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The sulfite-activated oxidation of reduced pyridine nucleotides by peroxidase
B1OCHIMICA ET B1OPHYSICA ACTA
03
THE SULFITE-ACTIVATED OXIDATION OF REDUCED PYRIDINE
NUCLEOTIDES BY PEROXIDASE S. J. KLEBANOFF
The Rocke[eller Institute, New York, N . Y . (U.S.A.)
(Received August 2oth, I96o)
SUMMARY I. The aerobic oxidation of sulfite ions is initiated by peroxidase in the presence of Mn ++ or certain phenolic compounds such as thyroxine or estradiol. An inhibition of sulfite oxidation by the peroxidase-thyroxine-O~ system was produced by epinephrine, glutathione, sodium thiosulfate, ascorbic acid, catalase and reduced diphosphopyridine nucleotide. 2. The oxidation of reduced diphosphopyridine nucleotide (or reduced triphosphopyridine nucleotide) by horseradish peroxidase, Mn ++ and oxygen is stimulated by sulfite ions. Hemoglobin, myoglobin, myeloperoxidase, a uterine homogenate and relatively large concentrations of inorganic iron can replace horseradish peroxidase in this reaction. Sulfite was most effective when cacodylate, phosphate, and to a lesser extent, arsenate buffers were employed, and was less effective in the presence of Tris, glycylglycine, or imidazole buffers at pH 7.0. The sulfite-activated oxidation of reduced diphosphopyridine nucleotide was inhibited by copper, hydrogen peroxide, sodium thiosulfate, glutathione, epinephrine, and ascorbic acid. 3. Sulfite and thyroxine, or sulfite and estradiol have a synergistic effect on the oxidation of reduced diphosphopyridine nucleotide by the Mn÷+-peroxidase-O, system. Sulfite ions have been employed in this way to magnify the response of the uterine enzyme to thyroxine and estradiol.
INTRODUCTION Many reactions catalyzed by peroxidase are stimulated by phenolic compounds. Among the phenolic compounds which are active in this way are thyroxine and certain of its analogues 1, ~ and phenolic estrogens 2,a. In the course of a study of the influence of thyroxine and estradiol on the Mn +÷ dependent aerobic oxidation of D P N H by peroxidase, it was observed that sodium sulfite had a marked stimulatory effect on that reaction. This prompted a study, first of the aerobic oxidation of sulfite by peroxidase, second of the oxidation of DPNH induced by the aerobic oxidation of sulfite by peroxidase and finally of the influence of thyroxine, estradiol and related compounds on these reactions. A preliminary report of this work has appeared elsewhere 4. Abbreviations: DPNH, TPNH, reduced di- and triphosphopyridine nucleotide respectively; DPN, diphosphopyridine nucleotide; TPN, triphosphopyridine nucleotide. I3iochim. Biophys. Acta, 48 (1961) 93 -1o3
94
s . I . KLEBANOFF METHODS AND MATERIALS
Oxygen uptake was determined b y standard Warburg manometric techniques. The oxidation of D P N H (or TPNH) was determined at 25 ° by the fall in absorbancy at 34 ° m/z using a Ca W MI4 recording spectrophotometer and fused quartz cells I cm in length. The blank was air unless otherwise indicated. The regeneration of D P N H from DPN was performed as follows : on the completion of the oxidation of D P N H by the peroxidase system, the p H was increased to 8.6 by the addition of sodium hydroxide and pyrophosphate buffer. Ethanol (3oo /~moles) and alcohol dehydrogenase (20/,g) were added and the increase in absorption at 34 ° m/, was determined. Horseradish peroxidase ( R Z I . I ) was obtained from Worthington Biochemical Corporation and myeloperoxidase was prepared as previously described 1. The uterine enzyme was prepared as follows: uteri from mature rats weighing approx. 30o g (Carworth farms) were homogenized in o.I M phosphate buffer p H 7.o with a Virtis "45" homogenizer.The 5 % homogenate was centrifuged at 5oo × g for 15 min and the supernatant fluid employed within 5 h of sacrifice. Crystalline myoglobin (horse heart), hemoglobin (bovine), alcohol dehydrogenase (yeast, ioo,ooo units/mg) and catalase (liver 3,ooo units/mg) were obtained from Sigma Chemical Company. Other special reagents were obtained as previously reported1, 2. The thyroxine analogues were very kindly supplied b y Drs. R. C. KROC, A. W. RUDDY and R. MELTZER of the Warner Lambert Research Institute and were dissolved in 0.oo2 N sodium hydroxide. Ethanolic solutions of the estrogens were employed except for the manometric experiments where aqueous solutions, prepared according to the method of MCSHAN AND MEYER5, were employed. The term sulfite is used in the text and in the figures to denote the mixture of sulfite and bisulfite ions which are present in solution at the particular p H employed. At the p H of most of the experiments (7.o) the species in solution is predominantly (98 %) sulfite ion. In the legends to the figures the stock solution, either sodium sulfite or sodium bisulfite, from which the aliquot was taken is indicated. A stock solution of sodium bisulfite prepared daily was employed in the majority of the experiments. RESULTS
Aerobic oxidation of sulfite by peroxidase The autoxidation of sulfite in o.I M phosphate buffer p H 7.0 was prevented b y the addition of Versene to the reaction mixture at a final concentration of I- IO-~ M. Mn ++ had a stimulatory effect on the autoxidation of sulfite even in the presence of considerably higher molar concentrations of Versene. When a relatively low concentration of Mn÷+ was employed, a marked increase in the rate of oxygen uptake was evident on the addition of peroxidase to the reaction mixture (Fig. Ia). Little or no effect of peroxidase was observed in the absence of Mn ++ under these conditions. The data presented in Fig. I a indicate that sulfite ions m a y be included among those substances which are oxidized b y peroxidase, Mn++ and oxygen in the absence of added H~O2. The stimulatory effect of thyroxine on the aerobic oxidation of sulfite in the presence of peroxidase is demonstrated in Fig. lb. In this experiment no Mn ++ was added to the reaction mixture in order to minimize the oxygen uptake in the absence Biochim. Biopkys. Acla, 48 (I961) 9 3 - t o 3
SULFITE AND REDUCED PYRIDINE NUCLEOTIDE OXIDATION
05
200 o
Mn++ + oxidose
175
Complete system
150
125 o
3_
0
100
75
50
25
/
~
Io
( Without peroxidose " (thyroxine
Peroxidose
20
30
40
I0 Time
20
30
40
(rain)
Fig. I. The aerobic oxidation of sulfite initiated b y peroxidase. The main c o m p a r t m e n t of the V~Tarl)urg vessels contained 2oo # m o l e s of Sorensen's p h o s p h a t e buffer p H 7.o, o.2 /zmole of disod i u m versenate and w a t e r to a final v o l u m e of 2.o ml. I n a, 2o /zmoles of s o d i u m bisulfite were added to the sidearm of all the flasks, and o.T /~mole of MnC12 and ~oo #g of horseradish peroxidase were added to the m a i n c o m p a r t m e n t w h e r e indicated. I n b, the complete s y s t e m contained o.I tzmole of t h y r o x i n e in one sidearm, 2o t~moles of s o d i u m bisulfite in the second sidearm and Ioo k~g of horseradish peroxidase in the m a i n c o m p a r t m e n t in addition to p h o s p h a t e buffer and Versene as indicated above. The center well contained 0.2 ml of 20 % K O H . The reaction was b e g u n b y the addition of the c o n t e n t s of the sidearms to the m a i n c o m p a r t m e n t . T e m p e r a t u r e , 37°; a t m o s p h e r e , air.
