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10, O C L O U L K
1967
Studies on Ascorbic Acid. I. Factors Influencing the Ascorbate-Mediated Inhibition of Catalase" Charles W. M. O r r t
The inhibition of catalase by ascorbate, and ascorbate plus Cu2+,has been studied. In the presence of Cu2+,the inhibition is very rapid. It is independent of substrate concentration and pH, but is strongly influenced by temperature. The temperature dependence reflects, a t least in part, the rate of the reaction between Cu2+ and ascorbate. The inhibition can be prevented if solutions of sufficiently high ionic strength are used ( ~ / 2= 0.1), but increases rapidly to a maximum when the ionic strength is reduced (x/2 = 0.002). Dimethyl sulfoxide reduces the inhibition by 50%. In-
hibition studies with ascorbate alone have demonstrated clearly that ascorbate, by itself, inhibits catalase. The inhibition is potentiated by EDTA and by iron, and a synergistic action has been found when both iron and EDTA are used with ascorbate. Dimethyl sulfoxide reduces the inhibition by ascorbate. I t is concluded that free-radical transients, known to be generated during the oxidation of ascorbate, are responsible for the inhibition of catalase. The most likely candidates are .OH and .O?H; possibly both are involved
F
Materials and Methods
ABFTRACT:
oulkes and Lemberg (1948) concluded from a manometric study of the inhibition of catalase by ascorbate that Cu2+ was a necessary participant in the reaction. They did not, however, propose a mechanism for the reaction. The same authors have suggested that a ferric-ascorbate complex occurs between catalase and ascorbate (Lemberg and Foulkes, 1948). This view was challenged by Chance (1950) who demonstrated spectrophotometrically that an enzymatically inactive catalase species (complex 11) is formed when ascorbate and catalase are incubated together. Chance concluded that ascorbate autoxidation occurred during the prolonged incubation of catalase with ascorbate resulting in a slow, continuous evolution of H 2 0 2 .Under these conditions, complex I1 is formed. More recently, it has been suggested (Orr, 1966) that the inhibition of catalase by ascorbate in the presence of Cu2+ could be due t o the production of transient free-radical species formed in the CuZT-catalyzedautoxidation of ascorbate. I n this report, data in support of this concept are considered and, in conjunction with data in a subsequent paper (Orr, 1967) where the relationship of these results t o those of Chance (1950) are discussed, it is suggested that the inhibition of catalase not only by Cu2+and ascorbate, but also by ascorbate alone (where any effect of Cu2+is excluded), can be ascribed to freeradical attack of the protein.
* From the Industrial Hygiene Research Laboratories, I.C.I., Ltd., Alderley Edge, Cheshire, England. Receiced Jirne 12, 1967. t Present address: The McCollum-Pratt Institute, Department of Biology, The Johns Hopkins University, Baltimore, Md.21218.
The materials and methods have been described in a previous communication (Orr, 1966). Chemicals which were not referred to in the Experimental Section of the previous report were all analytical reagent grade quality.
Experimental Section The Effect oj Substrate Concentrution, Tetirperuture, und Inhibitor Concentrution on the Kinetics of the Inhibition. Previously it was shown (Orr, 1966) that the inhibition of catalase by 2 x lop4 M ascorbate is greatly potentiated by 2 X 10-6 M Cuz+. At concentrations of Cu2+ higher than this the inhibition is almost too rapid to measure adequately under the conditions of the assay. The parameters of the inhibition have been examined in greater detail. The effect of varying the temperature, and the concentrations of H2O2and ascorbate, a t a constant Cu2+ concentration (2 X 10-6 M) was measured. The data from these experiments are shown in Table I. Quite clearly, the inhibition is unrelated to the concentration of substrate; over a 2.5-fold range of H 2 0 2concentrations, the inhibition for a given set of conditions remains unchanged. The inhibition is markedly temperature dependent. When catalase solutions were incubated for 10 min at 0" (ice) in the presence of Cu2+ and ascorbate, and assayed immediately, essentially no inhibition occurred. However, it has been found by spectrophotometric assay of ascorbate at 265 mp, that the reaction between Cuy+and ascorbate at 0" is very slow. After 10-min incubation at 0 almost no ascorbate has been oxidized, since the spectrum remains essentially unchanged. O,
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TABLE I : The Effect of Temperature, Ascorbate Concentration, and Substrate Concentration in the Presence of 2 x 10-6 M Cu2+on the Inhibition of Catalase after 10-min Incubation.
