Production of superoxide anion by N,N-bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane buffer during oxidation of oxyhemoglobin by nitrite and effect of inositol hexaphosphate on the oxidation

Biochimica et Biophysica Acta, 709 (1982) 187-193

187

Elsevier Biomedical Press BBA31393

PRODUCTION OF SUPEROXIDE ANION BY N,N-BIS(2-HYDROXYETHYL)IMINOTRIS(HYDROXYMETHYL)METHANE BUFFER DURING OXIDATION OF OXYHEMOGLOBIN BY NITRITE AND EFFECT OF INOSITOL HEXAPHOSPHATE ON THE OXIDATION HIROAKI KOSAKA * and ITIRO TYUMA

Department of Physicochemical Physiology, Medical School, Osaka University, Nakanoshima, Osaka (Japan) (Received April 14th, 1982) (Revised manuscript received July 26th, 1982)

Key words: Oxyhemoglobin; Nitrite," ESR; Superoxide anion; Inositol oxidation

Oxidation of oxyhemoglobin by nitrite is characterized by the presence of a lag phase followed by an autocatalysis. As reported previously (Kosaka, H., lmaizumi, K. and Tyuma, I. (1982) Biochim. Biophys. Acta 702, 237-241), in phosphate buffer nitrite produced an ESR signal at g 2.005 (hereafter referred to as the g 2 radical). The g 2 radical produced NO~ from NO2--, then NO~ oxidized oxyhemoglobin. Superoxide dismutase did not modify the oxidation. On the other hand in N,N-bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane (histris) buffer, superoxide dismutase markedly elongated the lag phase and accelerated the autocatalysis, indicating Oz-- production. Bistris scavenged the g 2 radical. O 2- was generated by the reduction of O z by a radical derived from bistris. Inositol hexaphosphate inhibited the oxidation by decreasing H202 production from oxyhemoglobin.

Introduction Addition of nitrite to an oxyhemoglobin solution transforms the hemoglobin into methemoglobin. The reaction is complex and the kinetics are characterized by the presence of a lag period followed by an autocatalytic phase [1-7]. The reaction, in the presence of inositol hexaphosphate, proceeded linearily with much retardation, while the oxidation by ferricyanide was hastened as much as ten times by the addition of inositol hexaphosphate [8]. The major difference between the two MetHb formers is that nitrite is known as

* Present address: Division of Environmental Health Research, Osaka Prefectural Institute of Public Health, 69-3, 1-chome, Nakamichi, Higashinari-Ku, Osaka, Japan Abbreviation: bistris, N,N-bis(2-hydroxyethyi)iminotris(hydroxymethyl)methane. 016%4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

one electron reducing agent while ferricyanide is the oxidizing agent [9]. To elucidate the mechanism of the autocatalysis, the oxidation process was followed by ESR spectroscopy and a radical was detected at g 2.005 (referred to as the g 2 radical) [10]. The characteristic of the radical was very similar to the MetHb radical [11] generated on mixing MetHb and H 2 0 2. Recently Jung et al. [12] also detected similar radical during the oxidation of HbO 2 by nitrite. We have further shown that the autocatalysis was delayed by the addition of KCN or catalase. The oxidation was not modified by superoxide dismutase in phosphate buffer. Then we proposed the chain reaction mechanism for the oxidation which includes the production of H202, the g 2 radical and NO 2. On the other hand, Tomoda et al. [13] reported that 0 2 was produced during the oxidation of HbO 2 by nitrite in bistris buffer, and

188

assumed that 0 2 and N O are responsible for the autocatalytic process. In this paper we have performed the similar study in bistris buffer and have now shown that 0 2- was generated from the reaction between bistris and the g 2 radical. Further, inositol hexaphosphate inhibited the oxidation by decreasing H 2 0 2 production from HbO 2.

