Unit 5 developing an enzyme assay introduction



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UNIT 5

DEVELOPING AN ENZYME ASSAY

Introduction:


Later this semester, we will be putting together some of the chromatography and electrophoresis techniques to develop a purification scheme for an enzyme, lactate dehydrogenase. Before an enzyme can be purified, an enzyme assay must be in place in order to detect which fraction the enzyme is partitioning into during the purification steps. This enzyme assay must be highly quantitative and reproducible so that you can assess the yield of enzyme that a particular purification step gives you. A quantitative enzyme assay is also useful for determining enzyme purity following purification steps. This is done by comparing enzyme activity in each fraction to the total protein in that fraction, using a protein assay for the total protein.
In this lab exercise, we will study the enzyme lactate dehydrogenase and develop a quantitative assay for its activity. We will also conduct a protein assay to determine its specific activity, an index of its purity. We will use the spectrophotometer for both the enzyme and the protein assay.

Lactate Dehydrogenase


Virtually every living cell processes glucose in order to obtain energy in the form of ATP. This process is called glycolysis, the lysis or splitting of glucose. In addition to the desired ATP—a net of 2 ATP per molecule of glucose—glycolysis yields the product, pyruvate (CH3COCOO-) and the byproduct, NADH.

Cells that have sufficient oxygen go on to convert the pyruvate to acetyl CoA, which in turn enters into the Kreb’s cycle in the mitochondrion. The Kreb’s cycle product, NADH, is later the substrate of the electron transport system for generating more ATP by oxidative phosphorylation through enzymes located within the inner membrane of the mitochondria.


In the absence of oxygen, cells undergo fermentation. The primary purpose of fermentation is to remove the hydrogens from the NADH to reform NAD. This NAD is then recycled as a substrate in glycolysis. Without this regeneration of NAD during fermentation, glycolysis would halt and the organism would die.
Fermentation in mammals occurs in muscle fibers during vigorous exercise. Under less strenuous conditions, muscles have sufficient oxygen to keep the Kreb’s cycle and the oxidative phosphorylation in their mitochondria going. This generates enough energy to power the muscles. However, when muscles are used heavily, there is not enough oxygen being delivered to satisfy the needs of the muscles. Under these conditions, the cells switch over to lactic acid fermentation. The pyruvate is converted to lactic acid, CH3CHOHCOO- with the hydrogens coming from the reduced NADH. This regenerates the NAD, which is so necessary to the continuation of glycolysis, and therefore the continuing use of the muscles.
The molecule involved in the lactic acid fermentation in mammalian muscles is lactate dehydrogenase (LDH). The reaction that it catalyzes is:


LDH


CH3CCOO- + NADH CH3CHCOO- + NAD+


O

OH

pyruvate lactate


This is a reversible reaction: when the heavy muscular use is over, and the cells are relaxing, the lactate that has built up within the system is converted back to pyruvate and the rest of aerobic respiration can continue as before. The LDH catalyzes this reverse reaction as well.
LDH is found in all types of muscles, although there are different isozymes in each muscle type. Thus the heart muscle isozyme (H isozyme) is different from the skeletal muscle isozyme (M isozyme). This may be used diagnostically: the presence of cardiac isozymes in the blood stream is an indicator of a heart attack.

Spectrophotometry

The spectrophotometer is a tool most commonly used for protein and enzyme assays because it can be an easy way to measure the concentration of a solute in a solution. Many solutes will absorb light of a specific wavelength, and the amount of light absorbed is proportional to the concentration of the solute. When solutes do not absorb light sufficiently for measurement, reacting the solute with something that forms a colored complex can make the solute detectable by spectroscopy. Reaction of the solute with such a chromophore can also be used to specifically detect that solute against a background of contaminants that would interfere with a direct assay of the solute.
In this lab exercise, we will assay the enzyme lactate dehydrogenase by the rate it produces the product NADH, which absorbs light strongly at a wavelength of 340 namometers. We will also assay for protein concentration using the Bradford assay, in which protein binds to Coomassie Blue dye to form a colored complex that absorbs light strongly at a wavelength of 595 namometers.
For a more complete discussion of the use of the spectrophotometer, review Chapter 19 “Measurements Involving Light A: Baisc Principles and Instrumentation” and Chapter 20 “Measurements Involving Light B: Applications and Methods” in your lab textbook, Basic Laboratory Methods for Biotechnology by Seidman & Moore.


