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A LABORATORY DEMONSTRATION OF BLOOD CATALASE R. QUENTIN BLACKWELL and LEONARD S. FOSDICK Northwestern University Dental School, Chicago, Illinois
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recent calorimetric method of Landahl' for the heat more rapidly than it can be lost from the system determination of the blood catalase level is a simple to the environment; hence the resultant temperature and rapid procedure, suitable not only for research rise. Since the degree of acceleration of the reaction purposes2 but also for a laboratory experiment in bio- is, within certain limits, proportional to the catalase chemistry. The method is especially interesting be- activity, it is possible to estimate that activity from cause it demonstrates graphically, by means of tem- the amount of heat liberated. To simplify the method perature rise, the remarkable effect which the presence Landahl measured not the heat liberated, but the of a small amount of enzyme can have in accelerating maximum temperature rise of the solution under the rate of a chemical reaction. We have found it standardized conditions. The standardization of conexpedient to demonstrate the experiment to the bio- ditions include: ( a ) pH, nature, and amount of buffer, chemistry students when they begin their laboratory (b) amount of HrOl, (c) amount of blood, (d) nature of thermal insulation, ( e ) heat capacity of the final soluwork on enzymes. In the Landahl method a measured amount of tion, container, and thermometer. hydrogen peroxide solution is added to a buffer solution containing a small amount of blood, the solutions are THE METHOD mixed thoroughly, and the temperature rise of the The original article by Landahl' should be consulted mixture is noted. The decomposition of hydrogen for the discussion of the various modifications v.hich peroxide: he studied. The procedure to be described below, a slight modification from Landahl, proved to be most satisfactory for our purposes. (The equipment, part is an exothermal reaction. In the presence of a cata- of which appears in Figures 1 and 2, and solutions lyst, especially in the presence of catalase, the derom- ment,ioned in the procedure will be described briefly position reaction proceeds a t a rate sufficient to liberate after the procedure.) Preparations lo Re Made Prior to Drawing Blood ' LANDAHL, H . D., P m c . SOC.E z p . Rid. Med., 84, 74-9 (1953). Sample. *ENGLANDER, H.R.,I. L. SHKLAIR, R. Q. BLACKIVELL, AND L. S.FosnIcK, 3. Pe72odolzl.,26, 233 (1055). ( 1 ) Add exactly 4.50 ml. of pH 7.0 buffer solution to each of a numher of n1a.stic tubes, stopper, and place in a thermostatted water bath a t 25°C. (2) Place a suitable sample of 30 per cent H 2 0 2 in the 25°C. water bath. (3) Shake down the mercury in the clinical thermometers and place them along with an Anschiitz thermometer in the 25'C. water bath. (4) Check the temperature of the wooden block and bring it to 25°C. by use of large, water-filled test tubes (previously equilibrated to 25'C. in the bath) immersed in the holes with the cork collars removed. Figure 1. Equipment Used far Cetelsse Determination 588
NOVEMBER, 19%
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Figure 2.
Comparison of Regular Clinical T h e r m o m e t e r with Special T y p e Used in This Work
Remove the tubes and replace the cork collars when the experiment is ready to begin. Experimental Procedure. (1) Prick the finger or ear lobe of the patient and obtaiu exactly 0.01 ml. of hlood in each of two 10microliter pipets for duplicate determinations (use the rubber tube and mouthpiece as used with regular blood pipets). (2) Immerse the tip of the pipet in the buffer solution, draw some solution into the pipet, expel the contents by blowing, wash the pipet by repeated drawing and expulsion of solution. (This must be done immediately to prevent the clotting of blood in the capillary of the pipet.) Repeat the operation with the second pipet in a second tube of buffer. (3) Insert t,he tuhes containing the 4.50 ml. of buffer plus the 0.01 ml. of hlood into the wooden block support. (4) Insert the Anschiitz thermometer into each tube, stir well, and record each temperature to the nearest 0.l0C. This temperature should be in the range 25 =t0.5"C., and preferably a t 2 5 T . (5) Add one small drop of capryl alcohol to each tube (to prevent loss of solut,ion by foaming when t,he oxygen evolution begins). (6) With a pipet add exactly 0.5 ml. of 30 per cent Hz02 to each tuhe, immediately insert a clinical thermometer, and stir steadily for 15 seconds. (7) Allow the tubes to stand 10 minutes or mow and record the maximum temperature reading. (8) Subtract t,he initial temperature reading (ohtained with the Anschutz thermometer) from the fiual one read from the rlinical thermometer to obtain the temperature rise. MATERIALS AND SOLUTIONS
The plastic tubes used were Lusteroid-brand celluloid centrifuge tubes, 13 X 95 mm., weight approximately 2.5 grams (Figure 1). The wooden blork mas made of hardwood, 5L/nX 86 / 8 X 3l/% in. It has fifteen one-in. holes, 3L/4in. deep, spaces 11/%in. between centers. Collars for the holes were made from No. 14 corks by cutting off the tops to leave 7/8 in. of height, and boring holes to provide a loose fit for the plastic tubes. The collars were inserted to a depth of about 3/8 in. into the holes in the wooden hlock.
