocity in

units of enzyme activity (µmol/min) for each concentration of enzyme. The table associated

with this file appears below, but the title appears only on the graph because it does not fit

above the table.

Table 3: Dependence of Initial Rate on Enzyme Concentration

cuvette # vol. ADH (µL) Enzyme conc.

(µg/mL) slope

(1/sec) vo(µmol/min) 1

2

3

4

5

4. The Logger Pro file for Figure 3 will plot the initial velocity (vo) versus enzyme

concentration (µg/mL). Follow the instructions for creating this graph and see an example

(Figure 3) in the appendix to this lab.

5. Choose the best amount of enzyme (µL) to use in Part III of this exercise. You need an

amount that will give a reaction rate that is slow enough that absorbance values do not

exceed 0.8 during the course of the run, but fast enough so that the rate can be reduced and

you will still have a reaction rate that can be measured. ©Barbara T. Nash, Ph.D., Mercy College September 2012 140

Methods Part III: Determination of the kinetic constants, Km and Vmax for NAD+ at

+ infinite [ethanol] and for ethanol at infinite [NAD ]

To evaluate the enzyme activity at different substrate concentrations, prepare an assay mix

containing approximately 10 Km for one substrate and without any of the second substrate.

Varying amounts of the second substrate are then added to the assay mix giving final

concentrations which range from approximately 0.4 Km to 5 x Km as follows:

+ A. Varying [NAD ]

+

Table C: Assay Mix for Varying [NAD ]

Stock solution

44.0 mM PPi, pH 8.8

1.00 M Ethanol

H2O

Total volume Volume for 1x Mix

500. µL

200. µL

167 µL

867 µL Volume for 10x Mix

5.0 mL

2.0 mL

1.67 mL

8.67 mL + Additions for Varying Amounts of [NAD ]

cuvette #

+ 20.0 mM NAD

H2O

Reagent F

ADH (1/200)

+

[NAD ] (mM) 1

100. µL

0 µL 2

40.0 µL

60.0 µL 3

20.00 µL

80.0 µL 4

12.50 µL

87.5 µL 5

10.00 µL

90.0 µL 6

8.00 µL

92.0 µL Enzyme Assay

1. After deciding on the amount of ADH you will use in this assay, calculate the amount of

Reagent F you will be using to keep the total volume of Reagents F and G (diluted ADH)

equal to 33 µL. Record these values on the table above.

2. Mix the assay mix well and transfer 867 µL to each of 6 cuvettes.

3. To one cuvette, add 100 µL of the NAD+ solution and the appropriate amount of Reagent F.

Cover with Parafilm and mix well.

4. Blank the spectrophotometer on this cuvette.

5. Add the appropriate amount of enzyme to this cuvette, cover with Parafilm, and mix well.

6. Read absorbance at 10-second intervals for 90 seconds. Record all values

7. Repeat steps 3, 5, and 6, using 40 µL, 20 µL, 12.5 µL, 10 µL, and 8 µL of NAD+, adding the

appropriate amount of H2O as indicated in the "additions" table.

8. Record your data in Table 4 and Figure 4 (Absorbance versus Time for Varying NAD+

Concentrations). Place odd-numbered runs on one graph (Figure 4a) and even-numbered runs

on another (Figure 4b). ©Barbara T. Nash, Ph.D., Mercy College September 2012 141

B. Varying [Ethanol]

Table D: Assay Mix for Varying [Ethanol]

Stock solution

44.0 mM PPi, pH 8.8

+ 20.0 mM NAD

H2O

Total volume Volume for 1x Mix

500. µL

200. µL

167 µL

867 µL Volume for 10x Mix

5.0 mL

2.0 mL

1.67 mL

8.67 mL Additions for Varying Amounts of [Ethanol]

cuvette #

1000 mM

Ethanol

H2O

Reagent F

ADH (1/200)

[Ethanol] (mM) 1

100. µL 2

50.0 µL 3

25.0 µL 4

12.50 µL 5

10.00 µL 6

8.00 µL 0 µL 50.0 µL 75.0 µL 87.5 µL 90.0 L 92.0 µL Enzyme Assay

1. Record the amount of Reagents F and G you will use in this assay on the table above as done

in Part A of this exercise.

2. Mix the assay mix well and transfer 867 µL to each of 6 cuvettes.

3. To one cuvette, add 100 µL of the ethanol solution and the appropriate amount of Reagent F.

Cover with Parafilm and mix well.

4. Blank the spectrophotometer on this cuvette.

5. Add the appropriate amount of enzyme to this cuvette, cover with Parafilm, and mix well.

6. Read absorbance at 10-second intervals for 90 seconds. Record all values

7. Repeat steps 3, 5, and 6, using 50 µL, 25 µL, 12.5 µL, 10 µL, and 8 µL of ethanol, ad...

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