Enzyme Assays

The activity of an enzyme is measured by the rate of the reaction that it catalyzes. This rate can be detected either by the measurement of product formation over time or by the measurement of the disappearance of substrate over time. The detection of substrate or product concentrations can be done in a number of ways. The easiest measurement case is when one or the other is a colored compound, in which case the concentration changes during enzyme catalysis can be directly followed by absorbance readings. When neither substrate nor product has any unique absorption characteristics that can be followed spectrophotometrically, enzymatic reaction rates must be followed by more difficult methods. Perhaps the most common method is the use of a radioactively-labeled substrate, in which case aliquots are taken during the time course of the enzyme reaction. By stopping the enzyme reaction over the time course of the reaction and assaying the radioactivity in product isolated from these aliqots, enzyme activity can be determined by the increase in product radioactivity over time.
Radiolabel enzyme reactions are more expensive, time-consuming, and hazardous than are spectrophotometric enzyme assays. Sometimes it is possible design a colorimetric enzyme assay by the use of alternative substrates that generate a colored enzyme-catalyzed product. A classic example of this an colorimetric enzyme assay for -galactosidase. The natural substrate for this enzyme is lactose, or -D-galactosidyl-D-glucose, which becomes hydrolyzed into the galactose and glucose products. Neither the substrate nor the products have a color that can be detected by spectrometry. If the artificial substrate para-nitrophenyl--D-galactose is used, the hydrolysis product para-nitrophenol has a yellow color that can be monitored at 520 nm absorbance.
Natural substrate: lactose (-D-galactosidyl-D-glucose)  D-galactose + D-glucose
Colorimetric assay: p-nitrophenyl--D-galactose  D-galactose + p-nitrophenol
When a colorimetric substrate or product is not possible, another approach is to couple the enzyme reaction to another enzyme reaction that does produce a colorimetric product. A classic example of this is to couple the first enzyme reaction to a second dehydrogenase reaction. A dehydrogenation reaction is easily followed by monitoring the appearance of NADH product at 340 nm. An example of a coupled reaction is an assay for hexose kinase, which catalyzes the reaction of D-glucose and ATP to form glucose-6-phosphate and ADP products:
D-glucose + ATP  D-glucose-6-P + ADP
None of the reactants or products have any unique colorimetric properties, but the D-glucose-6-phosphate product of the reaction is the substrate for a second enzyme, D-glucose-6-phosphate dehydrogenase to form a colorimetric product NADH whose formation can be monitored at 340 nm:
D-glucose-6-P + NAD  gluconate-6-P + NADH
By the way, these two enzymes (hexokinase and D-glucose-6-phosphate dehydrogenase are often used clinically to measure the concentration of glucose in the blood of diabetics.)
Once a method has been found to detect an enzyme-catalyzed reaction, the optimal buffering conditions for the assay must be determined. Enzyme activity is affected by a number of parameters, listed in the table below:


Parameters affecting rates of enzyme-catalyzed reactions


How rates are affected

The temperature

There is an optimal temperature: as temperature rises, chemical reactions rates increase until a temperature is reached that begins to denature the enzyme.

The pH

There is a pH optimum: a pH that is too low or too high can affect the chemistry of the active site and can affect the overall enzyme conformation.

The substrate concentration

As substrate concentration increases, the reaction rate increases until the enzyme active sites become saturated.

The cofactor concentration

Since cofactors participate in the enzyme-catalyzed reaction, the reaction rate increases as cofactor concentration increases until the enzyme active sites become saturated.

