CAUTION: The following procedure involves hazardous reagents.  If you attempt any of the procedures or experiments mentioned on this site, you do so entirely at your own risk. Biochemistry experiment: Colorimetric Assay for Catalase Activity C. Thorsten Introduction:   Enzymes are proteins that catalyze specific reactions in living organisms. In many cases, enzymes can be purified and studied in vitro. A problem presents itself, though: how do you know for certain you have a particular enzyme?  How would you distinguish that enzyme from all the other enzymes and proteins in solution?  The answer is to involve the enzyme in a reaction for which it is specific.  Ideally the procedure would revolve around formation of some colored end-product, or perhaps the de-colorizing or changing of a colored substance in a measurable fashion. The absorbance of this colored solution is measured at a specific wavelength and is compared to a set of standards; from this, one can calculate the enzyme's activity. Generally speaking, the colorimetric assay (and the wavelength at which to measure absorbance) will be different for each enzyme; the experimenter must decide well in advance what assay to use.  The following experiment is a simple introduction to spectrophotometric enzyme assays. For demonstration purposes, you can conduct it qualitatively:  that is, simply showing the general procedure, the formation of a colored product, the process of measuring absorbance, and so forth. On the other hand, a quantitative approach would focus on careful measurements. In other words, you can make this experiment as simple or as comprehensive as desired. You can take it to the level of exploring new schemes for catalase purification, for example. The possibilities are almost without limit here. In any case, it is wise to run through this experiment at least once before using it in a class. The colorimetric assay itself is based roughly on the method of A.K. Sinha (1972). I've found it necessary to fill in some "holes" in the procedure and to explain some details which the student may not readily assume. Of course, I've tried to phrase this write-up in a way that's easier for the beginner to follow than if he or she were reading directly from a scientific journal. Supplies: Spectrophotometer and glass cuvettes; Centrifuge; Potassium dichromate; Hydrogen peroxide solution; Glacial (98-100%) acetic acid; Phosphate buffer (0.01 M), pH 7.0; Raw potato or raw liver; Blender; Funnel and filter paper; Droppers; Test tubes; Small (ca. 50 mL) flask; Distilled / deionized water ("dH2O"); Stopwatch. Procedure: Note: This experiment can take as little as 20 minutes if it's set up ahead of time and done in a qualitative manner for demonstration purposes; however, in its ordinary form as a laboratory exercise it can take several hours. Preparing the reagents (but not the catalase) in advance will save some time. Preparing the Reagents: Prepare 50 mL of a 5% aqueous solution of potassium dichromate in distilled water. Slowly add 150 mL of glacial acetic acid to this. Place the resulting solution in a 250 mL reagent bottle and set aside. Do not spill this liquid!  It is toxic and corrosive. Prepare a 0.2 M solution of hydrogen peroxide. Hydrogen peroxide from drugstores is nominally 3% by weight; this corresponds to approximately 0.9 M H2O2 (True 3.0% H2O2 by weight would be about 0.88 molar; "3%" hydrogen peroxide U.S.P. can be anywhere from just over 2% to almost 4%). Prepare or obtain about 250 mL of 0.01 M phosphate buffer, pH 7.0. Enzymes are generally the most stable in a solution that's buffered near that of their original environment. The Standard Curve:  Sinha's procedure (1972) recommends using 6 different test tubes containing increasing amounts of H2O2 (10 up to 160 micromoles). To each of these is added 2 mL of the dichromate / acetic acid reagent. When a blue precipitate forms in each, do the following: 1.) Heat each test tube for 10 minutes in a boiling water bath to decompose the blue precipitate. This will leave a green solution of chromic acetate. 