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Effect of nitrite on respiration and oxidative phosphorylation
Int.
3.
Biochem., 1974, Vol. 5, pp. 349 to 352. Pergamon Press. Printed in Great Britain
EFFECT
OF NITRITE
ON RESPIRATION
349
AND OXIDATIVE
PHOSPHORYLATION K. S. CHEAH Agricultural Research Council, Meat Research Institute, Langford, Bristol BS18 7DY, U.K. (Received 16 August 1973) ABSTRACT I. The effect of nitrite on the mitochondrial oxidation of succinate and of ascorbate plus tetramethyl-p-phenylenediamine (TMPD) was investigated using tightly-coupled ox neck muscle mitochondria. 2. Both respiration and oxidative phosphorylation were inhibited by nitrite, the effect being more pronounced at a low pH. 3. The oxidation of ascorbate plus TMPD was more sensitive to nitrite than the oxidation of succinate at both pH 7.2 and at pH 6. I. 4. At pH 6.1, the State 3 respiration for the oxidation of ascorbate plus TMPD was completely inhibited by 3.75 pmoles nitrite per mg. protein as compared with 15.50 pmoles nitrite per mg. protein for the oxidation of succinate.
NITRITE is widely employed in the meat industry for the manufacture of cured meat products (Walters, 1971; Mohler, Baumann & Ebert, 1g71), and also in the spectrophotometric analysis of cytochrome a3 in mitochondria contaminated with either haemoglobin or myoglobin (Chance, 1957). In the presence of nitrite, neither haemoglobin nor myoglobin interferes with the Soret absorption spectrum of the cytochrome as-CO complex since these haem pigments are oxidized to their ferric forms by nitrite (Lemberg & Legge, 1g4g), and are thus unable to form their respective CO-complexes Nitrite has also been 1957). (Ch ante, briefly reported to inhibit the aerobic respiration of excised muscle (Walters, I 97 I), mitochondrial respiration and cytochrome oxidase (EC 1.9.3.1) activity (Walters & Taylor, I 965). In this report, a detailed study of the effect of nitrite on mitochondrial respiration and oxidative phosphotylation using ox neck muscle mitochondria is described. Nitrite was found to inhibit both respiration and oxidative phosphorylation, the effect being more pronounced at a low pH. MATERIALS AND METHODS Antimycin A, rotenone and the sodium salts of ADP and succinate were obtained from Sigma
Chemical Corp. ; sodium ascorbate, sodium nitrite and tetramethyl-p-phenylenediamine (TMPD) dihydrochloride from BDH Chemicals Ltd. ; crystalline Bacillus subtilis proteinase (Nagarse) from Teikoku Chemical Co.; all other reagents were of AnalaR grade. p-Trifluoromethoxycarbonylcyanide-phenylhydrazone (FCCP) was kindly supplied by Dr. P. Heytler. Mitochondria from the ox neck muscle were isolated using crystalline B. subtilis proteinase (Cheah & Cheah, 1971). Oxygen uptake was measured with a Clark oxygen electrode pellow Spring Biological Monitor (Model 53)] at 25’ C in a total volume of 2.5 ml. The reaction medium contained 1.0 mM EDTA, 30.0 mM KCl, 6.0 mM MgCI,, 75.0 mM sucrose and 20.0 mM KH,PO,. The ADP/O ratio and the respiratory control index (RC’I) were determined from the ‘electrode traces (Chance & Williams. 1456). and urotein was estimated by Folin-phenol”;eagent (Lowry, Rosebrough, Farr & Randall, 1951) using bovine serum albumin as standard. RESULTS
AND DISCUSSION
The effect of nitrite on mitochondrial respiration and oxidative phosphorylation was tested using succinate and ascorbate plus TMPD, each of which donated electrons to a different and specific site of the respiratory chain system (Lee, Sottocasa & Ernster, 1967). With both of these substrates, nitrite was more effective at a low than at a high pH as clearly illustrated for the oxidation of succinate (FIG. I) and of ascorbate plus TMPD (FIG. 2).