The product concentration

Enzymes only affect the rate of reaction and do not affect the equilibrium constant of the reaction. As equilibrium is approached between the substrate and product concentrations, the rate of reverse reaction approaches the rate of forward reaction.

To make an enzyme assay reliable, all of the above parameters must be carefully considered. If the assay done on one day can be expected to be reproducible on another day, the temperature, pH, substrate concentration, cofactor concentration, and product concentration must be the same each time. In developing an enzyme assay, the optimum conditions of temperature and pH will provide the most robust conditions and the best sensitivity for detecting low levels of the enzyme activity. Similarly, high concentrations of substrates and any necessary cofactors will give the maximum rate of reaction and the highest level of enzyme activity. For that reason, saturating amounts of substrate and cofactor is used.


In developing an enzyme assay all of these parameters must be determined by altering the buffering conditions until the maximum catalytic rate has been found. This is what we will do in this lab exercise.

Lab 5-A:

Preparation of Reagents

Introduction:


All biochemical reactions occur under conditions of strict control over the concentration of hydrogen ions. Life cannot exist when there are large fluctuations of the hydrogen ion also known as H+ or proton. Changes in pH, the measurement of the concentration of hydrogen ions, will affect the structure of proteins and therefore the activity of enzymes upon which life depends.
Buffers are extensively used by both living organisms in vivo and scientists in vitro to prevent these catastrophic fluctuations of pH. Buffers, or more properly buffer systems, are molecules that can donate or accept hydrogen ions in order to maintain the pH within a small range. For example, the blood pH is maintained at 7.4 by a carbonic acid-bicarbonate buffer. In this system carbonic acid (H2CO3) is the proton donor and bicarbonate (HCO3-) is the proton acceptor. When the pH is basic and H+ is low, the equilibrium is shifted and the carbonic acid will donate protons and make the pH more acidic. Conversely, when the pH is acidic, the bicarbonate will accept the excess protons restoring the pH of 7.4.
The original buffers used in the lab were made from simple weak acids and bases, such as acetic acid, phosphoric acid and citric acid. Today some of these buffers are still in use. Citrate buffers are often used in ELISA assays. However, these early buffers had limitations. They often changed their pH too much if the solution were diluted or if the temperature were changed. To counteract these problems, N.E. Good developed a series of buffers that are zwitterions, containing both negative and positive charges on a single molecule. In addition to being less sensitive to temperature or dilution, Good’s buffers also do not cross the plasma membrane. Some examples of Good’s buffers, now the preferred buffers in many labs, are the commonly used Tris, HEPES (often used in cell culture), MOPS (used in electrophoresis of RNA), CAPS, the buffer we will be using for the LDH assay, and bicine, the buffer for the ion exchange column in Unit 5.
Each buffer has a buffering range that is special to that buffer. The midpoint of this range is known as the pKa. The following table gives the pKa for several common Good’s buffers.


Buffer

pKa

CAPS

10.4

Bicine

8.35

HEPES

7.55

MOPS

7.20

Tris

8.30

In addition to the Good’s buffers, we will also be making a phosphate buffer. In this buffer, we will be making a given concentration of sodium phosphate monobasic and sodium phosphate, dibasic. Then we will combine them to produce the desired pH.



Safety:


Wear goggles when using acid or base to adjust the pH of the buffers.

Protocol:




Part I. Prepare the solutions for enzyme assays

Students may collaborate in the preparation of the following solutions for use by the entire class. Part of this collaboration is the sharing of the Media Prep Form describing the preparation of each solution in for individual lab reports. Each prepared solution should be labeled with its contents and concentrations, as well as a control number that cross references the solution to its Media Prep Form. Students using a solution should record its control number in their lab report.




1. Make up 50 mL 0.4 M CAPS buffer, pH 10.0. The formula weight of CAPS is 221.3 g/mole. Place in a bottle and label, complete a media prep form, and store in the refrigerator.