of thyroxine. Horseradish peroxidase could be replaced in this reaction by myeloperoxidase, and thyroxine by a number of thyroxine analogues as well as by certain estrogens (Table I). The oxygen uptake approached o.5 mole/mole of sulfite oxidized, varying from 8- 9 /~moles of oxygen taken up during the oxidation of 2o/xmoles of sulfite. The pH optimum was between 7.o and 7-5- Phosphate, imidazole, glycylglycine and cacodylate buffer at pH 7.o were found to have comparable activity in this system. Table II demonstrates the inhibitory effect of epinephrine, reduced glutathione, oxidized glutathione, sodium thiosulfate, ascorbic acid and catalase on the aerobic oxidation of sulfite in the presence of peroxidase and thyroxine. The inhibition by thiosulfate of the oxidation of sulfite by certain dog liver preparations has been reported 6. At low concentrations of ascorbic acid, the inhibition was seen to disappear after a lag period which decreased in length with a decrease in ascorbic acid concentration. It is probable that the lag period is the time required for the complete oxidation of ascorbic acid under these conditions. An inhibition of sulfite oxidation was produced by DPNH whether the oxidation of sulfite occurred simply in phosphate buffer pH 7.o, or in a reaction mixture which Biockim. Biophys. Acla, 48 (~06[) 93-1o3
96
s.J. KLEBANOFF
contained in addition to phosphate buffer pH 7.o, Versene, Mn++ and peroxidase, or Versene, thyroxine and peroxidase (Table lII). In the latter 2 systems the oxygen uptake increased again at high DPNH concentrations. However, the total oxygen uptake under these conditions was proportional to the DPNH concentration and TABLE I EFFECT
OF T H Y R O X I N E SULFITE
ANALOGUES OXIDATION
AND
CERTAIN
ESTROGENS
ON
BY PEROXIDASE
As in Fig. I b except t h a t the t h y r o x i n e analogues and estrogens (final concentration 5' IO-~ M) were s u b s t i t u t e d for L-thyroxine as indicated. A dditions
Oxygen uptake ~l/±o rain
None L-thyroxine D-thyroxine N-acetyl-L-thyroxine 3,3'5-triiodo-L-thyronine 3,3"5-triiodo-L-thyronine, m e t h y l ether 3,3'5' triiodo-DL-thyronine 3,5-diiodo-L-thyronine DL-thyronine 3,5-diiodo-L-tyrosine N-acetyl diiodo-L-tyrosine 3 monoiodo-L-tyrosine L-tyrosine L-phenylalanine Estradiol 17fl Diethylstilbestrol
o 7I 68 7° 47 16 66 5° 68 49 47 22 2o o 87 31
TABLE II EFFECT
OF I N H I B I T O R S
As for the complete s y s t e m in Fig. I b e x c e p t t h a t t h e inhibitors were added to the m a i n flask at the final c o n c e n t r a t i o n s indicated. The per cent inhibition was calculated from the oxygen u p t a k e during the initial i o mid of the experiment. Inhibitor
Inhibition (%)
Epinephrine 5" IO 6 M Epinephrine 5' 10 -5 M Epinephrine 5" I o -4 M Reduced glutathione 5' lO-5 M Reduced g l u t a t h i o n e 5" lO-4 M Reduced g l u t a t h i o n e 5" lO-3 M Oxidized g l u t a t h i o n e 5" Io-5 M Oxidized glutathione 5' IO-4 M Oxidized glutathione 5' lO-3 M Sodium thiosulfate 5" lO-4 M Sodium thiosulfate 5" Io-3 M S o d i u m thiosulfate 5" lO-2 M Sodium sulfate 5" lO-2 M Ascorbic acid 5" IO-~ M Ascorbic acid 5" lO-5 M Ascorbic acid 5" lo-4 M Catalase 15o/*g/ml (45 ° u n i t s / m l ) Catalase 15o/~g/ml heated a t 95 ~for I o miD
o 37 92 o 90 ioo o 88 ioo 14 50 91 o o 94 ioo 91 2i
Biochim. Biophys..4eta,
48 (1961) 9 3 - t o 3
SULFITE AND REDUCED PYRIDINE NUCLEOT1DE OXIDATION
97
TABLE III INHIBITION OF SULFITE OXIDATION BY D P N H E a c h W a r b u r g flask c o n t a i n e d 2 o / t m o l e s of s o d i u m b i s u l f i t e i n one s i de a r m a n d D P N or D P N H in t h e a m o u n t s i n d i c a t e d in t h e second s i d e a r m . T h e c o n t e n t s of t h e m a i n c o m p a r t m e n t are ind i c a t e d u n d e r S y s t e m s A, B a n d C. T h e c e n t r e w e l l c o n t a i n e d 0.2 m l of 20 % K O H . T h e r e a c t i o n w a s b e g u n b y t h e a d d i t i o n of t h e c o n t e n t s of t h e s i d e a r m s t o t h e m a i n c o m p a r t m e n t . T e m p e r a t u r e , 37°; a t m o s p h e r e , air. S y s t e m A, a u t o x i d a t i o n in p h o s p h a t e buffer p H 7.0. T h e m a i n c o m p a r t m e n t c o n t a i n e d 200 # m o l e s of p h o s p h a t e buffer p H 7.0 a n d w a t e r t o a final v o l u m e of 2.o ml. S y s t e m B, o x i d a t i o n in t h e p resence of Mn ++ a n d p e r o x i d a s e . T h e m a i n c o m p a r t m e n t c o n t a i n e d 2oo # m o l e s of p h o s p h a t e buffer p H 7.0, 0.2 # m o l e of d i s o d i u m V e r s e n a t e , o.oi # m o l e of M n C1v IOO # g of h o r s e r a d i s h p e r o x i d a s e a n d w a t e r to a final v o l u m e of 2.0 ml. S y s t e m C, o x i d a t i o n in t h e p r e s e n c e of t h y r o x i n e a n d p e r o x i d a s e . As for s y s t e m B e x c e p t t h a t o.i / ,mol e of s o d i u m - L - t h y r o x i n e r e p l a c e d t h e Mn CI v DPNH or DP N
A mount. (#moles)
-DPNH DPNH DPNH DPNH DPNH DPNH DPNH DPNH DPN DPN I)PN I)PN DPN DPN I)PN DPN
-0.025 0.05 o.i 0. 5 1 ,o 2.o 5.0 1o.o o.o25 0.05 o.I o.5 i .o 2.0 5.o
0.700 II
Oxygen uptake (#Ilxo rain) System A
System B
System C
IOZ 72 38
54 55 45 26 2 .5 4 9 ]5 58 6t 67 55 60 56 62 58
80 78 81 72 35 4 8 30 47 78 75 76 78 79 80 75 78
1o
o o 2 2 2 i 18 125 115 129 lO8 112 106 lO7
lO.O
Without peroxidase sulfite , ~ Mn-t-+
~
o~oo \
-, 0.500\ 0.400
~
0.300 0.200
Complete system
0.1 O0
,
-
Time (min)
Fig. 2. Sulfite a c t i v a t e d o x i d a t i o n of D P N H . T h e c o m p l e t e s y s t e m c o n t a i n e d 200 # m o l e s of p h o s p h a t e buffer p H 7.o, 0.3 # m o l e of D P N H , o. 5 # m o l e of MnC1 v xoo # g of h o r s e r a d i s h p e r o x i d a s e , i . o # m o l e of s o d i u m bisulfite a n d w a t e r to a final v o l u m e of 3.0 ml. B i o c h i m . B i o p h y s . A cta, 48 (I961) 93-1o3
98
s . J . KLEBANOFF
probably represented the oxidation of D P N H rather than the oxidation of sulfite. No inhibition of sulfite oxidation was observed with DPN.
