Unit 5 developing an enzyme assay introduction
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Extraction and Assay of Lactate Dehydrogenase Activity
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.
CH3CCOO- + NADH CH3CHCOO- + NAD+
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!
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.
Part I. Enzyme extraction
Each group is to take 1/8 beef heart. Remove fat and connective tissue from a beef heart with scissors.
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).
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.
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.
Turn on the spectrophotometer and set the wavelength to 340 nm. Allow to warm up about 10 minutes.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
As above, subtract the rate of nonenzymatic NADH formation from that of the enzymatic NADH formation to identify the optimal temperature for LDH.
Report your results in a table.
Questions for Unit 5
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?
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).
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 = C2V2 equation 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
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:
The absorbance of a 2.2 x 10-5 M solution of NADH in a 1-cm cuvette at 340 nm.
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:
Go back to your tables in your spreadsheet and convert your slopes (A340/sec) to units of activity, using the equation above.
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.
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:
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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