  1. Make up 40 mL of 0.4 M Tris-Cl buffer, pH 9.0. Place in a bottle, label, and store in at room temperature.

3. Make 100 mL 1.0 M lactic acid (FW= 90.08 g/mole). Adjust the pH to 7.0 with NaOH before adjusting to the final volume. Label and refrigerate. This may already be made for you.


4. Make 20 mL 100 mM NAD+ with FW = 685.4 g/mole. Label and refrigerate.


  1. Make 200 mL 0.05 M sodium phosphate monobasic (120.0 g/mole) and 300 mL 0.05 M sodium phosphate, dibasicH2O (140.0 g/mole). These solutions may be prepared from the dry chemical or from stock solutions prepared earlier in the semester that have been properly documented and stored. Mix together the following ratios to make a series of buffers of different pH:


Final pH

50 mM Na2HPO4 (mL)

50 mM NaH2PO4 (mL)

6.0

6.15

43.85

7.0

30.5

19.5

8.0

47.35

2.65

Check the pH with a calibrated pH meter and adjust if necessary with the 50 mM solutions. Record any volume adjustments. Label and store in the refrigerator.



Part II. Prepare the spectrophotometer for kinetic analysis

Select the spectrophotometer that you will be using to measure reaction rates with. Study the operator manual to make sure you know how to select the correct wavelength and the absorption mode. You will making numerous measurements over short periods of time while measuring the reaction rates, so become familiar with any sophisticated programs that your instrument offers for collecting and graphing data for you. You will need graphs of the rate of reaction: absorbance (y-axis) and time (x-axis)


MEDIA PREP FORM Control #
Name of Solution/Media:
Amount prepared: Date:
Preparers(s):



Component

Brand/Item #

Storage conditions/ date received

FW or initial concentration

Amount used

Final concentration






































































Balance used



Calibration status

pH meter used



Calibration status


Initial pH



Final pH

Adjusted pH with


Prep temperature



Sterilization procedure

Storage conditions

Calculations/Comments:


MEDIA PREP FORM Control #
Name of Solution/Media:
Amount prepared: Date:
Preparers(s):


Component

Brand/Item #

Storage conditions/ date received

FW or initial concentration

Amount used

Final concentration






































































Balance used



Calibration status

pH meter used



Calibration status


Initial pH



Final pH

Adjusted pH with


Prep temperature



Sterilization procedure

Storage conditions

Calculations/Comments:



Lab 5-B:

Extraction and Assay of Lactate Dehydrogenase Activity

Introduction:


The H isozyme of lactate dehydrogenase, LDH, is found in heart muscle. Because LDH is located in the cytosol, it may be isolated with relative ease. In this protocol, we will be performing the crude isolation of LDH. However, it is not common to use a crude extract for activity assays because there are many contaminating proteins that will affect the activity results of your desired enzyme. A purified enzyme will give a more accurate, and usually higher, activity reading.
The first step is the homogenization of beef heart muscle tissue at 4oC. There are several methods for homogenization. In the case of animal tissue which lacks cell walls, a gentle approach can be taken: the tissue can be homogenized using a glass homogenizer. This is a glass tube with a closely fitted sintered glass pestle. This works well to homogenize small amounts of tissue, especially if the homogenizer is placed in an ice bucket and ice-cold buffer is used.
Centrifugation of the cell lysate will remove unbroken cells, dense cell organelles such as nuclei, and cellular debri. The supernatant containing the enzyme is a crude extract, containing thousands of types of proteins and other complex molecules. We will use the crude extract to develop a quantitative enzyme assay.
Lactate dehydrogenase is a reversible enzyme that catalyzes the reaction between pyruvate and lactate. When there is lactate and NAD+ present, the LDH will remove two hydrogens from the lactate to form NADH and pyruvate. This is a redox reaction; the lactate is oxidized to pyruvate and the NAD+ is reduced to NADH, which will absorb UV light at 340 nm. The amount of pyruvate produced is directly related to how much LDH is in the sample. Therefore the Absorbance340 is a function of the amount of LDH present.