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Materials and Methods I

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Human adult hemoglobin from flesh blood was freed from superoxide dismutase and catalase by a CM-sephadex column [14], and stripped of phosphates by the Dintzis column method [15]. MetHb was prepared by treating the HbO 2 with 5 equiv. of K3Fe(CN) 6 at 37°C for 30 min and passed through a Sephadex G-25 column. The concentration of hemoglobin was given on a monomer basis. Bistris, superoxide dismutase and catalase were from Sigma Chemical Co. and used without further purification. The activity of the enzymes was expressed in units (U). Superoxide dismutase was assayed by the method of McCord and Fridovich [16]. 1 unit of catalase was defined as that amount which decomposed 1.0/xM of H202 per min when acting on 0.02 M H202 in 0.05 M phosphate buffer at pH 7.0 and 25°C. Spectrophotometric measurements were carried out on a Hitachi 320L recording spectrophotometer with cell compartment thermostatically controlled at 25°C. The formation of MetHb was followed by measuring absorbance at 577 or 630 nm. ESR data were taken at 77 K as reported previously [10]. Results

Effect of bistris In bistris buffer, the oxidation process of HbO 2 by nitrite was very similar as in phosphate buffer with a lag period followed by an autocatalytic phase. In bistris buffer, however, the addition of superoxide dismutase prolonged the lag phase and accelerated the autocatalysis as shown in Fig. 1. Catalase decelerated the autocatalysis, but did not prolong the lag phase. Whereas, in the presence of superoxide dismutase, catalase inhibited both the lag and autocatalytic phases. These results indicate

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Fig. 1. Effect of supero~dde dismutase and catalase on the oxidation of 120 p M HbO 2 by 900 p M nitrite in 0.05 M bistfis

buffer conmirdng 0.1 M C I - , pH 7.0. Curves (1) and (7) are

control. Superoxidedismutase concentrations(U) are: (2) 2; (3) 230; (8) 25. Catalase concentrations (U) are: (4) 80; (5) 420. Curve (6) is catalase (210 U) plus superoxide dismutase (120 U). Solid and dashed curves are for HbO2 preparation contained 3 and 9% MetHb, respectively.

the involvement of H202 in the autocatalysis. Unexpectedly, as presented in curve 8 where the initial concentration of Hb 3÷ (9%) was 3 times as high as other experiments, superoxide dismutase did not prolong but shortened the lag phase. This result indicates the requirement of Hb 3÷ for the initiation of the autocatalysis. Furthermore the addition of KCN clearly delayed the oxidation, since K C N strongly binds to ferric iron atom in MetHb. It is very likely that the reaction of MetHb and H 202 will produce the g 2 radical as in phosphate buffer [10]. Actually, the g 2 radical but no N O H b was detected by ESR technique in bistris buffer. The concentration of the radical, however, was far lower than that in phosphate buffer. The oxidation velocity was also decreased with the increase of bistris concentration as shown in Fig. 2B. Similarly, in the mixture of MetHb and H202, the MetHb radical concentration in 0.05 M bistris buffer was 15% of that in 0.05 M phosphate buffer at pH 7.0. Fig. 2A depicts that the concentration of the MetHb radical decreased with the increase in bistris concentration. The reaction of ferrimyoglobin (Mb 3÷ ) and H202 was known to form a spectrophotometrically detectable red compound (peroxide compound, ferrylmyoglobin, Mb 4÷) [17,18], and a free radical

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Fig. 2. A: Effect of bistris concentrations on the signal height of MetHb radical, produced by mixing 0.9 mM Hb 3+ and 0.9 mM H202 at pH 7.0. B: Comparison of the effect of buffer salts on the oxidation velocities of 60 #~M HbO 2 by 3 mM nitrite at pH 7.4, evaluated as the half-life of the reaction, tl/2 (rain). e, bistris buffer containing 0.1 M CI-; O, iris buffer containing 0.1 M Cl- ; A phosphate buffer, pH 7.4 is chosen as the common range of these buffers. Concentration of nitrite is 3 mM because the oxidation is too slow in 0.1 M bistris.

