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A magneto-kinetic study of the reaction between ferrimyoglobin and methyl hydrogen peroxide
313
GLUCOSE METABOLISM BY BRAIN
6 H. KEWlTZ AND I. B. WILSON, Arch. Biochem. Biophys., 60 (1956) 261. 7 E. SCHOFFI~NIELS, I. B. WILSON AND D. •ACHMANSOHN, Biochim. Biophys. Acla, 27 (1958) 629. s I. ]3. WILSON, Biochim. Biophys. Acta, 27 (1958) 196. 9 j. j . GHOSH AND J. H. QUASTEL, Nature, 174 (1954) 28. 10 E . ANNAU, I. BANGA, B. G6zsY, ST. HUSZ~K, K. LAKI, B. STRAUB AND A. SZENT-GYORGYI, Z. physiol. Chem., Hoppe Seyler's, 236 (1935) I. 11 R. A. PETERS, Brit. Med. Bull., 9 (1953) 116. 12 C. MARTIUS AND D. NITZ-LITZOW, Biochim. Biophys. Acta, 12 (1953) 134. 13 A. R. SCHULZ AND H. Goss, Biochim. Biophys. Acta, 20 (1956) 443. i4 F. C. G. HOSKIN, Can. J. Biochem. and Physiol., 34 (1956) 743. 15 R. V. COXON AND R. J. ROBINSON, Proc. Roy. Soc. (London), B, 145 (1956) 232. 16 L. H. COHEN AND ~V. t{. NOELL,Federation Proc., 18 (1959) 28. 17 j. BERNSOHN, I. NAMAJUSKA AND L. S. G. COCHRANE, Arch. Biochem. Biophys., 62 (1956) 274. 18 D. NACHMANSOHN,Chemical and Molecular Basis o/Nerve Activity, Academic Press, New York, 1959.
Bioehim. Biophys. Acta, 4 ° (196o) 3o9-313
A M A G N E T O - K I N E T I C S T U D Y OF T H E R E A C T I O N B E T W E E N FERRIMYOGLOBIN
AND METHYL HYDROGEN
A R T H U R S. B R I L L * , A N D E R S E H R E N B E R G * * AND H E N D R I K
PEROXIDE DEN HARTOG***
E. R. Johnson Foundation Jor ~Iedical Physics, University o/Pennsylvania, Philadelphia, Pa. (U.S.A.) (Received August 26th, 1959)
SUMMARY
I. The reaction between ferrimyoglobin and methyl hydrogen peroxide has been studied with a new instrument for measuring rapid changes in the magnetic susceptibility of dilute aqueous solutions of proteins. 2. A comparison of the magnetically and spectrophotometrically obtained rate data shows differences, increasing toward lower temperatures, which can be explained in part by the production of free radicals. 3. The molar susceptibility at 20°C of the myoglobin compound formed is 330o" IO-6 emu with a standard error of 5oo • IO-6 emu.
INTRODUCTION
The reactions of pertinent to the They have been years. The recent
the hemoproteins with peroxides are of interest in themselves and elucidation of the mechanisms of peroxidase and catalase action. studied spectrophotometrically and magnetometrically for m a n y development of rapid spectrophotometric techniques 1 has permitted
* Present address: D e p a r t m e n t of Engineering Physics, Rockefeller Hall, Cornell University, Ithaca, N e w York, U.S.A. ** Present address: Biochemical D e p a r t m e n t , Medical Nobel Institute, Stockholm, Sweden. *** P r e s e n t address: N a t u u r k u n d i g L a b o r a t o r i u m , University of A m s t e r d a m , A m s t e r d a m , The Netherlands.