LDH


CH3CCOO- + NADH CH3CHCOO- + NAD+

OH

O

pyruvate lactate


In this lab we will extract enzyme from beef heart cells and develop an assay for the LDH activity in the crude extract. Note that in a real application of enzyme assays, a crude extract is rarely assayed because there are too many contaminating proteins that can interfere with the progress of the assay, and thus skew the results!

Safety:


Wear gloves while working with both assays.

The Bradford reagent will stain clothing; wear a lab coat or old clothes.

Make sure the spectrophotometer cord is not frayed and the spectrophotometer is not near water.

Protocol:

Part I. Enzyme extraction

  1. Each group is to take 1/8 beef heart. Remove fat and connective tissue from a beef heart with scissors.




  1. Cut up the beef heart into fine pieces. Weigh out 1.0 g in a weigh boat and transfer to a tissue homogenizer. Rinse any remaining heart pieces from the weigh boat to the homogenizer with 2 mL cold sodium phosphate buffer (0.05 M, pH 7).




  1. Homogenize for 10-20 strokes to break up the cells and release the cytoplasmic components. Keep the homogenizer on ice so that the extraction buffer stays as close to 4oC as possible during homogenization. If material gets lodged at the bottom of the homogenizer, you need to push the piston in with more force. Be sure to twist the piston as you push it in so that the tissue is well homogenized by the shear forces that it makes. Avoid introducing any air in the solution. You will know when homogenization is complete when you can no longer see visible clumps of tissue.



  1. Divide the homogenate into two microcentrifuge tubes, counterbalance, and centrifuge 10,000xg for 10 minutes at 4oC. Collect the supernatant. Discard the pellet by transferring the supernatant to a fresh microcentrifuge tube. Keep this crude extract on ice at all times until ready to use.



Part II. Optimal pH for LDH enzyme assay

In this part, you will incubate your enzyme with a high level of NAD and l-lactate at room temperature. To determine the best pH for the enzyme reaction, you will compare reaction rates in buffers of different pH. You will be check pH 6, 7, and 8 with the phosphate buffers that you prepared in Lab 5A, and pH 9 and 10 with the Tris and CAPS buffers prepared at the same time.





  1. Turn on the spectrophotometer and set the wavelength to 340 nm. Allow to warm up about 10 minutes.




  1. Prepare a “pH Master Mix” of the l-lactate and NAD so that they are added together in each reaction. You will be checking five different buffers, and you will need enough master mix for at least five reactions. Usually a master mix also has a little extra in case you end up needed to repeat any of the reactions. Therefore your master mix will contain 10X the amounts listed.




Reagent

mL/assay

mL/10 assays

100 mM NAD+

0.10 mL

1.0 mL

1.0 M lactic acid, pH 7

0.20 mL

2.0 mL




  1. Pipet into a UV-transparent semimicrocuvette 0.8 mL of one of your prepared varied pH buffers and pipet 0.2 mL of the “pH Master Mix” into it. Cover with parafilm and gently invert the cuvette several times to mix.




  1. Place the cuvette in the spectrophotometer and blank the spectrophotometer to zero absorbance. Record a baseline of nonenzymatic formation of NADH over a period of about 60 seconds.

NOTE: If your spectrophotometer does not have a program to measure rates of absorbance change, you will need to record time points manually. To do this, make a table of 10 second increments and record absorbance readings to correspond to the time points. When you have collected all absorbance data, you will need to make a graph of the data (time in seconds for the x-axis, absorbance at 340 nm for the y-axis) to determine the slope.




  1. Working quickly and efficiently, pipet 0.02 mL of your LDH crude extract into the cuvette and immediately cover with parafilm and gently invert several times to mix. Return the cuvette as quickly as possible to the spectrophotometer. Immediately record the rate of NADH formation over a period of at least 120 seconds, or until the rate drops off. You will know when the rate has dropped off when a graph of absorbance (y-axis) and time (x-axis) is no longer linear. Make your absorbance measurements as often as possible over this 2 minute time period.