Sulfite activated oxidation of D P N H D P N H (or T P N H ) is oxidized rapidly in a reaction mixture which contains peroxidase, Mn++, sulfite, oxygen and phosphate buffer p H 7.0 under the conditions employed in Fig. 2. The D P N H oxidized in this way could be readily regenerated by the alcoholalcohol dehydrogenase system. Sulfite rapidly disappears from the reaction mixture during the oxidation of D P N H under these conditions. A second aliquot of D P N H , added following the complete oxidation of the initial aliquot, is oxidized at the same rate as observed in the absence of sulfite. Only on the addition of a second aliquot of sulfite is the rate of oxidation of D P N H increased to that observed initially. The amount of sulfite routinely employed in this study (I.O /*mole) was the amount required for the complete oxidation of 0.3/,mole of D P N H at p H 7.0. Sulfite rapidly disappeared under these conditions despite the presence of D P N H in the reaction mixture which has been found under milder oxidative conditions (see Table I I I ) to inhibit sulfite oxidation. Mn ÷+ could not be replaced in this reaction by equal concentrations of Mg ++, Cu ++, Co ++, Ba ÷+, Ca ++, Cd ++, Fe ++ or Zn ÷+. A fresh stock solution of sodium sulfite or sodium bisulfite could be employed interchangeably whereas sodium metabisulfite (sodium pyrosulfite) was twice as active on a molar basis. Sodium sulfite could not be replaced by IO times the concentration of sodium sulfate (or sodium bisulfate) or sodium thiosulfate. D P N H could be replaced by equal concentrations of T P N H . An oxygen requirement was indicated by the absence of D P N H oxidation in an atmosphere of nitrogen and a phosphate requirement was suggested by the decrease in the rate of oxidation of D P N H which resulted from a decrease in the phosphate buffer concentration. A similar effect of phosphate buffer was observed when the ionic strength was maintained with either sodium chloride or potassium chloride. A marked stimulatory effect of sulfite on D P N H oxidation was observed with phosphate, cacodylate and to a lesser extent arsenate buffer, whereas sulfite was much less effective in the presence of Tris, glycylglycine or imidazole buffer at p H 7.0. Caeodylate buffer was more effective than phosphate buffer at a concentration of 200 /*moles/ 3.0 ml of reaction mixture. However, the rate of oxidation of D P N H fell more rapidly with a decrease in cacodylate concentration than with a decrease in phosphate concentration. At concentrations of buffer below IOO vmoles/3.o ml of reaction mixture, the rate of oxidation of D P N H in phosphate buffer was more rapid than in cacodylate buffer. Both the rate of oxidation of D P N H and the total amount of D P N H oxidized was increased with a fall in p H from 8.0 to 7.0 when 0.03 M Sorensen's phosphate buffer was employed. As the p H was decreased from 7.0 to 5.7, the total amount of D P N H oxidized was increased slightly with little change in the rate of oxidation. Myeloperoxidase, hemoglobin or myoglobin could substitute for horseradish peroxidase in the sulfite activated oxidation of D P N H under the conditions employed in Fig. 3. Inorganic iron also was active in relatively high concentrations (Fig. 3d). The oxidation of D P N H in the presence of hemoglobin or myoglobin was associated with an initial lag period which was abolished by a low concentration of H~O 2 and prolonged b y the addition of catalase.
Biochim. Biophys. Acla, 48 (1961) 93-1o3
SULFITE AND REDUCED PYRIDINE NUCLEOTIDE OXIDATION
Without Mn+f sulfite
0.700
9 ~)
Without Mn +f
0.600 o
0.500 globm
Myeloperoxidase
0.400 0.300 zL E 0.200 0 ro 0.1 O0 I
I
Complete
Complete
system
system i
I
I
1
¢ -6 ._o o
Without sulflte
Without Mn ++
0.700
ik
0.600
d ,,Mn
0.500
.
_
~ o b i n
~cNoride
0.400 Complete 0.300
system
0.200 Complete ~ystem
0.100
I L
I 2
L. 3
l I
L 2
I
3
Time (rain)
Fig. 3. Effect of myeloperoxidase, hemoglobin, myoglobin, and ferrous chloride. As in Fig. 2 except that the horseradish peroxidase w a s replaced by o.i ml of a myeloperoxidase solution (absorbancy 43o/absorbancy 390 = 1.55; absorbancy at 43 ° m# ~ 0.980) in a, by 2o0 #g of hemoglobin in b, by 2o0 #g of myoglobin in c, and by o.1 ffmole of ferrous chloride in d.
Inhibitors Cupric chloride at concentrations equimolar to that of Mn ++ in Fig. 2 (I.7- Io-4M), completely inhibited the sulfite activated oxidation of D P N H . H20 2 at concentrations equal to or greater than the sulfite concentration decreased both the rate and the total amount of D P N H oxidized, which suggested a competitive oxidation of sulfite by H20 2. An inhibition of the sulfite activated oxidation of D P N H was produced by sodium thiosulfate at concentrations of 3" lO-6 M or greater, by glutathione, either oxidized or reduced, at concentrations of 3" 1°-5 M or greater and by epinephrine or ascorbic acid at concentrations of 6- lO -7 M or greater. Crystalline catalase at concentrations up to 500 ~ g / 3 . 0 ml of reaction mixture did not inhibit the sulfite activated oxidation of D P N H in contrast to the inhibitory effect of catalase on the aerobic oxidation of sulfite ions (see Table II). Biochim. Biophys. Acla, 48 (I96~) 93--io3
IOO
s . J . KLEBANOFF
Stimulators
Table IV demonstrates the effect of thyroxine, certain thyroxine analogues, and estrogens on the oxidation of D P N H in the presence and absence of sulfite ions. In this experiment both the Mn ++ and peroxidase concentration was decreased until I.O/zmole of sodium sulfite alone had no effect on D P N H oxidation. A stimulatory effect of 0.05 tzmole of the thyroxine analogue or estrogen was still evident in a number of instances. A combination of sulfite ions and the thyroxine analogue or estrogen resulted in a considerable increase in the rate of oxidation of DPNH. The conversion of triiodothyronine to its methyl ether, or the conversion of thyroxine to N-acetyl thyroxine, decreased, but did not abolish, the stimulatory effect of these compounds on the oxidation of D P N H in the presence of sulfite. Triiodothyroacetie acid had considerably less a c t i v i t y t h a n triiodothyronine. Estradiol in equimolar concentrations was less active than thyroxine and the effect of estradiol was abolished by conversion to the 3 acetate. The synergistic effect of sulfite and thyroxine, and sulfite and estradiol on the oxidation of D P N H by Mn ÷+, peroxidase and oxygen, suggests a means by which a hormone sensitive peroxidase present in small amounts in tissues m a y be demonstrated. This is illustrated in Fig. 4. The phenol activated D P N H oxidase of uterus, which is considered 7 to be identical with the uterine peroxidase described by LUCAS et al. 8 is stimulated by estradiol 9 and thyroxinO ° under certain conditions. Little or no effect of the hormones on the oxidation of D P N H was observed when the assay was performed at p H 7.0 and when the homogenate (5 %) was prepared from a normal adult animal not previously injected with estrogen (Fig. 4). However, a marked effect of estradiol or thyroxine was observed when sulfite ions also were added to the assay system (Fig. 4)- Similar results were observed when T P N H was employed instead of D P N H . An oxidation of D P N H is observed in the absence of the uterine preparation under the conditions employed in Fig. 4. This non-enzymic oxidation of D P N H in the presence of Mn++, 03, sulfite, and phosphate buffer p H 7.0 was found to be greatly stimulated by the presence of cobaltous ions in the reaction mixture and to be sensitive to thyroxine n. Caution should be exercised in the interpretation of an apparent stimulation by thyroxine of an enzymic oxidation of D P N H under conditions in which a thyroxine sensitive non-enzymic oxidation of D P N H might be expected. DISCUSSION FRIDOVICH AND HANDLER12-14 have made use of the aerobic oxidation of sulfite as a sensitive detector of free radicals in a number of enzyme reactions based on the fact that the aerobic oxidation of sulfite is a chain reaction which is initiated by free radicals. They report on the initiation of sulfite oxidation by horseradish peroxidase in the presence of peroxide and a peroxidizable substrate 14. The aerobic oxidation of sulfite is shown here to be initiated by peroxidase in the absence of added H20 2 if either Mn ++ or a phenolic compound such as thyroxine or estradiol is added to the reaction mixture (Fig. I). That H20 2 is formed and utilized in the course of this Ieaction is suggested by the inhibitory effect of catalase (Table II). These data, and those o f FRIDOVICHAND HANDLER J 4 suggest that free radicals are formed in certain peroxidase catalyzed reactions and indeed, YAMAZAKIet al. is, 16 have identifed free radicals Biochim. Biophys. 3cta, 48 (1961) 93-io3
SULFITE AND REDUCED PYRIDINE NUCLEOTIDE OXIDATION
IOI
TABLE IV E F F E C T OF T H Y R O X I N E , T H Y R O X I N E ANALOGUES AND ESTROGENS
The reaction m i x t u r e contained 200 ffmoles of p h o s p h a t e buffer p H 7.0, 0. 3 ffmole of D P N H , o.oi /~mole of MnC12, 5fig of horseradish peroxidase a n d w a t e r to a final v o l u m e of 3.o ml. Sodium bisulfite (i.o ffmole), t h y r o x i n e (o.o 5 ffmole), t h y r o x i n e analogues (0.05 #mole) and estrogens (0.05/,mole) were added as indicated. E t h a n o l was added w i t h the estrogens at a final concentration of 1 . 7 %. Absorbancy change per minute at 340 ml* Amdogue
+ analogue,
+ analogue,
sulfite
+ sulfite
-
-
L-thyroxine N-acetyl-L-thyroxine 3,3'5-triiodo-L-thyronine 3,3'5-triiodo-L-thyronine, m e t h y l ether 3,3'5'-triiodo-DL-thyronine 3,5-diiodo-L-thyronine DL-thyronine 3,3'5-triiodothyroacetic acid 3,3'5-triiodothyroacetic acid m e t h y l ether 3,5-diiodo-L-tyrosine N-acetyl 3,5-diiodo-L-tyrosine 3-monoiodo-L-tyrosine L-tyrosine Estradiol 17fl Estradiol 170~ Estradiol 17-acetate Estradiol 3-acetate
o.o6o o.o8o o.o75 0.005 o. 15 ° o.t oo 0.060 0.04o 0.005 0.o0.5 0.005 o.ooo o.ooo o.o7o
o.38o o. 17 ° o.35o 0.065 0.760 o.24o 0.440 0.070 0.020 0.025 o.ol o o.ol o o.ooo o.i 9o
0.000
O. 145
o.o45 o.ooo
o.i 3 ° o.ooo
b Without surf ire ',' e s l r a d i o l Mn +¢ " ulerine preperotion
~
W~thout thyroxine
0.700
i sulfite Mn ++ ' ulerlne preperotiofl
0.600 E ¢)
i
0.500
•~ 0.400 c
0.300
3
system
I Complete
0.200
Complete
syslern
oJoo
L
i
Time (rain)
2
5
Fig. 4- The synergistic effect of sulfite and t h y r o x i n e or sulfite and estradiol on D P N H oxidation b y the uterine enzyme. The complete s y s t e m contained 200 # m o l e s of p h o s p h a t e buffer p H 7.o, 0. 3 /~mole of D P N H , 0 . 5 / , m o l e of MnC12, o.2 ml of a 5 % uterine p r e p a r a t i o n , o.i /,mole of t h y r oxine in a ; o. I # m o l e of estradiol 17fl and ethanol at a final c o n c e n t r a t i o n of 3.3 % in b ; I .o #mole of s o d i u m bisulfite and w a t e r to a v o l u m e of 3.o ml. The b l a n k contained all c o m p o n e n t s except D P N H .
