II. IMPROVED METHOD OF ISOLATION; INHIBITION AND INACTIVATION; REACTION WITH OXYGEN. BY ERWIN HAAS, CARTER J. HARRER, AND T. It.

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1 CYTOCHROME REDUCTASE II. IMPROVED METHOD OF ISOLATION; INHIBITION AND INACTIVATION; REACTION WITH OXYGEN BY ERWIN HAAS, CARTER J. HARRER, AND T. It. HOGNESS (From the George Herbert Jones Chemical Laboratory of the University of Chicago, Chicago) (Received for publication, January 10, 1942) A previous paper (1) reported the isolation of an enzyme, cytochrome c reductase, which can be rapidly reduced and oxidized by dihydrotriphosphopyridine nucleotide and ferricytochrome c, respectively. The reductase thus establishes a link in the chain of respiratory enzymes. The preparations of the enzyme obtained previously contained a small amount of impurities, especially a few per cent of a hemin compound; moreover, there was a rather low yield in some of the isolation steps. Modifications of the previously described procedure increased the yield 8-fold and resulted in a 98 per cent pure reductase, free of hemin. Isolation of Enzyme Yeast was washed, dried, and autolyzed, as previously described (1). Table I indicates the amount of enzyme which could be extracted, under optimum conditions, from five different sources. The figures of Table I represent minimum values of the enzyme concentration in yeast, since probably only a fraction of the total enzyme present could be extracted. In the method for the isolat,ion of the enzyme (l), Steps 1 to 4 remain unchanged. Directions for We modified steps follow. Step 5. Adsorption on Aluminum Hydroxide Gel-5 gm. of the enzyme preparation obtained after Step 4 are dissolved in 200 ml. of water and the ph is adjusted to about 9 with 4 ml. of N potassium hydroxide. y-aluminum hydroxide gel (2) is added in fractions, until the supernatant solution becomes colorless. Approximately 7 gm. of aluminum hydroxide are required. The enzyme is eluted from the aluminum hydroxide by twice adding 120 ml. of a solution which is 64 per cent saturated with respect to ammonium sulfate and 0.1 N with respect to ammonium hy- 1 This hemin compound, in the reduced form, has the or-band at 557 mp, the Soret band at 420 mp. The presence of this hemin compound in yeast was confirmed in a recent communication by Bach, Dixon, and Keiliu (Nature, 149, 21 (1942)). They isolated a new soluble cytochrome component, bl, from yeast, which, on spectrometric evidence, seems to be identical with the one previously observed here (1). 341

2 342 CYTOCHROME REDUCTASE. II droxide. The elution is continued by adding two 160 ml. portions of a solution which is 40 per cent saturated with ammonium sulfate and 0.1 N with respect to ammonium hydroxide. The enzyme is precipitated from the combined eluates by increasing the ammonium sulfate concentration to 65 per cent saturation and adjusting the hydrogen ion concentration to ph 4.5 (560 ml. of eluate + 90 gm. of solid ammonium sulfate + 70 ml. of 2 N acetate buffer, ph 4.5). The enzyme is separated by centrifugation, dissolved in 150 ml. of water, and the solution brought to ph 9 with 5 ml. of N ammonium hydroxide. This solution contains 1200 mg. of protein: W = 40; purity = Step 6. Adsorption on Calcium Phosphate Gel-Tricalcium phosphate gel (about 17 gm.) is added fractionally to the enzyme solution until the supernatant becomes colorless. After centrifugation the combined pre- Cytochrome Reductase Extracted from Yeast I Source material Time of extraction, room temperature Concentration Fleischmann s bakers yeast.. Keeley s beer yeast*. Drewry s ale yeast?. Westminster ale yeastf.... Canadian ale yeasti * Keeley Brewing Company, Chicago. t Drewry s, Ltd., South Bend, Indiana. $ Westminster Brewing Company, Chicago. 0 Canadian Brewers, Ltd., Toronto. hrs. gm. enzyme per Rg. dry yeas cipitate is washed with 400 ml. of water and the enzyme is eluted with three 200 ml. portions of 0.2 M phosphate, ph 6.1. The enzyme is precipitated from the combined eluate in order to remove phosphate which interferes in the next purification step. To 600 ml. of the eluate, 310 gm. of solid ammonium sulfate are added (saturation = 80 per cent), and the solution is centrifuged. The precipitate, which contains the enzyme, is dissolved in 70 ml. of 0.03 N ammonium hydroxide. The solution now contains 340 mg. of protein: W = 67; purity = Step 7. Adsorption on Aluminum Hydroxide Repeated-Portions of aluminum hydroxide gel are again added to the enzyme solution until the supernatant liquid is colorless (about 3 gm. of aluminum hydroxide required). The enzyme is eluted from the adsorbent by washing three times with 60 ml. of a solution which is 64 per cent saturated with ammonium sulfate and 0.1 N with respect to ammonium hydroxide. In 180 ml. of the