  1. If the rate drops off before the 120 second period is over, you have too much enzyme activity to measure well. In this case, you need to repeat steps 3-5 with a smaller amount of added enzyme. If the amount of enzyme added must be less than 10 microliters, you may want to dilute your LDL crude extract by half so that you can pipet a larger volume and still get a slower enzymeatic rate.




  1. Repeat steps 3-5 for each of your prepared buffers until you have a reaction rate for each of the pH values (pH 6, 7, 8, 9 and 10).




  1. To identify the optimal pH of your enzyme reaction, you need to estimate the rate of reaction from the slopes of your graphs of absorbance (y-axis) and time (x-axis). Before comparing the slopes under different pH conditions, it is important that you subtract the nonenzymatic reaction rates measured before addition of the heart extract. So take the slope of the line after adding enzyme and subtract the slope of the line before adding enzyme. Compare the differences of these numbers under the different buffering conditions.



Part III. Optimal temperature for LDH enzyme assay

In this part, you will incubate your enzyme with a high level of NAD and l-lactate at the optimal pH that you discovered in Part II. To determine the best temperature for the enzyme reaction, you will compare reaction rates in water baths of different temperatures.




  1. Prepare a “Temperature Master Mix” of the l-lactate and NAD so that they are added together in each reaction. Since now you know the best buffer to use, you will include this buffer in your master mix.




Reagent

ML/assay

mL/5 assays

100 mM NAD+

0.10 mL

0.5 mL

1.0 M lactic acid

0.20 mL

1.0 mL

Optimal pH buffer

0.8 mL

4.0 mL




  1. Pipet 1.2 mL of “Temperature Master Mix” into 4 different test tubes. Allow each tube to equilibrate to the following temperatures: 4oC (in an ice bath), room temperature (record the temperature), 37o C, and 55oC.




  1. After about 3 minutes of temperature equilibration, transfer the 45-55 degree master mix to a cuvette and immediately measure the baseline nonenzymatic rate of NADH formation for 60 seconds. Add an appropriate amount of LDL crude extract (determined in Part II), cover with parafilm, gently and quickly invert to mix, and immediately measure the rate of enzymatic NADH formation for 120 seconds at that temperature.




  1. Repeat the nonenzymatic and enzymatic rates of NADH formation for all temperatures. If the air is humid, watch out for any condensation on the faces of the cuvette since this condensation will read as an absorbance in the spectrophotometer.




  1. As above, subtract the rate of nonenzymatic NADH formation from that of the enzymatic NADH formation to identify the optimal temperature for LDH.




  1. Report your results in a table.

Questions for Unit 5



Lab 5-A:


1. What is the final concentration of the phosphate buffers that you prepared, at pH 6, 7, and 8?

2. Why was phosphate not used to create a pH 10 buffer?





  1. Buffers are made by making the salt of a weak acid or a weak base. These weak acids and bases buffer best within 1 pH unit of their pKa. Explain how phosphate can buffer over such a wide pH range: (5.5-8.5).


Lab 5-B:


1. Calculate the final concentrations of NAD and lactate in each of the reaction mixtures that you made in the enzymatic reactions during the Parts I-III sections of this lab exercise. Recall that you started out with 100 mM NAD and 2.0 M lactate, but diluted these solutions to a final concentration in 1.2 mL of reaction mixture. The C1V1 = C2Vequation can be used to make these calculations.

2. Make a table in an Excel spreadsheet for each of the experiments you did to measure enzyme activity, one for the optimal pH, one for the optimal temperature, and one for each pH. In your table, list the slope of your enzyme progress curve (change in absorbance over unit time) BEFORE AND AFTER addition of the enzyme to the cuvette. Be sure to include the correct units of measurement, in this case A340/sec.