13iochim. Biophys. Acta, 48 (I961) 93 lO3
102
S . J . KLEBANOFF
generated from substrates by peroxidase by means of electron paramagnetic resonance spectroscopy. Once initiated, sulfite oxidation is maintained as a chain reaction through the action of highly reactive intermediates formed either from the stepwise reduction of oxygen or the stepwise oxidation of sulfite ions. The inhibition of sulfite oxidation by D P N H (Table III) and the oxidation of D P N H induced by the aerobic oxidation of sulfite in the presence of peroxidase (Fig. 2) suggest that DPNH reacts with the same component of the sulfite oxidation system which is essential for the propagation of the chain reaction and is itself oxidized in the process. A similar mechanism was advocated by ALYEAAND BACKSTROMfor the inhibitory effect of certain alcohols on the oxidation of sodium sulfite 17. The oxidation of DPNH becomes a sensitive detector of the free radical chain initiating event under these conditions. Thyroxine, estradiol as well as certain other phenolic compounds have a stimulatory effect on a number of peroxidase catalyzed reactions 1-3. Thyroxine and estradiol are substrates for peroxidase 18,19 and are essential, under certain conditions for the initiation of sulfite oxidation by peroxidase. This is at least suggestive evidence for the conversion of thyroxine and estradiol to free radical intermediates (possibly phenoxy radicals) under the influence of peroxidase and is compatible with a role for the free radicals in the stimulatory effect of thyroxine, estradiol as well as certain other phenolic compounds on peroxidase catalyzed reactions, as proposed previously by a number of investigators3,2°, 21. In the absence of the protective effect of certain hydrogen donors, thyroxine and estradiol are oxidized further by peroxidase and H20 ~ to forms which are no longer active either biologically*, 82 or as stimulators of certain peroxidase catalyzed reactions ~9. MAYRARQUE-KODJAet al. 18 have reported on the extensive structural changes observed on the exposure of thyroxine to peroxidase and H202 and at least 5 chromatographically distinct products of estradiol oxidation by H~O~ and horseradish peroxidase are demonstrable (J. ENSINCKAND S. J. KLEBANOFI;, unpublished data). The synergistic effect of sulfite and thyroxine, or sulfite and estradiol on D P N H oxidation in the presence of peroxidase (Table IV) suggests a means by which the stimulatory effect of thyroxine or estradiol on this peroxidase system may be magnified. That this may prove useful in the search for thyroxine or estradiol sensitive peroxidase systems in mammalian tissues is suggested by the potentiation by sulfite ions of the hormonal effect on the oxidation of D P N H or T P N H by the uterine enzyme (Fig. 4)ACKNOWLEDGEMENTS
I am grateful to Dr. R. M. ARCHIBALD for his encouragement and support and to Miss K. HOULDER for her valuable technical assistance. This work was supported in part by research grant A 2772 from the National Institutes of Health. REFERENCES 1 S. J. KLEBANOFF,J. Biol. Chem., 234 (1959) 2437. S. J. KLEBANOFF,J . Biol. Chem., 234 (1959) 2480. 3 H. G. WILLIAMS-ASHMAN,M. CASSMANAND M. KLAVINS, Nature, 184 (1959) 427. S. J. KLEBANOFF, Federation Proc., 19 (196o) 31. * The inactivation of thyroxine by the HzO~-peroxidase system was demonstrated by the tadpole metamorphosis test (S. J. KLEBANOFF, unpublished data).
Biochim. Biophys. Acta, 48 (1961) 93-1o 3
S U L F I T E AND R E D U C E D
PYRIDINE
NUCLEOTIDE
OXIDATION
103
5 W. a . MCSHAN AND R . K. MEYER, Arch. Biochem., 9 (1946) 165. 8 I. FR~DOVlCH AND P. HANDLER, J. Biol. Chem., 223 (1956) 321. 7 V. P. HOLLANDER AND J. R . BEARD, 42rid meeting o/the Endocrine Society, A b s t r a c t No. 39, 1960. 8 F. V. LUCAS, H . A. NEUFELD, J. G. UTTERBACK, A. P. MARTIN AND E . STOTZ, J. Biol. Chem., 214 (1955) 775. 9 S. TEMPLE, V. P. HOLLANDER, N. HOLLANDER AND M. L. STEPHENS, J. Biol. Chem., 235 (196o) 15o4. 10 A. T. nEVER, C. M. SOKATCH AND J. H . ANGI1N, Federation Proc., 19 (196o) 175. 11 S. J. KLEBANOFF, First International Congress o/Endocrinology, Copenhagen, 196o. 12 I. FR1DOVICH AND P. HANDLER, J. Biol. Chem., 233 (1958) 1578. 13 I. FRIDOVlCH AND ]~. HANDLER, Biochim. Biophys. Acta, 35 (19.59) 546. 14 I. FRIDOV1CH AND P. HANDLER, Federation Proc., I 9 (196o) 29. 15 I. YAMAZAK1, H . S. MASON AND L. PIETTE, Biochem. Biophys. Research, 1 (I959) 336. 16 I. YAMAZAKI, H . ,%. MASON AND L. PIETTE, Federation Proc., 19 (196o) 33. 17 H . N. ALYEA AND H . L. V. BACKSTROM, J. ~,~m. Chem. Soc., 51 (1929) 90. 18 A. MAYRARQUE-KODJA, S. BOUCHILLOUX AND S. LISSITZKY, Bull. s6c. chim. biol., 4 ° (1958) 815. 19 S. J. KLEBANOFF, Biochim. Biophys. Acla, 44 (196o) 5Ol. 20 n . CHANCE, Arch. Biochem., 24 (1949) 389. 21 T. AKAZAWA AND E . E . CONN, J. Biol. Chem., 232 (1958) 403 • ~,2 S. J. ]{LEBANOFE AND S. J. SEGAL, J. Biol. Chem., 235 (196o) 52.