3 HAAS, HARRER, AND HOGNESS 343 combined eluates, 140 mg. of enzyme are finally obtained; W = 155; purity = The enzyme is precipitated at ph 4.5 by increasing the ammonium sulfate concentration to 80 per cent saturation and is collected by centrifugation. The precipitate is treated with 0.30 ml. of 2 N ammonium hydroxide to adjust the ph to about 9. By evaporating in a high vacuum the suspension can be frozen and stored at 0 over phosphorus pentoxide (as drying agent). No decrease in activity was observed after 23 months of storage. Reaction with Oxygen Cytochrome reductase can be reoxidized by cytochrome c (Equation 1) or by atmospheric oxygen, according to Equation 2. CR.H2 + 2CyFe CR + 2CyFe++ + 2H+ (1) CR-H, ) CR + Hz02 (2) At low oxygen pressure the velocity of Reaction 2 is given by -dojdt = k3 (CR.Hz) (02). The manometric experiment outlined in Table II was performed to determine the reaction rate of cytochrome c reductase with oxygen. A comparison of k3 (oxygen) with kz (ferricytochrome c) shows that cytochrome reductase will react about 7 X lo5 times faster with ferricytochrome c than with oxygen. This fact indicates that the direct reaction of the cytochrome reductase with oxygen is very probably without physiological significance. Comparison with Old Yellow Enzyme-Both the old yellow enzyme of Warburg and Christian (3) and cytochrome reductase have alloxazine mononucleotide as their prosthetic group. However, the two enzymes are not identical with respect to enzymatic activity and other physical properties shown in Table III, in which kl, kz, and ks represent specific velocity constants for the following reactions. - - d(cyfe+++) dt d(tpn-ii,) dt = kl (alloxaxine) (TPN.HJ = kz (dihydroalloxazine) (ferricytochrome) -- do2 = i& (dihydroalloxazine)(oxygen) dt K designates the dissociation constant K= alloxazine X protein enzyme Since the prosthetic groups are identical, the protein must be responsible for the different properties of the two enzymes. Replacement of the pro-

4 344 CYTOCHBOME REDIJCTASE. II 25 ; center cup, 0.1 ml. of 2 N KOH. - II Reaction with Oxygen - Experiment 1. Air Experiment 2. Air Experiment 3. Oxygen X 2.25 ml. water M phosphate, ph ml M HCN mg. triphosphopyridine nucleotide mg. Zwischenferment.. 10 glucose-6-phosphate I 1.95 X lo-* mole cytochrome reductase X IO-* mole X 10m8 mole Oxygen uptake?&a. c.mm. c.mm. c.11zm a02 Experiment I-v = x = 1.63 X lo-5 (mole X liter-l X min.-l) * = (02) (C R.H,) (02) = 2.7 X lo-* (mole X liter-f) (CR.Hz) = 7.5 X 10P (mole X liter- ) = 0.80 X lo4 (liter X min.? X mole-l) Comparison of Cytochrome Reductase and Old Yellow Enzyme Rate constant k = liter X minute-l X mole-l; dissociation constant K = liter-l mole. III Enzyme Cytochrome c reductase... Old yellow enzyme.. Triphosphopyridine nucleotide kl* 85 x X lo6 -- Reaction with Cytochrome c Oxygen / I I Dissociation constant kz ka K 53,000 X lo5 0.8 X lo4 / 1 X X lo5 10 X lo X lo-9 Absorption maximum, x * Details concerning the determination of Icl and k, will be submitted in a communication dealing with the kinetics of t.he enzymatic reactions. tein of the old yellow enzyme by that of the reductase increases the activity towards cytochrome and triphosphopyridine nucleotide tremendously but simultaneously diminishes the activity towards oxygen. The autoxi-