CHEAH
3.50 300 pi
266
ADP
_ /
RCI = -ye-
f 5.22
266 T-i-
= 5.22
(*NO;)
= 1.79
2 pM Rotenone = 1.72
<*NO27
f 5.65 = 2 -58
(*NO;)
= 1.79
= 1.39
tyM
I+NO$
FCCP
\ electrode traces showing the effect of nitrite on the oxidation of succinate at pH 7.2 and at pH 6.1. The numbers, adjacent to the electrode traces, refer to respiratory activities expressed in natoms 0 per minute per mg. protein. The concentration of nitrite in the electrode traces is expressed in pmoles nitrite per mg. protein. Total protein: A, x.33 mg. ; B, 1-64 mg. Final pH: A, 7.2; B, pH 6.1. Other experimental details are described in the text. -1
lmin+
FIG. I .-Oxygen
The oxidation of succinate at pH 7-2 (FIG. I, Trace A) was unaffected by nitrite at a concentration of 3-75 pmoles nitrite per mg. protein. The values for the State 3 respiration, the RCI and the ADPjO ratio did not alter in the presence and absence of nitrite. However, at pH 6~1 the State 3 rate, the RCI and the ADP/O ratio for the oxidation of succinate were affected (Fro. I, Trace B) even though a slightly lower concentration of nitrite (2.75 ~moles per mg. protein) was used. The oxidation of ascorbate plus TMPD was also more sensitive to nitrite at pH 6 I than at pH 7.2 (FIG. 2). For a complete inhibition
of
the
State
3
respiration
of
ascorbate plus TMPD, 2632 pmoles nitrite per mg. protein was required at pH 7.2 (Trace A), compared with only about 3-75 pmoles nitrite per mg. protein at pH 6-1 (Trace B). The inhibition of nitrite on the ADP stimulated respiration could also be relieved by the uncoupler FCCP (Trace B), as observed for the oxidation of succinate (FIG. I, Trace B) . The inhibitory effect of the various concentrations of nitrite on the succinoxidase system of the ox neck muscle mitochondria at pH 7-2 and at pH 6.1 is shown in FIG. 3. At pH 6-r and at a concentration of about 15-i pmoles nitrite per mg. protein the State 3 respiration (A), the RCI (B) and the
EFFECT OF NITRITF. ON RESPIRATION
1974,5
_
351
A 100 *‘.
ti
pti 6.1
.L-
a*:’
4mM Ascorbote +
2 pg Antimycin
. ./
A
0.2mM
.r..I’ ,...
.,.‘..i‘ ..i“ * ,_.I ,,...‘,. , ,
TMPD
26.32pmoles
NOi
5
10
15
NoNO
m
48pM
60
.’
FIG. 2,-Oxygen electrode traces showing the effect of nitrite on the oxidation of ascorbate plus TMPD at PH 7.2 and at pH 6.1. Experimental details are described in FIG. I. Total protein: A, 1.33 mg.; B, Ierg mg. FinalpH: A, 7.2; B, 6.1.
5 No
ADP/O
at pH 6, I a much lower concentration of nitrite was required to block completely the State 3 respiration for the oxidation of
ascorbate plus TMPD than at pH 7.2. FIG. 4 illustrates the effect of the various concentrations of nitrite affecting the oxidation of ascorbate plus TMPD at pH 7.2 (A) and at pH 6.1 (B). Complete inhibition of the State 3 respiration, the RCI and the ADP/O ratio was attained at about 3.75 pmoles nitrite per mg. protein at pH 6.1, compared with about 26.0 at pH 7.2.
pmoles
nitrite
per mg.