intermediate (MetHb radical) was detected by ESR technique during the reaction [ 11 ]. King and Winfield [19] suggested that "Mb4+ , a compound containing quadrivalent iron and a free radical, might be a precursor of Mb 4+ . Bistris inhibited the production of the peroxide compound as well as the MetHb radical. Fig. 3 illustrates the peroxide compound minus MetHb difference spectra. In 0.05 M bistris buffer the concentration of the peroxide compound was lower than that in 0.05 M phosphate buffer. In 1 M bistris buffer (Fig. 3C), further decrease of the concentration was observed and the production of HbO 2 was recognized during the decomposition as indicated by the appearance of a peak at 577 nm. On addition of inositol hexaphosphate (Fig. 3D), the concentration of the peroxide compound in-

(rim)

Fig. 3. Difference spectra (the peroxide compound minus MetHb) for the reaction of 120/tM MetHb with 103/~M H202 at pH 7.0 and for that in the presence of inositoi hexaphosphate. (A), in 0.05 M phosphate buffer; (B), in 0.05 M bistris buffer; (C), in 1 M bistris buffer; (D), in i M bistris buffer containing 480 #M inositol hexaphosphate. Conditions for automatic repeated scanning are: A and B, 60 nm/min; C and D, 600 nm/min. Figures attached to each curve denote the number of scannings.

creased and the peak of HbO 2 disappeared. Fig. 4 depicts the formation and spontaneous decomposition of the peroxide compound. Formation rate seems to be similar in all the buffers, but the decomposition rate increases with the increase in i

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Fig. 4. Effect of bistris and superoxide dismutase (370 U) on the formation and decomposition of the peroxide compound measured at 630 nm. Conditions are as in Fig. 3. Curve 1: in 0.05 M phosphate buffer with or without superoxide dismutase. Bistris concentration is: curve (2), 0.025 M; (3), 0.05 M; (4), 0.5 M; (5), 1 M. Solid curves are without superoxide dismutase. Dashed curves are with superoxide dismutase.

190 TABLE I C O M P A R I S O N O F T H E R A T E C O N S T A N T OF F O R M A T I O N A N D D E C O M P O S I T I O N O F T H E P E R O X I D E C O M P O U N D O F M E T H E M O G L O B I N IN D I F F E R E N T B U F F E R S Reaction conditions are as in Fig. 6. Spectrophotometric measurements of kinetics of formation and decomposition of the peroxide compound were made at 630 n m by the use of extinction coefficients of 3.70 and 1.52 m M - I . c m - I for MetHb and the peroxide compound, respectively. The rate constants were obtained according to the method of Yonetani and Schleyer [20]. Phosphate (0.05 M)

Concentration of bistris (M) 0.025

Formation rate ( M - 1. s - 1) Control With inositol hexaphosphate Decomposition rate ( s - l ) Control With inositol hexaphosphate

0.05

2.1- 10 2

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Fig. 5. Effect of inositol hexaphosphate on the oxidation of 120 # M HbO 2 by 220/AM nitrite in 0.05 M phosphate buffer, pH 7.0, in the presence of the following concentration of H202 (/tM): (1) none; (2) 1.7; (3) 8.6. Solid curves are without inositol hexaphosphate. Dashed curves are with inositol hexaphosphate (480 ~M). The reaction wag followed by measuring the absorbance at 577 rim.

7.2-10- 3

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nant [22-24]. EDTA enhances the reactivity of Fe 3 +, whereas diethylenetriamine pentaacetic acid blocks Fe 3÷ reactivity [22]. However, in 1 mM neither EDTA nor the pentaacetic acid modified the oxidation of HbO 2 by nitrite in both the buffers. Furthermore, OH" scavengers such as 84 mM ethanol, 120 mM methanol, 10 mM tert-butyl alcohol, 3 mM benzoate or 4 mM formate did not modify the oxidation. Another OH" scavenger, Tris [25] also did not affect the oxidation as indicated in Fig. 2B.