5 HAAS, HARRER, AND HOC~NESS 345 dation of free alloxazine mononucleotide is inhibited when it combines with protein and one would therefore expect an enzyme With a small dissociation constant to react slowly with oxygen. The results of Table III confirm this relationship between dissociation of the prosthetic group and reactivity towards atmospheric oxygen. Denaturation of Cytochrome c Red&use-From the preceding data, it is apparent that the old yellow enzyme is not identical with cytochrome reductase and the question arises whether it could be a degradation product of the cytochrome reductase. In this case the denaturation of the reductase should result in a decreased activity with cytochrome but increased activity with oxygen. We have followed both reactions with enzyme preparations which were denatured by keeping them for several weeks at low temperature or for a few minutes at elevated temperature in M phosphate buffer, ph 7.2. The reaction with cytochrome was IV Denaturation of Cytochrome c Reductase Incubation Activity towards oxygen Activity towards cytochrome c _.._- per cent per cent days at 3. 64* 8.8t 10 min ~.~- ~- k = ; X 2.3 X log $ *kso = 1.1 X 1OP (min.-l) tkp = 6.0 X 1OF (min.?) k5p = (min.-l) k6,p = (min.-l) measured spectrophotometrically, as described previously (1) ; the reaction with oxygen, as described in Table II. The results are given in Table IV; k represents the velocity constants for the inactivation of the enzyme towards cytochrome and oxygen, respectively. The results of Table IV indicate that denaturation of the enzyme diminishes its activity towards both oxygen and cytochrome and therefore the old yellow enzyme cannot be considered as a degradation product of cytochrome reductase. The activity of the enzyme towards cytochrome is destroyed to a greater extent than that towards oxygen. This fact indicates that the mechanism of oxidation by cytochrome differs from that in which oxygen takes part,. Inhibition by Substituted Phenols Krahl and Clowes (4) and Krahl, Keltch, and Clowes (5), after finding that the respiration of fertilized Arbacia eggs is inhibited by certain substituted phenols, showed that with cell-free systems neither the cyto-

6 346 CYTOCHROME REDUCTASE. II chrome oxidase system nor certain dehydrogenase systems are affected by these substances. The catalytic activity of flavoproteins, however, is inhibited. To obtain information concerning the effect of 2,4-dinitro-o-cyclohexylphenol on the individual steps involved in the respiratory process, we have investigated its effect on the various isolated components of the system which involves cytochrome c, cytochrome reductase, triphosphopyridine nucleotide, Zwischenferment, and glucose-6-phosphate. With this information we then tried to correlate the inhibitory effect on isolated systems with that on the respiration of intact bakers yeast. Inhibition of Cytochrome Reductase2-Cytochrome reductase was incubated with the substituted phenol for 15 minutes (0.001 M phenol, M phosphate buffer, ph 8.3, temperature 25 ). The enzymatic activity was determined by the specific cytochrome reductase test previously described (1). The result of the experiment showed that under the specified conditions the enzymatic action of cytochrome reductase was 70 per cent inhibited. Triphosphopyridine Nucbotide (TPN) and Xubstituted Phenols-The specific test for TPN, described in a previous publication (6), can be used to study the effect of organic compounds on its catalytic activity. Despite incubation in a M solution of 2,4-dinitro-o-cyclohexylphenol, buffered with phosphate, no effect on the catalytic activity of TPN could be observed. In the activity test the velocity-determining reaction is the oxidation of dihydro-tpn. Therefore one can conclude that dihydro- TPN is not affected by this substituted phenol. Inhibition of Zwischenferment-The rate of reaction of Zwischenferment was measured by ultraviolet spectroscopy in a manner similar to the method of Negelein and Haas (7). The reduction of TPN is indicated by an increase in the light absorption at h 340 mp. Under the conditions specified in Table V, the rate of reduction of TPN is a function of the Zwischenferment concentration. As the substituted phenols exhibit a strong light absorption in the ultraviolet region, it is necessary to use very thin absorption cells during this test. Zwischenferment was incubated for 15 minutes at 25 (0.001 M phenol M phosphate, ph 8.3). Details concerning the analytical test are given in Table V. The suppression of the reduction of triphosphopyridine nucleotide indicates that M 2,4-dinitro-o-cyclohexylphenol produces a 90 per cent inhibition of Zwischenferment. Inhibition of Respiration-The action of the substituted phenol on the 2 Dr. Krahl suggested this experiment in a private communication and kindly supplied the substituted phenols.