Temp ("C)
I
hl fA/ M/ 'IO 50 100
'$50
1
I
00
MOLARITY
M/lOOO
OF T R l S
FIGURE 1: The effect of ionic strength on the inhibition of catalase by Cu'+ and ascorbate. The assay was run at 37" for 10 min at 2 x 10-6 M C U ~and + 2 X 10- ' M
ascorbate.
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The temperature dependence of the reaction can therefore be related to the rate of the catalysis of ascorbate by Cu2+,which under these conditions is maximal at 37 O and 2 X lop5M ascorbate. The Effect of pH on the Inhibition. The effect of pH on the inhibition of catalase was tested using Sorenson phosphate buffers. These were prepared after shaking the respective phosphate solutions with 8-hydroxyquinoline and subsequent extraction of the 8-hydroxyquinoline with chloroform. No effort was made t o test the effect of p H above 8 since it has been shown (Chance, 1954) that H202 decomposes spontaneously in this p H range. Catalase was incubated in ~ / 1 0 0Sorenson's phosM ascorbate and 2 X phate buffer with 2 X M Cu2+ at p H 7.75, 7.3, 6.85, and 6.3. After 10-min incubation at 37", a n aliquot of each was assayed. No effect of p H could be demonstrated; the inhibition in all cases was approximately 40 %. The Effect of Ionic Strength on the Reaction. I t has been claimed (Foulkes and Lemberg, 1948) that an increase in the ionic strength of phosphate buffer from ~ / 2 1 5 0to ~ / 1 0 5completely prevents the inhibition of catalase by ascorbate. However, their data indicate that catalase in the absence of ascorbate is 40% less active in ~ / 1 0 5phosphate. No explanation for this effect or for the reduced inhibition is given; however, it is reasonable to suggest that residual Cu2+,apparently present in Foulkes and Lemberg's preparation, is effectively removed from solution at the higher phosphate concentration. It has been found here that Tris, which does not chelate Cu2+ (Good et al., 1966), completely prevents the inhibition of catalase by Cu2+ and ascorbate when the incubation is run a t ~ / 1 0Tris (pH 7.0). I n fact, a t this molarity, the activity of catalase was slightly higher in the presence of C U ~and + ascorbate. As the molarity of Tris is decreased the inhibition increases rapidly and is maximal at ~ / 5 0 (Figure 0 1).
Concn of Ascorbate (M)
Inhibn ~~
Concn of H20z(pmoles) 42 65 66 105 ~
_
_
37 25 0
2
x
10-6
15 2 2 . 3 25 30 29.5 25 25 23 0 0 7.5 0
37 25 0
2
x
10-5
66.2 6 1 . 1 63.7 70 5 7 . 4 56 57.7 5 7 . 9 0 0 1 4 0
37 25 0
2
x
10-4
6 9 . 7 69.8 70.7 76 59 5 3 . 5 59.5 5 3 . 5 7 5 0 14 0
The Effect of EDTA on the Inhibition of Catalase by Ascorbate. It has always been difficult to rule out the possibility that C U ~is+present in trace amounts ( 10P M), and that this level of contamination is responsible for the inhibition of catalase in supposedly uncontaminated solutions of ascorbate. I n interpreting their results, Foulkes and Lemberg (1948) conclude that the presence of contaminating Cu2+ is essential for the inhibition of catalase by ascorbate. They draw this conclusion from the effect of the chelating agent diethyl dithiocarbamate' on the reaction. At a concentration of 2 X M, DEDTC clearly reduces the inhibition by ascorbate, but by no means completely. If their data are calculated as per cent inhibition after incubation of catalase with 2 x low4M ascorbate, the inhibition in the presence of DEDTC is still 40% after 20-min incubation at 37". In view of these results, it was felt that if contaminating C U ~was + present and responsible for the ascorbate effect found here, a reduction in the per cent inhibition would be expected if DEDTC was included in the incubation mixture. I t was found that a t 2 X M ascorbate, the degree of inhibition of catalase after 10 and 20 rnin of incubation was identical in the presence or absence of 2 X M DEDTC (at this concentration, DEDTC has no effect on catalase activity). From these data, it can be concluded that ascorbate alone inhibits catalase. However, as another check to demonstrate that the inhibition in the absence of Cu*+ was due only t o ascorbate, EDTA was included in the reaction. Incubation mixtures were prepared containing catalase, 2 X M ascorbate, and 2 x M EDTA (sodium salt) in ~ / 1 0 0Tris buffer at pH 7.0. The incubation