2.) Cool to room temp. and add to each tube enough dH2O to make the volume up to 3 mL. 3.) Transfer 3 mL of the first test tube's contents to a clean cuvette and measure the absorbance at 570 nanometers in the spectrophotometer. Repeat this for the remaining five tubes, using a cleaned cuvette each time. 4.) Using the data obtained, plot a graph of Absorbance at 570 nm (y-axis) versus micromoles of H2O2 in the cuvette (x-axis). The best-fit "curve" through your data points should be a straight line, assuming all goes well. You should probably use a modern spreadsheet program to do the linear regression for you... otherwise, you'll need to use pen-and-paper linear-regression methods to come up with the equation of the best-fit line, a procedure we won't get into here. Catalase Preparation:  Don't do this until you've prepared all other solutions and done the "the standard curve" part first. Enzymes, once freed from their native environment, tend to lose their activity. This can be slowed by (1) doing the enzyme preparation as late in the procedure as possible, (2) using a buffer at physiological pH (see above), and (3) keeping the enzyme preparation cold.1 Catalase is present in a number of readily-available sources. A raw potato or other tuber is the favorite source for classrooms; raw liver or raw meat can be used instead. Purified catalase can be purchased from biological supply houses as well. If you're starting with a potato, simply homogenize some chilled, raw pieces in a blender with cold, distilled H2O, pour it through some filter paper into a clean beaker, and centrifuge out the remaining solids. For our modified experiment, centrifugation speed is not critical2; pick a medium setting and let it run for five to ten minutes. Using a dropper, siphon off the clear liquid and put it in a test tube (leaving all solids behind) and immediately put it in the refrigerator or stick it into a beaker full of ice. Enzyme Dilution:  This is necessary to get the enzyme concentration within a range that can be measured properly by the method. Too much enzyme will act so rapidly that there won't be enough H2O2 left to detect with the colorimetric assay. Finding the proper dilution of your enzyme preparation will involve some trial and error; dilutions of up to 1:1000 may be required (three 1:10 dilutions in series). At each dilution step you can test the activity of the enzyme on H2O2 (jump ahead to the Assay procedure, but instead use this "temporary" preparation to see how much H2O2 is decomposed before adding the dichromate / acetic acid reagent; if the absorbance is within your spectrophotometer's range of detection, you may be able to stop diluting.)3 Sinha (1972) found it necessary to add 0.5% bovine serum albumin (BSA) to stabilize the catalase at such high dilutions. The BSA will not interfere with the assay, but it should be subtracted from the result if and when you do a determination of total protein in the solution (see step 8 of Assay, below). The Assay: Once you've made your graph of Absorbance at 570 nm vs. micromoles of H2O2, you can use aliquots of your enzyme preparation to test how much H2O2 the enzyme destroys in various time intervals; alternatively, you can use differing amounts of enzyme preparation and a fixed time interval. If you've found the proper dilution for the enzyme, the former is suggested. Its procedure is as follows: 1.) From the H2O2 solution, add enough to a small flask so that there are 800 micromoles (0.0008 moles) H2O2 in it. This corresponds to 4 mL of a 0.2 M solution of H2O2. 2.) Add 5 mL of the phosphate buffer to this. 3.) Add 1 mL of the enzyme preparation that was diluted according to the instructions, above. Swirl the flask gently, but don't actually shake it or you'll throw off your results. 4.) Withdraw 1.0 mL of this reaction mixture and inject it into 2.0 mL of dichromate / acetic acid reagent. This time you don't have to worry about gentle swirling- you can go ahead and agitate it. The goal here is to halt the enzymatic reaction completely.  Using different test tubes, repeat this procedure at 60 second intervals. 5.) Heat each test tube for 10 minutes in a boiling water bath to decompose the blue precipitate and produce a green solution. 6.) Measure the absorbance at 570 nanometers in the spectrophotometer. 7.) Using the "standard curve", determine how much H2O2 was left in the solution when the enzyme was stopped with acetic acid. 8.) Optionally, determine how much protein is in the solution and relate this to enzyme activity. The method of Lowry, et al. (1951) or of Bradford (1976) and Spector (1978) is suggested. If you don't have the time or desire to perform these methods, try measuring the total mass of solids in the evaporated enzyme preparation and relating this to activity. Obviously, there will be other proteins, carbohydrates, and nucleic acids in such a residue, so you won't be able to know how much catalase is really in there unless you've either gone through a whole