protein
The State 4 respiration, with ascorbate plus TMPD as the electron donor, was sensitive to a high concentration of nitrite at pH 7.2 (see FIG. 2, Trace A). This inhibitory effect by nitrite
on the cytochrome
oxidase
, 40
Per mg Protein)
a*’
pH 6.1
1
fi
ratio (C) were completely blocked’ Complete inhibition was not observed in either the State 3 respiration, the RCI or the ADP/O ratio when the nitrite concentration was increased by o-5-fold at pH 7.2. As in the case of the oxidation of succinate,
, 35
.*
100
0,
f
i / 25 30
(pmofer
.d-
5
, 20
10 NO2
15 20
25 30 35
40
[~mofes per mg protein)
I
C
_ 100 0s u % occ
:k<’ 4’ *’
8060-
2
z_
pH 6.1
,.I
40 -
:*”
,*,..._*....~.~ ,’ ..L+’ K ,.+H7. 2 .,.....‘.~ 20 _ ,,.p:. ,ll,_.., ~._._... ‘.I..‘.,W,,,
‘ ,..,‘ :,:.= 5
, I / I f / 10 15 20 25 30 35
NoNO
/ 40
( pmofer pet mg protein)
FIG. 3.-Effect
of various concentrations of nitrite on the succinoxidase system at pH 7.2 and at pH 6.1. The State 3 rate (A), the RCI (B) and the ADP/O ratio (C) were calculated from the electrode traces. The data are an average value from at least three separate ADP additions. Other details are described in FIG. I.
activity was more pronounced at pH 6-x than at pH 7.2. Complete inhibition of the cytochrome oxidase activity was obtained with about 20.0 itmoles nitrite per mg. protein at pH 6-r.
CHEAH
352
Int.
3. Biochem.
REFERENCES
_
. State
100
;;--”
80
$
60
$ z -
40
3
A
rate
*f
, .‘: .I
RCI
.
20
5
10
15
NaN02
O:
80
$
60
2 -
40
(pmoles
B
100
20
,. ,’
,*. I
/..“‘,
. State
I
d ,’ a’
,-
30
per mg protein)
I,:i W’ f , ; I ;: , <’
20 -
25
3
rate
. RCI . ADP 0 /
i / .i / .
. /d’
..3
-r’ 1.25
NaN02
, 2.5
I
I
3-75
(pmoles
5.0 per
mg protein
1
FIG. +-Effect of various concentrations of nitrite on the oxidation of ascorbate plus TMPD atpH7.2andatpH6.1.
ACKNOWLEDGEMENT The author would like to thank Cheah for technical assistance.
Mrs.
A.
M.
CHANCE, B., & WILLIAMS, G. R. (rg56), ‘ The respiratory chain and oxidative phosphorylation ‘, in Advances in Enzymology (ed. Nord), Vol. 17, pp. 65-134. New York: Interscience. CHANCE, B. (Ig57), ‘ Techniques for the assay of the respiratory enzymes ’ in Methods in Enzymology (ed. Colowick & Kaplan), Vol. IV, pp. 273-329. New York & London: Academic Press. CHEAH, K. S., & CHEAH, A. M. (IgTI), ‘ Postmortem changes in structure and function of ox muscle mitochondria. I. Electron microscopic and polarographic investigations ‘, 3. Bioenerg., 2, 85-92. LEE, C. P., SOTTOCASA, G. L., & ERNSTER, L. (1g67), ‘ Use of artificial electron acceptors for abbreviated phosphorylating electron transport: flavin-cytochrome c ‘, in Methods in Enzymology (ed. Estabrook & Pullman), Vol. X, pp. 33-37. New York & London: Academic Press. LEMBERG, R., & LEGGE, J. W. (Ig4g), Hematin compounds and bile pigments, pp. 389-390. New York: Interscience. LOWRY, 0.. ROSEBROUGH. N. 1.. FARR. A. L.. & RANDALL; R. J. (1951); ‘ Pr%ein measurement with the Folin-phenol reagent ‘, 3. Biol. Chem., ‘93,265~-175. MOHLER, Kl., BAUMANN, M., & EBERT, H. ( 197 I), ‘ Reactions of nitrite with blood and meat ‘, in Proc. 17th Eur. Meetine of Meat Workers, DLL IgS--1o& Meat Rese&& Institute (61I%h September), Bristol. WALTERS, C. L., & TAYLOR, A. McM. (Ig65), ‘The reduction of nitrite by skeletal-muscle mitochondria ‘, Biochim. Biophys. Acta, g6,522-524. WALTERS, C. L. (IgTI), ‘ Chemical and biochemical implications of nitrite during curing ‘, in Proc. 17th Eur. Meeting of Meat Workers, pp. I82-194, Meat Research Institute (610th September), Bristol. Key Word Index: Ox neck muscle mitochondria, oxidative phosphorylation, respiratory nitrite, control, ADP/O ratio, succinoxidase, cytochrome oxidase (us).