Biochim. Biophys. Acta, 48 (1961) 9 3 - l O 3
03
THE SULFITE-ACTIVATED OXIDATION OF REDUCED PYRIDINE
NUCLEOTIDES BY PEROXIDASE S. J. KLEBANOFF
The Rocke[eller Institute, New York, N . Y . (U.S.A.)
(Received August 2oth, I96o)
SUMMARY I. The aerobic oxidation of sulfite ions is initiated by peroxidase in the presence of Mn ++ or certain phenolic compounds such as thyroxine or estradiol. An inhibition of sulfite oxidation by the peroxidase-thyroxine-O~ system was produced by epinephrine, glutathione, sodium thiosulfate, ascorbic acid, catalase and reduced diphosphopyridine nucleotide. 2. The oxidation of reduced diphosphopyridine nucleotide (or reduced triphosphopyridine nucleotide) by horseradish peroxidase, Mn ++ and oxygen is stimulated by sulfite ions. Hemoglobin, myoglobin, myeloperoxidase, a uterine homogenate and relatively large concentrations of inorganic iron can replace horseradish peroxidase in this reaction. Sulfite was most effective when cacodylate, phosphate, and to a lesser extent, arsenate buffers were employed, and was less effective in the presence of Tris, glycylglycine, or imidazole buffers at pH 7.0. The sulfite-activated oxidation of reduced diphosphopyridine nucleotide was inhibited by copper, hydrogen peroxide, sodium thiosulfate, glutathione, epinephrine, and ascorbic acid. 3. Sulfite and thyroxine, or sulfite and estradiol have a synergistic effect on the oxidation of reduced diphosphopyridine nucleotide by the Mn÷+-peroxidase-O, system. Sulfite ions have been employed in this way to magnify the response of the uterine enzyme to thyroxine and estradiol.
INTRODUCTION Many reactions catalyzed by peroxidase are stimulated by phenolic compounds. Among the phenolic compounds which are active in this way are thyroxine and certain of its analogues 1, ~ and phenolic estrogens 2,a. In the course of a study of the influence of thyroxine and estradiol on the Mn +÷ dependent aerobic oxidation of D P N H by peroxidase, it was observed that sodium sulfite had a marked stimulatory effect on that reaction. This prompted a study, first of the aerobic oxidation of sulfite by peroxidase, second of the oxidation of DPNH induced by the aerobic oxidation of sulfite by peroxidase and finally of the influence of thyroxine, estradiol and related compounds on these reactions. A preliminary report of this work has appeared elsewhere 4. Abbreviations: DPNH, TPNH, reduced di- and triphosphopyridine nucleotide respectively; DPN, diphosphopyridine nucleotide; TPN, triphosphopyridine nucleotide. I3iochim. Biophys. Acta, 48 (1961) 93 -1o3
94
s . I . KLEBANOFF METHODS AND MATERIALS
Oxygen uptake was determined b y standard Warburg manometric techniques. The oxidation of D P N H (or TPNH) was determined at 25 ° by the fall in absorbancy at 34 ° m/z using a Ca W MI4 recording spectrophotometer and fused quartz cells I cm in length. The blank was air unless otherwise indicated. The regeneration of D P N H from DPN was performed as follows : on the completion of the oxidation of D P N H by the peroxidase system, the p H was increased to 8.6 by the addition of sodium hydroxide and pyrophosphate buffer. Ethanol (3oo /~moles) and alcohol dehydrogenase (20/,g) were added and the increase in absorption at 34 ° m/, was determined. Horseradish peroxidase ( R Z I . I ) was obtained from Worthington Biochemical Corporation and myeloperoxidase was prepared as previously described 1. The uterine enzyme was prepared as follows: uteri from mature rats weighing approx. 30o g (Carworth farms) were homogenized in o.I M phosphate buffer p H 7.o with a Virtis "45" homogenizer.The 5 % homogenate was centrifuged at 5oo × g for 15 min and the supernatant fluid employed within 5 h of sacrifice. Crystalline myoglobin (horse heart), hemoglobin (bovine), alcohol dehydrogenase (yeast, ioo,ooo units/mg) and catalase (liver 3,ooo units/mg) were obtained from Sigma Chemical Company. Other special reagents were obtained as previously reported1, 2. The thyroxine analogues were very kindly supplied b y Drs. R. C. KROC, A. W. RUDDY and R. MELTZER of the Warner Lambert Research Institute and were dissolved in 0.oo2 N sodium hydroxide. Ethanolic solutions of the estrogens were employed except for the manometric experiments where aqueous solutions, prepared according to the method of MCSHAN AND MEYER5, were employed. The term sulfite is used in the text and in the figures to denote the mixture of sulfite and bisulfite ions which are present in solution at the particular p H employed. At the p H of most of the experiments (7.o) the species in solution is predominantly (98 %) sulfite ion. In the legends to the figures the stock solution, either sodium sulfite or sodium bisulfite, from which the aliquot was taken is indicated. A stock solution of sodium bisulfite prepared daily was employed in the majority of the experiments. RESULTS
Aerobic oxidation of sulfite by peroxidase The autoxidation of sulfite in o.I M phosphate buffer p H 7.0 was prevented b y the addition of Versene to the reaction mixture at a final concentration of I- IO-~ M. Mn ++ had a stimulatory effect on the autoxidation of sulfite even in the presence of considerably higher molar concentrations of Versene. When a relatively low concentration of Mn÷+ was employed, a marked increase in the rate of oxygen uptake was evident on the addition of peroxidase to the reaction mixture (Fig. Ia). Little or no effect of peroxidase was observed in the absence of Mn ++ under these conditions. The data presented in Fig. I a indicate that sulfite ions m a y be included among those substances which are oxidized b y peroxidase, Mn++ and oxygen in the absence of added H~O2. The stimulatory effect of thyroxine on the aerobic oxidation of sulfite in the presence of peroxidase is demonstrated in Fig. lb. In this experiment no Mn ++ was added to the reaction mixture in order to minimize the oxygen uptake in the absence Biochim. Biopkys. Acla, 48 (I961) 9 3 - t o 3
SULFITE AND REDUCED PYRIDINE NUCLEOTIDE OXIDATION
05
200 o
Mn++ + oxidose
175
Complete system
150
125 o
3_
0
100
75
50
25
/
~
Io
( Without peroxidose " (thyroxine
Peroxidose
20
30
40
I0 Time
20
30
40
(rain)
Fig. I. The aerobic oxidation of sulfite initiated b y peroxidase. The main c o m p a r t m e n t of the V~Tarl)urg vessels contained 2oo # m o l e s of Sorensen's p h o s p h a t e buffer p H 7.o, o.2 /zmole of disod i u m versenate and w a t e r to a final v o l u m e of 2.o ml. I n a, 2o /zmoles of s o d i u m bisulfite were added to the sidearm of all the flasks, and o.T /~mole of MnC12 and ~oo #g of horseradish peroxidase were added to the m a i n c o m p a r t m e n t w h e r e indicated. I n b, the complete s y s t e m contained o.I tzmole of t h y r o x i n e in one sidearm, 2o t~moles of s o d i u m bisulfite in the second sidearm and Ioo k~g of horseradish peroxidase in the m a i n c o m p a r t m e n t in addition to p h o s p h a t e buffer and Versene as indicated above. The center well contained 0.2 ml of 20 % K O H . The reaction was b e g u n b y the addition of the c o n t e n t s of the sidearms to the m a i n c o m p a r t m e n t . T e m p e r a t u r e , 37°; a t m o s p h e r e , air.