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bistris concentration as reported in Table I. Furthermore Fig. 4 shows that superoxide dismutase did not modify the process in phosphate buffer, but increased the concentration of peroxide compound in bistris buffer. Since the oxidation of HbO 2 by nitrite in bistris buffer was retarded on addition of both superoxide dismutase and catalase, it is assumed that the Haber-Weiss reaction will occur [21 ]. The rate of the reaction is very slow. There are recent reports on Fe 3+ contamination and on the catalysis of the Haber-Weiss reaction by the contami-

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Fig. 6. Effect of inositol hexaphosphate on the formation and decomposition of the peroxide compound and the reduction by nitrite. Conditions are as in Fig. 4. Curves (1), 0.05 M phosphate buffer; (2), 0.05 M bistris buffer; (3), 1 M bistris buffer. Solid curves are without inositol hexaphosphate. Dashed curves are with 480 # M inositol hexaphosphate. 29 # M nitrite is added at the time indicated by the arrow in curve 1.

191

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Fig. 7. The o x i d a t i o n of H b O 2 by nitrite in 0.05 M p h o s p h a t e buffer in the presence a n d absence of 480 # M inositol h e x a p h o s p h a t e . The c o n c e n t r a t i o n of nitrite ( # M ) : (1) 260; (2) 320; (3) 410; (4) 580. C o n d i t i o n s are as in Fig. 5.

In bistris buffer, the effect of the addition of catalase or KCN on the oxidation of HbO z by nitrite was similar to that in phosphate buffer, indicating the involvement of the g 2 radical. The marked effect of superoxide dismutase, however, was contrasted with their ineffectiveness in phosphate buffer. Moreover in bistris buffer, the oxidation was far slower than that in phosphate buffer. Above differences indicate that in bistris buffer the g 2 radical is scavenged by the buffer salt and 0 2 produced. In Fig. 3C, HbO 2 was detected at high bistris concentration. This result can be explained by assuming the following reactions:

Effect of inositol hexaphosphate As in bistris buffer [8], inositol hexaphosphate markedly inhibited the autocatalysis also in phosphate buffer as depicted in Fig. 5. Isosbestic points were observed during the oxidation. The inhibition by inositol hexaphosphate was recovered on addition of H202. This result implies that the following reaction [10] is inhibited by inositol hexaphosphate: 2H +

+ H b O 2 + N O 2 ~ Hb 3+ + H 2 0 2 + N O ~

(t)

In the mixture of MetHb and H 2 0 2 , inositol hexaphosphate accelerated the formation of the peroxide compound to a similar extent in all the buffers, but did not change the spontaneous decomposition rate of the compound as indicated in Fig. 6 and Table I. The following reaction [10] is also indicated in Fig. 6 (curve 1): H b 4+

+NOr

+ H + -* Hb3+

+NO 2 +H20

(2)

where inositol hexaphosphate did not change the velocity of the reduction of the peroxide compound by nitrite. Fig. 7 depicts the effect of inositol hexaphosphate in the presence of different concentrations of nitrite. As clearly seen, the decrease in nitrite concentration increased the inhibition of the autocatalysis by inositol hexaphosphate. This indicates that inositol hexaphosphate inhibits reaction 1, leading to the decrease of H202 and the g 2 radical concentrations.