3.
Biochem., 1974, Vol. 5, pp. 349 to 352. Pergamon Press. Printed in Great Britain
EFFECT
OF NITRITE
ON RESPIRATION
349
AND OXIDATIVE
PHOSPHORYLATION K. S. CHEAH Agricultural Research Council, Meat Research Institute, Langford, Bristol BS18 7DY, U.K. (Received 16 August 1973) ABSTRACT I. The effect of nitrite on the mitochondrial oxidation of succinate and of ascorbate plus tetramethyl-p-phenylenediamine (TMPD) was investigated using tightly-coupled ox neck muscle mitochondria. 2. Both respiration and oxidative phosphorylation were inhibited by nitrite, the effect being more pronounced at a low pH. 3. The oxidation of ascorbate plus TMPD was more sensitive to nitrite than the oxidation of succinate at both pH 7.2 and at pH 6. I. 4. At pH 6.1, the State 3 respiration for the oxidation of ascorbate plus TMPD was completely inhibited by 3.75 pmoles nitrite per mg. protein as compared with 15.50 pmoles nitrite per mg. protein for the oxidation of succinate.
NITRITE is widely employed in the meat industry for the manufacture of cured meat products (Walters, 1971; Mohler, Baumann & Ebert, 1g71), and also in the spectrophotometric analysis of cytochrome a3 in mitochondria contaminated with either haemoglobin or myoglobin (Chance, 1957). In the presence of nitrite, neither haemoglobin nor myoglobin interferes with the Soret absorption spectrum of the cytochrome as-CO complex since these haem pigments are oxidized to their ferric forms by nitrite (Lemberg & Legge, 1g4g), and are thus unable to form their respective CO-complexes Nitrite has also been 1957). (Ch ante, briefly reported to inhibit the aerobic respiration of excised muscle (Walters, I 97 I), mitochondrial respiration and cytochrome oxidase (EC 1.9.3.1) activity (Walters & Taylor, I 965). In this report, a detailed study of the effect of nitrite on mitochondrial respiration and oxidative phosphotylation using ox neck muscle mitochondria is described. Nitrite was found to inhibit both respiration and oxidative phosphorylation, the effect being more pronounced at a low pH. MATERIALS AND METHODS Antimycin A, rotenone and the sodium salts of ADP and succinate were obtained from Sigma
Chemical Corp. ; sodium ascorbate, sodium nitrite and tetramethyl-p-phenylenediamine (TMPD) dihydrochloride from BDH Chemicals Ltd. ; crystalline Bacillus subtilis proteinase (Nagarse) from Teikoku Chemical Co.; all other reagents were of AnalaR grade. p-Trifluoromethoxycarbonylcyanide-phenylhydrazone (FCCP) was kindly supplied by Dr. P. Heytler. Mitochondria from the ox neck muscle were isolated using crystalline B. subtilis proteinase (Cheah & Cheah, 1971). Oxygen uptake was measured with a Clark oxygen electrode pellow Spring Biological Monitor (Model 53)] at 25’ C in a total volume of 2.5 ml. The reaction medium contained 1.0 mM EDTA, 30.0 mM KCl, 6.0 mM MgCI,, 75.0 mM sucrose and 20.0 mM KH,PO,. The ADP/O ratio and the respiratory control index (RC’I) were determined from the ‘electrode traces (Chance & Williams. 1456). and urotein was estimated by Folin-phenol”;eagent (Lowry, Rosebrough, Farr & Randall, 1951) using bovine serum albumin as standard. RESULTS
AND DISCUSSION
The effect of nitrite on mitochondrial respiration and oxidative phosphorylation was tested using succinate and ascorbate plus TMPD, each of which donated electrons to a different and specific site of the respiratory chain system (Lee, Sottocasa & Ernster, 1967). With both of these substrates, nitrite was more effective at a low than at a high pH as clearly illustrated for the oxidation of succinate (FIG. I) and of ascorbate plus TMPD (FIG. 2).