of thyroxine. Horseradish peroxidase could be replaced in this reaction by myeloperoxidase, and thyroxine by a number of thyroxine analogues as well as by certain estrogens (Table I). The oxygen uptake approached o.5 mole/mole of sulfite oxidized, varying from 8- 9 /~moles of oxygen taken up during the oxidation of 2o/xmoles of sulfite. The pH optimum was between 7.o and 7-5- Phosphate, imidazole, glycylglycine and cacodylate buffer at pH 7.o were found to have comparable activity in this system. Table II demonstrates the inhibitory effect of epinephrine, reduced glutathione, oxidized glutathione, sodium thiosulfate, ascorbic acid and catalase on the aerobic oxidation of sulfite in the presence of peroxidase and thyroxine. The inhibition by thiosulfate of the oxidation of sulfite by certain dog liver preparations has been reported 6. At low concentrations of ascorbic acid, the inhibition was seen to disappear after a lag period which decreased in length with a decrease in ascorbic acid concentration. It is probable that the lag period is the time required for the complete oxidation of ascorbic acid under these conditions. An inhibition of sulfite oxidation was produced by DPNH whether the oxidation of sulfite occurred simply in phosphate buffer pH 7.o, or in a reaction mixture which Biockim. Biophys. Acla, 48 (~06[) 93-1o3
96
s.J. KLEBANOFF
contained in addition to phosphate buffer pH 7.o, Versene, Mn++ and peroxidase, or Versene, thyroxine and peroxidase (Table lII). In the latter 2 systems the oxygen uptake increased again at high DPNH concentrations. However, the total oxygen uptake under these conditions was proportional to the DPNH concentration and TABLE I EFFECT
OF T H Y R O X I N E SULFITE
ANALOGUES OXIDATION
AND
CERTAIN
ESTROGENS
ON
BY PEROXIDASE
As in Fig. I b except t h a t the t h y r o x i n e analogues and estrogens (final concentration 5' IO-~ M) were s u b s t i t u t e d for L-thyroxine as indicated. A dditions
Oxygen uptake ~l/±o rain
None L-thyroxine D-thyroxine N-acetyl-L-thyroxine 3,3'5-triiodo-L-thyronine 3,3"5-triiodo-L-thyronine, m e t h y l ether 3,3'5' triiodo-DL-thyronine 3,5-diiodo-L-thyronine DL-thyronine 3,5-diiodo-L-tyrosine N-acetyl diiodo-L-tyrosine 3 monoiodo-L-tyrosine L-tyrosine L-phenylalanine Estradiol 17fl Diethylstilbestrol
o 7I 68 7° 47 16 66 5° 68 49 47 22 2o o 87 31
TABLE II EFFECT
OF I N H I B I T O R S
As for the complete s y s t e m in Fig. I b e x c e p t t h a t t h e inhibitors were added to the m a i n flask at the final c o n c e n t r a t i o n s indicated. The per cent inhibition was calculated from the oxygen u p t a k e during the initial i o mid of the experiment. Inhibitor
Inhibition (%)
Epinephrine 5" IO 6 M Epinephrine 5' 10 -5 M Epinephrine 5" I o -4 M Reduced glutathione 5' lO-5 M Reduced g l u t a t h i o n e 5" lO-4 M Reduced g l u t a t h i o n e 5" lO-3 M Oxidized g l u t a t h i o n e 5" Io-5 M Oxidized glutathione 5' IO-4 M Oxidized glutathione 5' lO-3 M Sodium thiosulfate 5" lO-4 M Sodium thiosulfate 5" Io-3 M S o d i u m thiosulfate 5" lO-2 M Sodium sulfate 5" lO-2 M Ascorbic acid 5" IO-~ M Ascorbic acid 5" lO-5 M Ascorbic acid 5" lo-4 M Catalase 15o/*g/ml (45 ° u n i t s / m l ) Catalase 15o/~g/ml heated a t 95 ~for I o miD
o 37 92 o 90 ioo o 88 ioo 14 50 91 o o 94 ioo 91 2i
Biochim. Biophys..4eta,
48 (1961) 9 3 - t o 3
SULFITE AND REDUCED PYRIDINE NUCLEOT1DE OXIDATION
97
TABLE III INHIBITION OF SULFITE OXIDATION BY D P N H E a c h W a r b u r g flask c o n t a i n e d 2 o / t m o l e s of s o d i u m b i s u l f i t e i n one s i de a r m a n d D P N or D P N H in t h e a m o u n t s i n d i c a t e d in t h e second s i d e a r m . T h e c o n t e n t s of t h e m a i n c o m p a r t m e n t are ind i c a t e d u n d e r S y s t e m s A, B a n d C. T h e c e n t r e w e l l c o n t a i n e d 0.2 m l of 20 % K O H . T h e r e a c t i o n w a s b e g u n b y t h e a d d i t i o n of t h e c o n t e n t s of t h e s i d e a r m s t o t h e m a i n c o m p a r t m e n t . T e m p e r a t u r e , 37°; a t m o s p h e r e , air. S y s t e m A, a u t o x i d a t i o n in p h o s p h a t e buffer p H 7.0. T h e m a i n c o m p a r t m e n t c o n t a i n e d 200 # m o l e s of p h o s p h a t e buffer p H 7.0 a n d w a t e r t o a final v o l u m e of 2.o ml. S y s t e m B, o x i d a t i o n in t h e p resence of Mn ++ a n d p e r o x i d a s e . T h e m a i n c o m p a r t m e n t c o n t a i n e d 2oo # m o l e s of p h o s p h a t e buffer p H 7.0, 0.2 # m o l e of d i s o d i u m V e r s e n a t e , o.oi # m o l e of M n C1v IOO # g of h o r s e r a d i s h p e r o x i d a s e a n d w a t e r to a final v o l u m e of 2.0 ml. S y s t e m C, o x i d a t i o n in t h e p r e s e n c e of t h y r o x i n e a n d p e r o x i d a s e . As for s y s t e m B e x c e p t t h a t o.i / ,mol e of s o d i u m - L - t h y r o x i n e r e p l a c e d t h e Mn CI v DPNH or DP N
A mount. (#moles)
-DPNH DPNH DPNH DPNH DPNH DPNH DPNH DPNH DPN DPN I)PN I)PN DPN DPN I)PN DPN
-0.025 0.05 o.i 0. 5 1 ,o 2.o 5.0 1o.o o.o25 0.05 o.I o.5 i .o 2.0 5.o
0.700 II
Oxygen uptake (#Ilxo rain) System A
System B
System C
IOZ 72 38
54 55 45 26 2 .5 4 9 ]5 58 6t 67 55 60 56 62 58
80 78 81 72 35 4 8 30 47 78 75 76 78 79 80 75 78
1o
o o 2 2 2 i 18 125 115 129 lO8 112 106 lO7
lO.O
Without peroxidase sulfite , ~ Mn-t-+
~
o~oo \
-, 0.500\ 0.400
~
0.300 0.200
Complete system
0.1 O0
,
-
Time (min)
Fig. 2. Sulfite a c t i v a t e d o x i d a t i o n of D P N H . T h e c o m p l e t e s y s t e m c o n t a i n e d 200 # m o l e s of p h o s p h a t e buffer p H 7.o, 0.3 # m o l e of D P N H , o. 5 # m o l e of MnC1 v xoo # g of h o r s e r a d i s h p e r o x i d a s e , i . o # m o l e of s o d i u m bisulfite a n d w a t e r to a final v o l u m e of 3.0 ml. B i o c h i m . B i o p h y s . A cta, 48 (I961) 93-1o3
98
s . J . KLEBANOFF
probably represented the oxidation of D P N H rather than the oxidation of sulfite. No inhibition of sulfite oxidation was observed with DPN.