purification routine or you've purchased your catalase from a supply house. Discussion: The dichromate / acetic acid reagent can be thought of as a "stop bath" for catalase activity. As soon as the enzyme reaction mixture hits the acetic acid, its activity is destroyed; any hydrogen peroxide which hasn't been split by the catalase will react with the dichromate to give a blue precipitate of perchromic acid. This unstable precipitate is then decomposed by heating to give the green solution. As Sinha admits, a problem with the dichromate assay is that sugars and certain amino acids will also react; however, the 1972 paper indicates that these molecules shouldn't have significant effects at the concentrations we'll be dealing with. Naturally, we can avoid the matter of interference altogether if we use as pure a catalase preparation as possible. Many scientific papers and at least a couple of books are devoted to the subject of purifying proteins; it's worth it to familiarize yourself with the basic principles. A good place to start is Protein Purification by Robert K. Scopes (1994). Catalase is only one of many readily-obtainable enzymes that can be assayed with a spectrophotometer and some fairly common lab reagents. Phosphatases are another favorite class of enzymes to study, but that's just an example. Consult the biochemical literature of the past 60 years or so, and you'll be sure to find numerous enzymes that can be assayed satisfactorily in the classroom. Notes: 1 The best temperature is just above freezing- otherwise, ice crystals that form can actually destroy enzyme activity unless the freezing is extremely rapid and thorough. Ordinary freezers can actually cause enzyme denaturation, not hinder it- though this varies depending on what else is in the solution besides the enzyme (salts, stabilizing reagents, other proteins, etc), among other factors. When ready to do the enzyme assay, you should let the enzyme mixture reach room temperature before adding the reagents.  Temperature has a marked effect on enzyme activity, and we don't want it varying between assay tubes if any kind of meaningful results are desired for this experiment.  Back to article 2 If time permits, pour an equal amount of homogenized potato into a few, separate centrifuge tubes and centrifuge each at a different speed (keeping the time constant).  See which speed gives you the highest enzyme activity in the clear solution that results.  Test the solid matter at the bottom of the tubes as well.  Do these solids have any catalase activity? Why or why not?  You can measure a solution's specific activity as micromoles H2O2 destroyed (per second) divided by the number of milligrams protein per ml of preparation. Obviously, the purer the preparation, the more of this protein is going to be actual catalase. Back to the article 3 Since you've already run the standards at this point, you can stop diluting when you get a reading that falls somewhere on your standard curve. Try to stay within these confines, since you can't be sure that absorbance readings outside this range will still be part of a linear function. In other words: we've made a standard curve that shows a linear relation for absorbance versus micromoles H2O2 in solution, but it is not safe to assume that the relation will be linear everywhere, or even anywhere, outside the range we've tested. If you want to prove linearity for a wider range of absorbances vs. concentrations, it's necessary to make a more comprehensive standard curve. Don't bother using more than about six points, though, unless you've got a surplus of time on your hands... Back to the article References: Bradford, M.M. "A Rapid and Sensitive for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding." Analytical Biochemistry 72, 248-254 (1976). Lowry, O.H., et al. "Protein Measurement with the Folin Phenol Reagent." Journal of Biological Chemistry 193, 265-275 (1951). Scopes, Robert K. Protein Purification: Principles and Practice. New York: Springer-Verlag, 1994. Sinha, A.K. "Colorimetric Assay of Catalase". Analytical Biochemistry 47, 389-394 (1972). Spector, T. "Refinement of the Coomassie Blue Method of Protein Quantitation." Analytical Biochemistry 86, 142-146 (1978). Copyright:  Articles, photographs, and other contents of this site are property of CR Scientific except where otherwise noted.  They may not be copied or distributed, in whole or in part, without prior written permission (click here for contact info). Articles / Experiments Index CR Scientific Catalog Main Page