CHEAH
3.50 300 pi
266
ADP
_ /
RCI = -ye-
f 5.22
266 T-i-
= 5.22
(*NO;)
= 1.79
2 pM Rotenone = 1.72
<*NO27
f 5.65 = 2 -58
(*NO;)
= 1.79
= 1.39
tyM
I+NO$
FCCP
\ electrode traces showing the effect of nitrite on the oxidation of succinate at pH 7.2 and at pH 6.1. The numbers, adjacent to the electrode traces, refer to respiratory activities expressed in natoms 0 per minute per mg. protein. The concentration of nitrite in the electrode traces is expressed in pmoles nitrite per mg. protein. Total protein: A, x.33 mg. ; B, 1-64 mg. Final pH: A, 7.2; B, pH 6.1. Other experimental details are described in the text. -1
lmin+
FIG. I .-Oxygen
The oxidation of succinate at pH 7-2 (FIG. I, Trace A) was unaffected by nitrite at a concentration of 3-75 pmoles nitrite per mg. protein. The values for the State 3 respiration, the RCI and the ADPjO ratio did not alter in the presence and absence of nitrite. However, at pH 6~1 the State 3 rate, the RCI and the ADP/O ratio for the oxidation of succinate were affected (Fro. I, Trace B) even though a slightly lower concentration of nitrite (2.75 ~moles per mg. protein) was used. The oxidation of ascorbate plus TMPD was also more sensitive to nitrite at pH 6 I than at pH 7.2 (FIG. 2). For a complete inhibition
of
the
State
3
respiration
of
ascorbate plus TMPD, 2632 pmoles nitrite per mg. protein was required at pH 7.2 (Trace A), compared with only about 3-75 pmoles nitrite per mg. protein at pH 6-1 (Trace B). The inhibition of nitrite on the ADP stimulated respiration could also be relieved by the uncoupler FCCP (Trace B), as observed for the oxidation of succinate (FIG. I, Trace B) . The inhibitory effect of the various concentrations of nitrite on the succinoxidase system of the ox neck muscle mitochondria at pH 7-2 and at pH 6.1 is shown in FIG. 3. At pH 6-r and at a concentration of about 15-i pmoles nitrite per mg. protein the State 3 respiration (A), the RCI (B) and the
EFFECT OF NITRITF. ON RESPIRATION
1974,5
_
351
A 100 *‘.
ti
pti 6.1
.L-
a*:’
4mM Ascorbote +
2 pg Antimycin
. ./
A
0.2mM
.r..I’ ,...
.,.‘..i‘ ..i“ * ,_.I ,,...‘,. , ,
TMPD
26.32pmoles
NOi
5
10
15
NoNO
m
48pM
60
.’
FIG. 2,-Oxygen electrode traces showing the effect of nitrite on the oxidation of ascorbate plus TMPD at PH 7.2 and at pH 6.1. Experimental details are described in FIG. I. Total protein: A, 1.33 mg.; B, Ierg mg. FinalpH: A, 7.2; B, 6.1.
5 No
ADP/O
at pH 6, I a much lower concentration of nitrite was required to block completely the State 3 respiration for the oxidation of
ascorbate plus TMPD than at pH 7.2. FIG. 4 illustrates the effect of the various concentrations of nitrite affecting the oxidation of ascorbate plus TMPD at pH 7.2 (A) and at pH 6.1 (B). Complete inhibition of the State 3 respiration, the RCI and the ADP/O ratio was attained at about 3.75 pmoles nitrite per mg. protein at pH 6.1, compared with about 26.0 at pH 7.2.
pmoles
nitrite
per mg.
protein
The State 4 respiration, with ascorbate plus TMPD as the electron donor, was sensitive to a high concentration of nitrite at pH 7.2 (see FIG. 2, Trace A). This inhibitory effect by nitrite
on the cytochrome
oxidase
, 40
Per mg Protein)
a*’
pH 6.1
1
fi
ratio (C) were completely blocked’ Complete inhibition was not observed in either the State 3 respiration, the RCI or the ADP/O ratio when the nitrite concentration was increased by o-5-fold at pH 7.2. As in the case of the oxidation of succinate,
, 35
.*
100
0,
f
i / 25 30
(pmofer
.d-
5
, 20
10 NO2
15 20
25 30 35
40
[~mofes per mg protein)
I
C
_ 100 0s u % occ
:k<’ 4’ *’
8060-
2
z_
pH 6.1
,.I
40 -
:*”
,*,..._*....~.~ ,’ ..L+’ K ,.+H7. 2 .,.....‘.~ 20 _ ,,.p:. ,ll,_.., ~._._... ‘.I..‘.,W,,,
‘ ,..,‘ :,:.= 5
, I / I f / 10 15 20 25 30 35
NoNO
/ 40
( pmofer pet mg protein)
FIG. 3.-Effect
of various concentrations of nitrite on the succinoxidase system at pH 7.2 and at pH 6.1. The State 3 rate (A), the RCI (B) and the ADP/O ratio (C) were calculated from the electrode traces. The data are an average value from at least three separate ADP additions. Other details are described in FIG. I.
