A. G. Spllttgerber, K. Mitchell. G. Dahle, M. puffer; and K. Blomquist Gustavus Adolphus College St. Peter, Minnesota 56082
I I I
The Kinetics and Inhibition of the Enzyme Methemoglobin Reductase A biochemistry experiment
It has been noted ~reviously(1) that there is a scarcity of convenient enzyme kinetic experiments for use in an undereraduate biochemistry laboratory. It would seem esexperience in sentiai that the student have some monitoring enzyme reactions and in using the Michaelis-Menten equation. In this regard, experiments published previously (1-4) are interesting and instructive. These experiments involve such hydrolytic enzymes as hacitracin (2), chymotrypsin (1, 3), and yeast invertase (4). This article describes the preparation and kinetics of a convenient oxidation-reduction enzyme system, methemoglohin reductase or diaphorase. The system is convenient because a crude enzyme extract is easily prepared, and a spectrophotometric assay method is possible. I t is not necessary to use the natural suhstrate, since the enzyme also has the ability to reduce certain autooxidizable dyes (5). The enzyme system obeys classical Michaelis-Menten kinetics both with reeard to suhstrate and with reeard to the required ~ ~ ~ ~ ' c o f a cInhibition tor. studies are easily made with salicylate ion, a competitive inhibitor (6). The Enzyme System
The erythrocyte contains several enzymes of which the natural suhstrate appears to he methemoglohin (7). These enzymes maintain hemoglobin in the active oxygen-carrying form. Some of these enzymes require a NADH cofactor, while others require NADPH. Current knowledge about the mechanism of the reduction of methemoglohin comes primarily from studies of the erythrocytes of patients suffering from hereditary methemoglohinemia (8). There is evidence that this inborn deficiency involves a NADH-dependent enzyme, the NADPH enzymes under normal conditions being relatively inactive in reducing methemoglohin.
Attempts have been made to purify and characterize the active enzyme with varying results (9-12). In some cases (9, IO), a flavin moiety has been found; in other cases (11, 12) no flavin was detected. In all cases it has been found that the purified enzymes are ineffective in reducing methemoglobin, presumably because an electron carrier necessary to the conversion has been lost in the purification procedure (7). The NADPH enzyme systems, inactive under normal conditions, may become active in the reduction of methemoglohin if an artificial electron carrier is added. This may account for the enhanced reduction of methemoglobin in the presence of autooxidizable dyes such as methylene blue (13). In addition to their natural suhstrate activity, all the methemoglohin reductases display diaphorase activity, the ability to reduce dyes such as 2,6-dichlorophenol indophenol (DCIP) at high rates. Because of the relative differences in rates of DCIP and methemoglohin reduction (9200:l) (5), the DCIP system is more useful as a student experiment. Preparation of the Enzyme
The enzyme extract is prepared by preferential ahsorption of the enzyme on DEAE cellulose (9) from a red cell hemolysate by the procedure which follows. Eight 50-ml centrifuge tubes are filled with whole blood and centrifuged a t 5000 rpm far 5 mi". The plasma is removed and replaced by 1% NaCl solution. The red cells are resuspended, centrifuged, and the NaCl layer removed. This washing procedure is repeated three times or until the supernatant is clear. The packed red cells fmm the last wash are combined with two volumes of distilled water and the mixture allowed to stand 30 min in the refrigerator. This forms a hydmlysate about 6% in hemoglobin. DEAE cellulose is equilibrated with 0.01 M sodium phosphate buffer a t pH 1.5 and filtered under suction. Ten grams of the filter cake is combined with 50 ml of the hemolysate and sufficient pH 7.5 buffer such that the slurry may be mechanically stirred. Stirring is carried out for about 1 hr in the refrigerator. The cellulose is filtered under suction and washed with 0.01 M pH 7.5 phosphate buffer until the hemoglobin is visually removed. Then approximately 2M) ml of a 0.1 M sodium phosphate buffer a t p H 5.5 containing 0.2 M NaCl is used to elute the enzyme fmm the cellulose. The resulting crude enzyme extract is stable in the refrigerator for several weeks.
Enzyme Assay All reactions are run in l-cm speetrophotometer cuvettes at the wavelength of maximum absorption of DCIP (600 nml i l l ) . The following quantities of various reagents are pipetted into the reference and sample cuvettes.
Reference
Figure 1. Methemoglobin reductsse kinetics with constant NADH and varying DCIP concentration, Lineweaver-Burk plots with and without inhibitor.
880 / Journal of Chemical Education
1m10.05 Mtris-HCI buffer, p H 7.5 0.5 m14 X 10-*M DClP 0.5 m18 X 10-'MNADH + 6 X I W 3 MEDTA 0.5 ml enzyme extract 0.5 ml water
Sample 1ml tris buffer 0.5 ml DCIP
0.5ml6 X 10-SMEDTA 0.5 ml enzyme extract 0.5 ml water
It should be noted that the reaction actually takes place in the reference cuvette. Since the dye is decolorized during the reac' tion, the absorbance in the sample cuvette appears to increase. Reaction is initiated by addition of NADH to the cuvette with shaking. Absorbance is initially adjusted to zero end readings are taken every 30 s. Similar runs are made using, respectively, 0.4, 0.3, 0.2, and 0.1 ml of DCIP, the euvette volume being maintained at 3.0 ml with additional water. Plots are made of absorbance versus time and the slopes are converted to reaction velocities (V) in moles/l-s using a rnillimolar absorhtivity value of 20.1 for the dye ( 1 1 ) .