NOTE: If your spectrophotometer was not able to give you these slope measurements, you will

need to plot your enzyme progress curve manually. From the absorbance readings, plot A340 (y-

axis) against time in seconds (x-axis). Find the slope of the lines during the linear range of the

curve.


3. Enzyme activity is expressed as the rate of product formation per unit time. Usually the units of activity are in micromoles of product formed per second. So far, our rates are expressed in change in absorbance per second, so we need to convert absorbance units to micromolar concentration of NADH. The conversion factor for such a calculation is called the molar extinction coefficient, or simply the absorptivity constant. (See Basic Laboratory Methods for Biotechnology by Seidman & Moore, Ch. 20 for a complete discussion of light absorption.)
The absorptivity constant for NADH is 6220 M-1cm-1 at 340 nm. This means that if you had a one molar solution of NADH in your cuvette (which is 1 cm wide), your solution would read an absorbance of 6220. Obviously, we cannot read such a high absorption, but we are also not using such a high concentration of NADH.
Calculate the following:

    1. The absorbance of a 2.2 x 10-5 M solution of NADH in a 1-cm cuvette at 340 nm.

    2. The concentration of NADH that reads 1 absorbance unit at 340 nm in a 1-cm cuvette.

4. To convert a rate of absorbance change at 340 nm in a 1 cm cuvette to the rate of NADH formed in micromolar , you must convert the rate of absorbance change at 340 nm to the NADH concentration in the 1 cm cuvette and the 1.2 mL reaction volume. The following equation will work:



Activity = amount of product formed / time

1 unit of activity (U) = 1 M/sec
enzyme activity (U) = [(A340/sec) / 6220 M-1] X 106 M/M X volume in cuvette (L)
So, assuming the final volume of reaction mixture in the cuvette was 1.2 mL, (or 0.0012 L)
Enzyme activity (U) = slope of progress line (sec-1) X (0.0012/.00622)
= slope of progress line (sec-1) X 0.194


Go back to your tables in your spreadsheet and convert your slopes (A340/sec) to units of activity, using the equation above.




  1. Your calculation of unit activity of your enzyme in the reaction mixture has related the change in NADH concentration per second in your cuvette. To make sure that the units of activity that you have just calculated are related to the amount of enzyme added to the reaction mixture, you must convert units activity to units of activity per mL of enzyme added.

Go back to your tables in the spreadsheet and divide the units of activity by the volume of enzyme (in mL) added to the 1.2 mL reaction mixture. So, if you had added 10 uL of enzyme to the cuvette, you should divide your units of activity in the cuvette by 0.010 mL to give you the units per mL of heart crude extract.





  1. Now that you have calculated the concentration (U/mL) of LDH enzyme activity in your crude extract, you can evaluate the effects of pH and temperature on enzyme activity.

Go to your spreadsheet and create graphs of each of the experiments that you performed. If you need help with creating graphs, refer back to instructions provided in Unit 4, or go to a tutorial on Excel graphing located at www.geospiza.com.


Be sure to title your graphs and to label the graphs with the correct units of measurement:


Title

x-axis

y-axis

Effects of pH on enzyme activity of LDL

(room temperature, __pH)



Buffer pH

LDL activity (U/mL)

Effects of temperature on enzyme activity of LDL

(____ pH)



Temperature (oC)

LDL activity (U/mL)


References


Berg, Tymoczko, & Stryer Biochemistry (5tth Ed) (2002), available at www.ncbi.nlm.nih.gov


Porter, S. Tutorial on Excel graphing. Available at www.geospiza.com
Price, N,C, (Ed.) Proteins Labfax. Academic Press. (1996)
Walsh, Gary, and Denis Headon. Protein Biotechnology. John Wiley & Sons. 1994
Worthington,C.C., Worthington Manual, Worthington Biochemical Corp., NJ 1988

BITC2411 Biotechnology Laboratory Instrumentation ACC Lab Manual, 2nd Edition

Unit 5. Developing an Enzyme Assay 2007



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