Biochim. Biophys. Acta, 4 ° (196o) 313-319
~I 4
A. ~:~. BIe,I L L , A- E H R E N B E R C ,
N. D E N
HARTOC
observation o f the kinetics of formation and decay of labile intermediate:.: as wel~ as the formation of stable compounds, and hence permitted the dct:<;minatio~ of th~ absorption spectra of t]~e short-Iived compounds. C~ANCF~ suggested ~)?at ~-: ]Zaukinebalance type magnetic susceptorneter be combined with a flow xystcm to achieve great time resolution J~; magnetic susceptibility measurements, so ki~;~t t]:~c maguetic m o m e n t of such labile intermediates could be determined. A most sensitive :magnetic susceptometer of this nature has been. developed a~d is described e]eswi~ercL Fig. x shows the performance of this instrument. Fig. ~ depicts a. flow experimer~t ,vhere t}~e magnetic susceptibility of the r e a d i o n mixture in half-cell ~ (whic]J is corL~tant i~> time for the constant flow rates ,Q5~ and. Qs) is compared with the m~_,gnetic susceptibility of the unreacted solution i~ half-cell z. \~:hile flow is in prog';e% ~.~teady-state reaction mixtures are observed with a time resolution of o.o5 sec or ]ee..s, co,' respo~?dJng to the time spent b y the mixture in the neighborhood of the mag~et. V;:he~ f%w i; stopped, the magnetic changes in the reaction mixture, now stat%~~ary :i~ the cell, are followed with a time resolution corresponding to t]~e e]ectr,micaU? adj~st:able response of the magnet~ In the experiments to be described, the respo~sc v.:'as ~omewhaf faster than in f:Jg. xA; for the fastest reaction tl~e u n d a m p e d angular fy, queney was ~ rad sec -~ and tl~e ratio of damping to criticaJ damping was 0.55i
i u Ion
A ~ : 5 5 X 1 0 - f l e.m.u.
Ai=15.5po Fig.
'
/.mogee!
half ceil I, conjoining i - - - ! , o f f .ei 2, Canto rdng. main so,u,ion ~_~--. reocf ~n m xt .... i
, secondary so~utmn ~
Qs, flow ro,t~ cf set:o-'ds '., .~.or~fio,-
I . Response of inst:rument t o a. era'rent
step in the directive force circuit (]eft), a,nd to a nickel-chloride conce~]i:ra,tJonstep (right) *.
]?~g, ?-, Schematic
of ion, e x p e r i m e n t .
The red compound %treed in the reaction between horse %rrimyoglobi~ and m e t h y l hydrogen peroxide was studied b y THEORELL AXD EUReN]~ER(Y~spcc!.rophoto-metrically, using a F,eckman apparatus, and magnetometriea]ly, usJ~g ti~ei_r special G o u y - t y p e balal~ce 4. Because of the instability of the compound at the ferrin~yoglobi> concentration of 65o ~3.f required for the G o u y susceptometer, some of the compound had reverted to ferrimyoglobiD during the relative]y long time required to make a measurement with this instrument. Correcting for the a m o u n t e4 ferrJmyoglobin present on the basis of spectrophotometric measurements, they obtail~ed %r the molar paramagnetic susceptibility of the compound a tentative value of 3ooo-~o ~ emu. T h e y also measured the mo]ar paramagnetic susceptibility ~)f the compound formed in the reaction between ferrimyoglobin and t I 2 0 2, ohtaining 35o0 , zo -'; enlu. Recent calculations of (;RIFfZ'rH'~ show t h a t a paramagnetic correction !.o the diamagnetism of the comparison hemoproteins should be made. Accordingly, these values are revised to 330o and 38oo • zo -a ernu. • F r o m this F i g u r e i t can be seen t]m.t t]~e p o o r t i m e resolution of t h e origins] flo-,v system
(see Fig. z4 of reff) ha.s bee]] remedied by the introductioJ~ of a. ceil of im]?~:ovc.ddesi..,}m
MAGNETO-KINETICS
OF A YERRIMYOGLOBIN REACTION
315