Typical Data, Reduction of DClP by Bovine Methemoglobin Reductase K m
Vmnx
Substrate
(mole/l)
(moleA-s)
NADH DCIP
5 . 7 X 10-3 1.15 X 10.'
8.7 X 10-8 1.75 X 10.'
KI ( S a l i e y k t e Ion)
4.4 X 4.9
10-2
x lo-'
A Lineweaver-~urkplot of 1 / V versus 1/(S) is made (Fig. I), ( S ) referring to DCIP concentration. This form of the Michaelis-Menten equation is given by
The plot should he a straight line with slope Km/Vmar and K, may he and y intercept l/Vmax,from which V,, found (the tahle). Under the reaction conditions outlined above, the highest substrate concentration (6.7 x M DCIP) yields a A(OD)/min value of fmm 0.02-0.03 depending on the enzvme extract. This is too small to measure well with, for example, a Spectronic 20 calorimeter, so that a more sophisticated instrument should be used. Also, the data of the tahle were obtained using an automatic pipet to fill the cuvettes. However, the use of ordinary pipets does not result in much sacrifice of accuracy. Inhibition by Salicylate
Five runs are made under the same conditions as in the nrevious assav exceut that 0.02 M sodium salicylate is present in each case. Reagent quantities are the same except that the 0.5 ml of water is replaced by 0.5 ml of 0.12 M sodium salicylate. Runs containing, successively, 0.5, 0.4, 0.3, 0.2, and 0.1 rnl of DCIP are made, and a second T d.n -R nlnt ~ ~.. ir. sknrennrerl. - .e.-w-e. -~- ~-e-~- . r.. -.-- .-. Inspection of the inhibitor and no-inhibitor plots (Fig. 1) shows that they have different slopes hut the same y intercept, hi^ situation corresponds to competitive inhi. bition, and the kinetic expression for this type of inhihition is a function of K ~ , the inhibitor constant, and (I), the inhibitor concentration 1IV
=
(K,"lV ,... ....." )I1 m,,"
+ (I)lK,l(l/(S)) + . .. .
11V , .,",, .... .
The slope of the inhibitor plot is thus seen to he larger by a factor (1 + (I)/KI) than that of the no-inhibitor plot. The inhibitor constant (the tahle) is easily determined algehraically fmm the slopes and the common intercept of the two plots. Cofactor Studies
The variation of rate with NADH concentration is studied with and without inhibitor. The assays are the same as in the preceding cases except that in successive runs, 0.5, 0.4, 0.3, 0.2, and 0.1 ml of NADH is used, constant cuvette volume being maintained by addition of 0.006 M EDTA. Results are shown in Figure 2 and the tahle. Inspection of Figure 2 shows that the inhibitor and no-inhibitor plots in this case have different slopes and different y intercepts, hut the x intercepts are the same. This indicates noncompetitive inhibition, the kinetic expression being given by
In this expression hoth the slope and y intercept are elevated by a factor (1 (I)/KI). The inhibitor constant (the table) is determined in the same manner as before.
+
+I", -1
A 0 INHIBITOR
Figure 2. Methemoglobin r e d u c t a s e kinetics with c o n s t a n t DCIP a n d varying NADH c o n c e n t r a t i o n Lineweaver-Burk plots with a n d withoul inhibitor.
-." The data presented in the table can he obtained in two 3-hr laboratory periods provided the reagents (except for NADH) are made up beforehand, Another laboratory peri0d is required for preparation of the enzyme extract. As shown in the figures, data from a single enzyme system show hoth competitive and noncompetitive inhibition. From this data, the student may speculate on the probable physical situation a t the active site of the enzyme. Many other kinetic experiments suggest themselves. Diaohorase is available as a commercial preparation (Nutritional Biochemicals) which gives resulti k i t h DCIP similar to the enzyme extract. Other substrates, e.g., methemoglohin, may be used in kinetic studies either alone or in the presence of the electmn mediators methylene blue or ferncyanide ion (10). Kinetic studies after further purification of the enzvme hv such methods as ael permeation chromatography (9, 10) may also he undertaken.' Literature Cited 111 Hurlhut, J . A . Ball. T. N . , Pound. H. C.. and Gravel. d. L.. J . CHEM. EDUC., i n..s,i. w i. n, . . ..a.i,. (21 Curragh. E.F., andThompson, D.J..Educ. inchem., 10. 17 11973). 131 Bender. M L . . Kezdy. F J . , and Wedler.F.C.. J. CHEMEDUC..44.84(19671. (41 Clark JI.. J . M.. "Exponmentsl Biochemiafry," W. H. Freeman. San Francisco.
1971.p. 113. (81 Jaife. E. R.. and Neumsnn, G.. in "Metabolism and Membrane Penneshility of Ewthrocvles Verlae. S t u t t ~ . and Thmmbocvtes, 1st International Svmomium." . . gafi. 1968, p . 83. 191 Scolf. E. M..andMcGrav. J.C., J. Blol Chsm.. 237.249 119621 (101 Kuma. F.. andInomata. H . J . Blol Chrm.. 247. 666(19721. (11) Surits, Y., Nomura. S.. and Yoneyama. Y . . J B i d Chsm.. 246,6W2(1971) (12) ~ ~ m ~ iF .kM.. i ~Kenuar. ~ . G. K.. and Kajita. A.. in "Methemoglobin Reductases. In Hereditary Disordrm of Erythmcyto Metabolism." Grune & Strafton, New York, 1968,p.67. (131 Warbuq, O., Kubowitz,F.. andChristian. W.. Blochem. 2..227,245(19301
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Volume 52, Number 10, October 1975 / 681