Pyrex tuhes, 25 X 150 mm., filled mith water and corked, were used to bring the wooden-block temperature to 25% (The tubes mere always carefully dried on the outside after removal from the m-ater hat,h and before insertion into the blork.) Initial temperature readings in the tubes were taken with t,he Anschiitz thermometer, range - 5 to 60°C., calihrated in 0.2"C. For the temperature rise it is much more convenient to use a maximum-temperature thermometer, .such as a cliuical thermometer, than to observe steadily an ordinary thermometer uutil the maximum temperature is reached. Landahl used ordinary clinical thermometers; however, they are usually made % ~ i tah range of 92-108'F. (equivalent to 33.342.2'C.) so that in the experiment a temperat,nre rise of a t least 8% is required to register a reading. I n anticipation of an occasional reading lower than this in the human and in blood other than human, we ordered special thermometers of the clinical type with a range of 2540°C. calibrated in 0.IoC." Figure 2 allows a comparison of the ordinary clinical thermometer (the smaller thermometer in the picture) with t,he special type. A small laboratory centrifuge is used routinely for "shaking do~vn" the mercury thread in the clinical thermometers. Ordinary 10-lambda micropipets were suitable for accurate measurement of the blood samples, but the hlood must not he alloved to stand in the pipets. The p H 7.0 phosphate buffer used was that recommeuded by Landahl (one part 0.1 M Na2HP04t,o one part 0.04 M NaHzP04). Instead of using one rnl.of this solution in each tube and adding 3.5 ml. of water' it is more convenient to dilute the buffer with water in those proportions (e. g. 1 liter buffer mith 3.5 liters water) and add 4.5 ml. of the diluted buffer to each tuhe. (For routine determiuatious it is convenient to fill 100 tubes a t a time, stopper, and keep refrigerated until used.) The 30 per cent hydrogen peroxide solut,ion (Superoxol) used mas used as obtained. RESULTS
Laudahl found that d h 58 normal individuals the meau temperature rise produced by the action of 0.01 ml. of human blood was about 12°C. Our results on normal subjects were as folloms: The special clinical-type thermometers were obtained from Rsseher and Betzold, Inc., Chicsgo, Illinois.
JOURNAL OF CHEMICAL EDUCATION
590
Number of subjects Mean tern eretu~e~ i s e Standard Beviatiin ' ltnnge
163
10.9'C. 1 .ST. '7.2-152°C.
~h~ slightly lower meall temperaturerise foulld ill our mork, if not due to sampling differences, may reflect procein part a differenre between the dures med (one notable difference in our procedure was the use of the larger clinical thermometers which
have a higher specific heat). I t serves to illustrate the necessity for each laboratory to establish its own calibration curve where temperature rise is plotted versus catalase activity (in suit,able units such as Keil unitas 0' QCat, etc.). For this purpose oue may use standardized catalase solutions or dry samples of catalase.' 4 ~ 0 t h standardized catalase solutions and cntiatalnre in ponder form w e svailrthl~irom Amour and Company, Chicago, Illinois.