activity was more pronounced at pH 6-x than at pH 7.2. Complete inhibition of the cytochrome oxidase activity was obtained with about 20.0 itmoles nitrite per mg. protein at pH 6-r.
CHEAH
352
Int.
3. Biochem.
REFERENCES
_
. State
100
;;--”
80
$
60
$ z -
40
3
A
rate
*f
, .‘: .I
RCI
.
20
5
10
15
NaN02
O:
80
$
60
2 -
40
(pmoles
B
100
20
,. ,’
,*. I
/..“‘,
. State
I
d ,’ a’
,-
30
per mg protein)
I,:i W’ f , ; I ;: , <’
20 -
25
3
rate
. RCI . ADP 0 /
i / .i / .
. /d’
..3
-r’ 1.25
NaN02
, 2.5
I
I
3-75
(pmoles
5.0 per
mg protein
1
FIG. +-Effect of various concentrations of nitrite on the oxidation of ascorbate plus TMPD atpH7.2andatpH6.1.
ACKNOWLEDGEMENT The author would like to thank Cheah for technical assistance.
Mrs.
A.
M.
CHANCE, B., & WILLIAMS, G. R. (rg56), ‘ The respiratory chain and oxidative phosphorylation ‘, in Advances in Enzymology (ed. Nord), Vol. 17, pp. 65-134. New York: Interscience. CHANCE, B. (Ig57), ‘ Techniques for the assay of the respiratory enzymes ’ in Methods in Enzymology (ed. Colowick & Kaplan), Vol. IV, pp. 273-329. New York & London: Academic Press. CHEAH, K. S., & CHEAH, A. M. (IgTI), ‘ Postmortem changes in structure and function of ox muscle mitochondria. I. Electron microscopic and polarographic investigations ‘, 3. Bioenerg., 2, 85-92. LEE, C. P., SOTTOCASA, G. L., & ERNSTER, L. (1g67), ‘ Use of artificial electron acceptors for abbreviated phosphorylating electron transport: flavin-cytochrome c ‘, in Methods in Enzymology (ed. Estabrook & Pullman), Vol. X, pp. 33-37. New York & London: Academic Press. LEMBERG, R., & LEGGE, J. W. (Ig4g), Hematin compounds and bile pigments, pp. 389-390. New York: Interscience. LOWRY, 0.. ROSEBROUGH. N. 1.. FARR. A. L.. & RANDALL; R. J. (1951); ‘ Pr%ein measurement with the Folin-phenol reagent ‘, 3. Biol. Chem., ‘93,265~-175. MOHLER, Kl., BAUMANN, M., & EBERT, H. ( 197 I), ‘ Reactions of nitrite with blood and meat ‘, in Proc. 17th Eur. Meetine of Meat Workers, DLL IgS--1o& Meat Rese&& Institute (61I%h September), Bristol. WALTERS, C. L., & TAYLOR, A. McM. (Ig65), ‘The reduction of nitrite by skeletal-muscle mitochondria ‘, Biochim. Biophys. Acta, g6,522-524. WALTERS, C. L. (IgTI), ‘ Chemical and biochemical implications of nitrite during curing ‘, in Proc. 17th Eur. Meeting of Meat Workers, pp. I82-194, Meat Research Institute (610th September), Bristol. Key Word Index: Ox neck muscle mitochondria, oxidative phosphorylation, respiratory nitrite, control, ADP/O ratio, succinoxidase, cytochrome oxidase (us).