Sulfite activated oxidation of D P N H D P N H (or T P N H ) is oxidized rapidly in a reaction mixture which contains peroxidase, Mn++, sulfite, oxygen and phosphate buffer p H 7.0 under the conditions employed in Fig. 2. The D P N H oxidized in this way could be readily regenerated by the alcoholalcohol dehydrogenase system. Sulfite rapidly disappears from the reaction mixture during the oxidation of D P N H under these conditions. A second aliquot of D P N H , added following the complete oxidation of the initial aliquot, is oxidized at the same rate as observed in the absence of sulfite. Only on the addition of a second aliquot of sulfite is the rate of oxidation of D P N H increased to that observed initially. The amount of sulfite routinely employed in this study (I.O /*mole) was the amount required for the complete oxidation of 0.3/,mole of D P N H at p H 7.0. Sulfite rapidly disappeared under these conditions despite the presence of D P N H in the reaction mixture which has been found under milder oxidative conditions (see Table I I I ) to inhibit sulfite oxidation. Mn ÷+ could not be replaced in this reaction by equal concentrations of Mg ++, Cu ++, Co ++, Ba ÷+, Ca ++, Cd ++, Fe ++ or Zn ÷+. A fresh stock solution of sodium sulfite or sodium bisulfite could be employed interchangeably whereas sodium metabisulfite (sodium pyrosulfite) was twice as active on a molar basis. Sodium sulfite could not be replaced by IO times the concentration of sodium sulfate (or sodium bisulfate) or sodium thiosulfate. D P N H could be replaced by equal concentrations of T P N H . An oxygen requirement was indicated by the absence of D P N H oxidation in an atmosphere of nitrogen and a phosphate requirement was suggested by the decrease in the rate of oxidation of D P N H which resulted from a decrease in the phosphate buffer concentration. A similar effect of phosphate buffer was observed when the ionic strength was maintained with either sodium chloride or potassium chloride. A marked stimulatory effect of sulfite on D P N H oxidation was observed with phosphate, cacodylate and to a lesser extent arsenate buffer, whereas sulfite was much less effective in the presence of Tris, glycylglycine or imidazole buffer at p H 7.0. Caeodylate buffer was more effective than phosphate buffer at a concentration of 200 /*moles/ 3.0 ml of reaction mixture. However, the rate of oxidation of D P N H fell more rapidly with a decrease in cacodylate concentration than with a decrease in phosphate concentration. At concentrations of buffer below IOO vmoles/3.o ml of reaction mixture, the rate of oxidation of D P N H in phosphate buffer was more rapid than in cacodylate buffer. Both the rate of oxidation of D P N H and the total amount of D P N H oxidized was increased with a fall in p H from 8.0 to 7.0 when 0.03 M Sorensen's phosphate buffer was employed. As the p H was decreased from 7.0 to 5.7, the total amount of D P N H oxidized was increased slightly with little change in the rate of oxidation. Myeloperoxidase, hemoglobin or myoglobin could substitute for horseradish peroxidase in the sulfite activated oxidation of D P N H under the conditions employed in Fig. 3. Inorganic iron also was active in relatively high concentrations (Fig. 3d). The oxidation of D P N H in the presence of hemoglobin or myoglobin was associated with an initial lag period which was abolished by a low concentration of H~O 2 and prolonged b y the addition of catalase.
Biochim. Biophys. Acla, 48 (1961) 93-1o3
SULFITE AND REDUCED PYRIDINE NUCLEOTIDE OXIDATION
Without Mn+f sulfite
0.700
9 ~)
Without Mn +f
0.600 o
0.500 globm
Myeloperoxidase
0.400 0.300 zL E 0.200 0 ro 0.1 O0 I
I
Complete
Complete
system
system i
I
I
1
¢ -6 ._o o
Without sulflte
Without Mn ++
0.700
ik
0.600
d ,,Mn
0.500
.
_
~ o b i n
~cNoride
0.400 Complete 0.300
system
0.200 Complete ~ystem
0.100
I L
I 2
L. 3
l I
L 2
I
3
Time (rain)
Fig. 3. Effect of myeloperoxidase, hemoglobin, myoglobin, and ferrous chloride. As in Fig. 2 except that the horseradish peroxidase w a s replaced by o.i ml of a myeloperoxidase solution (absorbancy 43o/absorbancy 390 = 1.55; absorbancy at 43 ° m# ~ 0.980) in a, by 2o0 #g of hemoglobin in b, by 2o0 #g of myoglobin in c, and by o.1 ffmole of ferrous chloride in d.
Inhibitors Cupric chloride at concentrations equimolar to that of Mn ++ in Fig. 2 (I.7- Io-4M), completely inhibited the sulfite activated oxidation of D P N H . H20 2 at concentrations equal to or greater than the sulfite concentration decreased both the rate and the total amount of D P N H oxidized, which suggested a competitive oxidation of sulfite by H20 2. An inhibition of the sulfite activated oxidation of D P N H was produced by sodium thiosulfate at concentrations of 3" lO-6 M or greater, by glutathione, either oxidized or reduced, at concentrations of 3" 1°-5 M or greater and by epinephrine or ascorbic acid at concentrations of 6- lO -7 M or greater. Crystalline catalase at concentrations up to 500 ~ g / 3 . 0 ml of reaction mixture did not inhibit the sulfite activated oxidation of D P N H in contrast to the inhibitory effect of catalase on the aerobic oxidation of sulfite ions (see Table II). Biochim. Biophys. Acla, 48 (I96~) 93--io3
IOO
s . J . KLEBANOFF
Stimulators
Table IV demonstrates the effect of thyroxine, certain thyroxine analogues, and estrogens on the oxidation of D P N H in the presence and absence of sulfite ions. In this experiment both the Mn ++ and peroxidase concentration was decreased until I.O/zmole of sodium sulfite alone had no effect on D P N H oxidation. A stimulatory effect of 0.05 tzmole of the thyroxine analogue or estrogen was still evident in a number of instances. A combination of sulfite ions and the thyroxine analogue or estrogen resulted in a considerable increase in the rate of oxidation of DPNH. The conversion of triiodothyronine to its methyl ether, or the conversion of thyroxine to N-acetyl thyroxine, decreased, but did not abolish, the stimulatory effect of these compounds on the oxidation of D P N H in the presence of sulfite. Triiodothyroacetie acid had considerably less a c t i v i t y t h a n triiodothyronine. Estradiol in equimolar concentrations was less active than thyroxine and the effect of estradiol was abolished by conversion to the 3 acetate. The synergistic effect of sulfite and thyroxine, and sulfite and estradiol on the oxidation of D P N H by Mn ÷+, peroxidase and oxygen, suggests a means by which a hormone sensitive peroxidase present in small amounts in tissues m a y be demonstrated. This is illustrated in Fig. 4. The phenol activated D P N H oxidase of uterus, which is considered 7 to be identical with the uterine peroxidase described by LUCAS et al. 8 is stimulated by estradiol 9 and thyroxinO ° under certain conditions. Little or no effect of the hormones on the oxidation of D P N H was observed when the assay was performed at p H 7.0 and when the homogenate (5 %) was prepared from a normal adult animal not previously injected with estrogen (Fig. 4). However, a marked effect of estradiol or thyroxine was observed when sulfite ions also were added to the assay system (Fig. 4)- Similar results were observed when T P N H was employed instead of D P N H . An oxidation of D P N H is observed in the absence of the uterine preparation under the conditions employed in Fig. 4. This non-enzymic oxidation of D P N H in the presence of Mn++, 03, sulfite, and phosphate buffer p H 7.0 was found to be greatly stimulated by the presence of cobaltous ions in the reaction mixture and to be sensitive to thyroxine n. Caution should be exercised in the interpretation of an apparent stimulation by thyroxine of an enzymic oxidation of D P N H under conditions in which a thyroxine sensitive non-enzymic oxidation of D P N H might be expected. DISCUSSION FRIDOVICH AND HANDLER12-14 have made use of the aerobic oxidation of sulfite as a sensitive detector of free radicals in a number of enzyme reactions based on the fact that the aerobic oxidation of sulfite is a chain reaction which is initiated by free radicals. They report on the initiation of sulfite oxidation by horseradish peroxidase in the presence of peroxide and a peroxidizable substrate 14. The aerobic oxidation of sulfite is shown here to be initiated by peroxidase in the absence of added H20 2 if either Mn ++ or a phenolic compound such as thyroxine or estradiol is added to the reaction mixture (Fig. I). That H20 2 is formed and utilized in the course of this Ieaction is suggested by the inhibitory effect of catalase (Table II). These data, and those o f FRIDOVICHAND HANDLER J 4 suggest that free radicals are formed in certain peroxidase catalyzed reactions and indeed, YAMAZAKIet al. is, 16 have identifed free radicals Biochim. Biophys. 3cta, 48 (1961) 93-io3
SULFITE AND REDUCED PYRIDINE NUCLEOTIDE OXIDATION
IOI
TABLE IV E F F E C T OF T H Y R O X I N E , T H Y R O X I N E ANALOGUES AND ESTROGENS
The reaction m i x t u r e contained 200 ffmoles of p h o s p h a t e buffer p H 7.0, 0. 3 ffmole of D P N H , o.oi /~mole of MnC12, 5fig of horseradish peroxidase a n d w a t e r to a final v o l u m e of 3.o ml. Sodium bisulfite (i.o ffmole), t h y r o x i n e (o.o 5 ffmole), t h y r o x i n e analogues (0.05 #mole) and estrogens (0.05/,mole) were added as indicated. E t h a n o l was added w i t h the estrogens at a final concentration of 1 . 7 %. Absorbancy change per minute at 340 ml* Amdogue
+ analogue,
+ analogue,
sulfite
+ sulfite
-
-
L-thyroxine N-acetyl-L-thyroxine 3,3'5-triiodo-L-thyronine 3,3'5-triiodo-L-thyronine, m e t h y l ether 3,3'5'-triiodo-DL-thyronine 3,5-diiodo-L-thyronine DL-thyronine 3,3'5-triiodothyroacetic acid 3,3'5-triiodothyroacetic acid m e t h y l ether 3,5-diiodo-L-tyrosine N-acetyl 3,5-diiodo-L-tyrosine 3-monoiodo-L-tyrosine L-tyrosine Estradiol 17fl Estradiol 170~ Estradiol 17-acetate Estradiol 3-acetate
o.o6o o.o8o o.o75 0.005 o. 15 ° o.t oo 0.060 0.04o 0.005 0.o0.5 0.005 o.ooo o.ooo o.o7o
o.38o o. 17 ° o.35o 0.065 0.760 o.24o 0.440 0.070 0.020 0.025 o.ol o o.ol o o.ooo o.i 9o
0.000
O. 145
o.o45 o.ooo
o.i 3 ° o.ooo
b Without surf ire ',' e s l r a d i o l Mn +¢ " ulerine preperotion
~
W~thout thyroxine
0.700
i sulfite Mn ++ ' ulerlne preperotiofl
0.600 E ¢)
i
0.500
•~ 0.400 c
0.300
3
system
I Complete
0.200
Complete
syslern
oJoo
L
i
Time (rain)
2
5
Fig. 4- The synergistic effect of sulfite and t h y r o x i n e or sulfite and estradiol on D P N H oxidation b y the uterine enzyme. The complete s y s t e m contained 200 # m o l e s of p h o s p h a t e buffer p H 7.o, 0. 3 /~mole of D P N H , 0 . 5 / , m o l e of MnC12, o.2 ml of a 5 % uterine p r e p a r a t i o n , o.i /,mole of t h y r oxine in a ; o. I # m o l e of estradiol 17fl and ethanol at a final c o n c e n t r a t i o n of 3.3 % in b ; I .o #mole of s o d i u m bisulfite and w a t e r to a v o l u m e of 3.o ml. The b l a n k contained all c o m p o n e n t s except D P N H .