7 HAAS, HARRER, AND HOGNESS 347 respiration of yeast was determined manometrically; the conditions are given in Table VI M 2,4-dinitro-o-cyclohexylphenol inhibits the respiration of bakers yeast 93 per cent. Inhibition of Zwischenferment by 9, Q-Dinitro-o-cyclohexylphenol Wave-length = 340 rnp; length of absorption cell = 0.10 cm.; temperature = 25O. V Experiment 1 Experiment ml M phosphate buffer, ph mg. potassium salt of glucosed-phosphoric acid mg. Zwischenferment (impure) triphosphopyridine nucleotide X 10V3 Mphenol Triphosphopyridine nucleotide reduced in 10 min. 9%. w I Effect of B,.&Dinitro-o-cyclohexylphenol on Respiration of Bakers Yeast Fleischmann s bakers yeast; temperature, 25 ; Center CUP, 0.1 Id. Of 2 N KOH; gas phase, air. - Side arm Experiment ml M phosphate, ph mg. yeast ml. H*O + 24 mg. glucose (added after 15 min. incubation) VI Experiment 2 12 mg. yeast 1 X lo+ M phenol,, Experiment X low3 M phenol > Oxygen uptake 20 I A summary of the inhibition experiments with intact yeast and with the isolated systems is presented in Table VII. The components of the isolated system are arranged in the order in which they react. There is no inhibition of the enzyme system which reacts between oxygen and cytochrome c. This observation by Krahl and Clowes has been confirmed by

8 348 CYTOCHROME REDUCTASE. II Dr. Bernard Block in our laboratory. Therefore it must be concluded that the interaction of the phenols takes place somewhere in the chain of enzymes between cytochrome c and glucose-6-phosphate. The results of Table VII indicate two possible points of interference by substituted phenols in the respiratory system, that is, cytochrome reductase and Zwischenjerment, only one of which is a flavoprotein. Since the inhibitory action of substituted phenols is not restricted to flavoproteins, no definite conclusion concerning the significance of these enzymes in the respiration of the living cell can be obtained from these inhibition experiments. Effect of Substituted Phenols on Isolated Intermediary Steps and on Respiration of Living Cells Enzyme system VII Inhibition by 1 X 10-S M 2.4-dinitro-o-cyclohexylphenol Oxygen I ccytochrome oxidase. 0 Cytochrome Triphosphopyridine c nucleotide i cytochrome Glucose-6-phosphate i Respiration of bakers yeast... Zwischenferment... SUMMARY reductase With an improved procedure of isolation, cytochrome reductase can be obtained with a purity of 98 per cent and with g-fold better yield than previously reported. 2. Since the enzyme reacts about lo6 times faster with cytochrome than with molecular oxygen, it must be concluded that the direct reaction of the reductase with oxygen is without physiological importance. 3. Denaturation of the enzyme diminishes its activity with cytochrome to a much greater extent than that with oxygen, thus indicating a different mechanism for the two reactions. Denatured cytochrome reductase and the old yellow enzyme of Warburg and Christian are not identical. 4. 2,4-Dinitro-o-cyclohexylphenol inhibits the respiration of intact yeast cells. In isolated systems the substituted phenol inhibits the enzymatic action of cytochrome c reductase and of Zwischenjerment, but it does not inhibit the enzymatic oxidation of cytochrome c. The inhibition of enzymatic reactions by this substituted phenol cannot be regarded as a specific flavoprotein inhibition

9 HAAS, HARRER, AND HOGNESS 349 We wish to acknowledge our indebtedness to the Rockefeller Foundation for a grant-in-aid which made this work possible, and to the Works Progress Administration for help during the course of this investigation. BIBLIOGRAPHY 1. Haas, E., Horecker, B. L., and Hogness, T. R., J. Bid. Chem., 136,747 (1940). 2. Willstiitter, R., and Kraut, H., Bet-. them. Ges., 66, 1117 (1923). 3. Warburg, O., and Christian, W., Biochem. Z., 264, 438 (1932); 266, 377 (1933). 4. Krahl, M. E., and Clowes, G. H. A., J. Gen. Physiol., 23, 413 (1940). 5. Krahl, M. E., Keltch, A. K., and Clowes, G. H. A., J. Biol. Chem., 136,563 (1940). 6. Haas, E., Harrer, C. J., and Hogness, T. R., J. Biol. Chem., 142, 835 (1942). 7. Negelein, E., and Haas, E., Biochem. Z., 282, 206 (1935).

10 CYTOCHROME REDUCTASE: II. IMPROVED METHOD OF ISOLATION; INHIBITION AND INACTIVATION; REACTION WITH OXYGEN Erwin Haas, Carter J. Harrer and T. R. Hogness J. Biol. Chem. 1942, 143: Access the most updated version of this article at Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts This article cites 0 references, 0 of which can be accessed free at tml#ref-list-1

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