H * + ' H b 4+ + A - X ---* H b 4+ + A + + X " H b 3+ + A + + X +

(3)

where A-X signifies bistris and is assumed to decompose into A ÷ and X" by "Hb4÷ . X" is a radical such as "CH2OH which has strong reducing properties and can react with oxygen to form a peroxy radical which can then decay to form 0 2 [26,27]. Then HbO 2 was produced: 0 2 +

X" "4-H b 3+ ~ H b O 2 + X +

(4)

The reduction of MetHb by 0 2 as a source of HbO 2 was not considered, since the rate of the reduction is slow [28] and the addition of superoxide dismutase did not inhibit the appearance of HbO 2 in Fig. 3C. The mechanism of oxidation of HbO 2 by nitrite in bistris buffer can be described as follows: 2H + + H b O 2 + N O 2 --* H b 3+ + H 2 0 2 + N O ~

(i)

H b 3+ + H 2 0 2 ---* "Hb4+ + H 2 0

(5)

H + + ' H b 4+ + A - X "-'* H b 4+ + A + + X " -* H b 3+ + A + + X + X'+O2~X

+ +02

(3)

(6)

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(7)

X" + H b O 2 + 2 H + ~ H b 3+ + H 2 0 2 + X +

(s)

0 2 +02

192 X ' + H b 3+ +02 ~ HbO2 +X +

(4)

"Hb4÷ +NO~- +H + ~ Hb4+ +NO~

(9)

Hb4+ +NO~- +H + ~ Hb3+ +NO~ +H20

(2)

HbO2 +NO~ --, Hb3+ +02 +NO 2

(10)

2NO2 + H 2 0 - , N204 + H 2 0 - , NO 2 +NO 3 +2H +

(11)

R e a c t i o n s 1, 2, 5, 9, 10 a n d 11 were r e p o r t e d previously in p h o s p h a t e buffer [10]. T h e a d d i t i o n of s u p e r o x i d e d i s m u t a s e increased the c o n c e n t r a t i o n of the p e r o x i d e c o m p o u n d in Fig. 4. This result can be e x p l a i n e d b y the r e m o v a l o f 0 2 b y s u p e r o x i d e d i s m u t a s e in reaction 6. Thus the eqiliblium was d i s p l a c e d to the right as in the case of q u i n o n e or n i t r o b l u e t e t r a z o l i u m [29-31], resulting in the increase of H202. In the lag p h a s e (Fig. 1), due to the low fraction of M e t H b , supero x i d e d i s m u t a s e can decrease X" b y the right shift of reaction 6 a n d thus the lag phase will elongate b y the i n h i b i t i o n of reaction 8. The rate c o n s t a n t of the o x i d a t i o n of HbO2 b y H202 was 22 M - I . s - 1 at 25°C in 0.05 M phosp h a t e buffer, p H 7.0. Because the rate c o n s t a n t of f o r m a t i o n of the p e r o x i d e c o m p o u n d was 240 M - l • s - t at the same c o n d i t i o n , the o x i d a t i o n of H b O 2 b y H 202 was not i n c l u d e d in the a b o v e scheme.

Effect of inositol hexaphosphate The o x i d a t i o n rate of H b O 2 b y H202 was increased to 55 M - 1 . s - I on a d d i t i o n of 480 # M inositol h e x a p h o s p h a t e . T h e i.v.crease of the rate of H202 a n d H b O 2 b y inositol h e x a p h o s p h a t e is similar to that of H202 a n d M e t H b (2.5-fold). Inositol h e x a p h o s p h a t e can cause a similar structural transition in H b O 2 [32] as the R ~ T transition observed in the ferric derivative [33]. V e r s m o l d et al. [34] r e p o r t e d that 2 , 3 - d i p h o s p h o g l y c e r a t e m a r k e d l y i n h i b i t e d the o x i d a t i o n of H b O 2 b y nitrite at 37°C. I n the case of one electron reducing agents such as nitrite, therefore, the rate of reaction 1 in the presence o f aUosteric effector is p r e s u m e d to decrease.

Acknowledgements T h e a u t h o r s t h a n k Dr. K a z u h i k o I m a i z u m i for his k i n d advice a n d suggestions. This w o r k was

s u p p o r t e d b y a G r a n t - i n - A i d for E n v i r o n m e n t a l Science f r o m the m i n i s t r y of E d u c a t i o n , Science a n d Culture of J a p a n .

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