13iochim. Biophys. Acta, 48 (I961) 93 lO3
102
S . J . KLEBANOFF
generated from substrates by peroxidase by means of electron paramagnetic resonance spectroscopy. Once initiated, sulfite oxidation is maintained as a chain reaction through the action of highly reactive intermediates formed either from the stepwise reduction of oxygen or the stepwise oxidation of sulfite ions. The inhibition of sulfite oxidation by D P N H (Table III) and the oxidation of D P N H induced by the aerobic oxidation of sulfite in the presence of peroxidase (Fig. 2) suggest that DPNH reacts with the same component of the sulfite oxidation system which is essential for the propagation of the chain reaction and is itself oxidized in the process. A similar mechanism was advocated by ALYEAAND BACKSTROMfor the inhibitory effect of certain alcohols on the oxidation of sodium sulfite 17. The oxidation of DPNH becomes a sensitive detector of the free radical chain initiating event under these conditions. Thyroxine, estradiol as well as certain other phenolic compounds have a stimulatory effect on a number of peroxidase catalyzed reactions 1-3. Thyroxine and estradiol are substrates for peroxidase 18,19 and are essential, under certain conditions for the initiation of sulfite oxidation by peroxidase. This is at least suggestive evidence for the conversion of thyroxine and estradiol to free radical intermediates (possibly phenoxy radicals) under the influence of peroxidase and is compatible with a role for the free radicals in the stimulatory effect of thyroxine, estradiol as well as certain other phenolic compounds on peroxidase catalyzed reactions, as proposed previously by a number of investigators3,2°, 21. In the absence of the protective effect of certain hydrogen donors, thyroxine and estradiol are oxidized further by peroxidase and H20 ~ to forms which are no longer active either biologically*, 82 or as stimulators of certain peroxidase catalyzed reactions ~9. MAYRARQUE-KODJAet al. 18 have reported on the extensive structural changes observed on the exposure of thyroxine to peroxidase and H202 and at least 5 chromatographically distinct products of estradiol oxidation by H~O~ and horseradish peroxidase are demonstrable (J. ENSINCKAND S. J. KLEBANOFI;, unpublished data). The synergistic effect of sulfite and thyroxine, or sulfite and estradiol on D P N H oxidation in the presence of peroxidase (Table IV) suggests a means by which the stimulatory effect of thyroxine or estradiol on this peroxidase system may be magnified. That this may prove useful in the search for thyroxine or estradiol sensitive peroxidase systems in mammalian tissues is suggested by the potentiation by sulfite ions of the hormonal effect on the oxidation of D P N H or T P N H by the uterine enzyme (Fig. 4)ACKNOWLEDGEMENTS
I am grateful to Dr. R. M. ARCHIBALD for his encouragement and support and to Miss K. HOULDER for her valuable technical assistance. This work was supported in part by research grant A 2772 from the National Institutes of Health. REFERENCES 1 S. J. KLEBANOFF,J. Biol. Chem., 234 (1959) 2437. S. J. KLEBANOFF,J . Biol. Chem., 234 (1959) 2480. 3 H. G. WILLIAMS-ASHMAN,M. CASSMANAND M. KLAVINS, Nature, 184 (1959) 427. S. J. KLEBANOFF, Federation Proc., 19 (196o) 31. * The inactivation of thyroxine by the HzO~-peroxidase system was demonstrated by the tadpole metamorphosis test (S. J. KLEBANOFF, unpublished data).
Biochim. Biophys. Acta, 48 (1961) 93-1o 3
S U L F I T E AND R E D U C E D
PYRIDINE
NUCLEOTIDE
OXIDATION
103
5 W. a . MCSHAN AND R . K. MEYER, Arch. Biochem., 9 (1946) 165. 8 I. FR~DOVlCH AND P. HANDLER, J. Biol. Chem., 223 (1956) 321. 7 V. P. HOLLANDER AND J. R . BEARD, 42rid meeting o/the Endocrine Society, A b s t r a c t No. 39, 1960. 8 F. V. LUCAS, H . A. NEUFELD, J. G. UTTERBACK, A. P. MARTIN AND E . STOTZ, J. Biol. Chem., 214 (1955) 775. 9 S. TEMPLE, V. P. HOLLANDER, N. HOLLANDER AND M. L. STEPHENS, J. Biol. Chem., 235 (196o) 15o4. 10 A. T. nEVER, C. M. SOKATCH AND J. H . ANGI1N, Federation Proc., 19 (196o) 175. 11 S. J. KLEBANOFF, First International Congress o/Endocrinology, Copenhagen, 196o. 12 I. FR1DOVICH AND P. HANDLER, J. Biol. Chem., 233 (1958) 1578. 13 I. FRIDOVlCH AND ]~. HANDLER, Biochim. Biophys. Acta, 35 (19.59) 546. 14 I. FRIDOV1CH AND P. HANDLER, Federation Proc., I 9 (196o) 29. 15 I. YAMAZAK1, H . S. MASON AND L. PIETTE, Biochem. Biophys. Research, 1 (I959) 336. 16 I. YAMAZAKI, H . ,%. MASON AND L. PIETTE, Federation Proc., 19 (196o) 33. 17 H . N. ALYEA AND H . L. V. BACKSTROM, J. ~,~m. Chem. Soc., 51 (1929) 90. 18 A. MAYRARQUE-KODJA, S. BOUCHILLOUX AND S. LISSITZKY, Bull. s6c. chim. biol., 4 ° (1958) 815. 19 S. J. KLEBANOFF, Biochim. Biophys. Acla, 44 (196o) 5Ol. 20 n . CHANCE, Arch. Biochem., 24 (1949) 389. 21 T. AKAZAWA AND E . E . CONN, J. Biol. Chem., 232 (1958) 403 • ~,2 S. J. ]{LEBANOFE AND S. J. SEGAL, J. Biol. Chem., 235 (196o) 52.
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