In an extensive series of experiments, GEORGE AND IRVINE6-1° have studied the reactions of ferrimyoglobin with hydrogen peroxide, methyl and ethyl hydrogen peroxides, and other strong oxidizing agents. They have shown that all the myoglobin compounds formed have the same absorption spectrum and state of oxidation, one equivalent higher than ferrimyoglobin. They have suggested that there is only one compound and that it contains iron in the quadrivalent state. Additionally they have shown that when the peroxides, which have two oxidizing equivalents, react with ferrimyoglobin, a transient oxidizing entity is produced which they suggest is a free radical. More recently G I B S O N , I N G R A M AND N I C H O L L S 11, utilizing electron spin resonance, have demonstrated the appearance of a free radical in the frozen reaction mixture of ferrimyoglobin and hydrogen peroxide. Their evidence indicates that the free radical is a product of a reaction of the transient oxidant rather than the peroxide, and is most likely situated in the globin where it is stabilized at the low temperature (9o°K) required for the experiments. Additionally, in experiments with the magnetic field set for a g-value of 6, they find a quenching of the five unpaired electron signal of ferrimyoglobin when the peroxide compound is formed, which implies a small magnetic moment for the compound. E X P E R I M E N T A L METHODS AND ANALYSIS OF DATA
The ferrimyoglobin, methyl hydrogen peroxide reaction has now been studied with the new rapid susceptometer. For reasons of temperature control, tile flow scheme of Fig. 2 was used in these experiments so that the unreacted ferrimyoglobin was in one half-cell, the reaction mixture in the second half-cell, and the difference in the magnetic susceptibilities was measured. Table I is a compilation of the experimental conditions and the data obtained. The ferrimyoglobin concentrations were measured TABLE
I
EXPERIMENTAL CONDITIONS AND DATA OBTAINED Expt.
[ F e r r i m y o g l o b i n Z × 106, M IMe O O H I × lO 6, 216r pH* T e m p e r a t u r e °C z]• X IO l l , e m u (volume magnetic suscept, change) h ( r a t e c o n s t a n t ) , sec -1 M -1
•
2
3
4
5
45-3
18.5
25.2
26.9
23.6
230o
21o
24 °
200
19o
8.36 25 --46.1 ( m e a n of 4) --
8.23
8.25
26 --21.1
21 --2o. 5 ( m e a n of 2)
--
37 °
8.29 13.9 -21o
8.28 25.2 --27. 9 lO3O
* I n a l l e x p e r i m e n t s o . I o 2"V/borate b u f f e r w a s used.
in terms of heroin by the pyridine hemochromogen method 12. Some heroin is destroyed in this reaction (see DISCUSSION). The concentrations given are for unreacted ferrimyoglobin solutions. The methyl hydrogen peroxide was kindly supplied by A. C. MAEHLY*. The hydrogen peroxide content, determined by the pertitanic acid complex * T h e a u t h o r s a r e p l e a s e d t o t h a n k DR. MAEHLY for h i s g e n e r o u s h e l p . Biochim.
Biophys.
A c t a , 4 ° (196o) 313 319
316
5 . s. B R I L L , ,4, E H R E N B E R G ,
H. D E X
HARTOG
m e t h o d , was 6 %. A correction was m a d e for the O.D. of this impm;ity, and ,:he c o n c e n t r a t i o n of the m e t h y l h y d r o g e n p e r o x i d e was m e a s u r e d spectrop~:o!:ometricaily at 230 m/* using an e x t i n c t i o n coefficient of 32 M - ~ cm q , Ti>: cl:m~ge :in n~ag~etic s u s c e p t i b i l i t y o b t a i n e d in e x p e r i m e n t 4 is considered !ess reliable tha,,~ o~ber d a t a and has not been inc!uded. The changes shown for expts. I and 3 aie the ~-~ea;~ -,;ah~e~ from identical reactions a n d c a r r y weights of 4. a n d 2 respectively. The rneth.od of o b t a i n i n g the r a t e c o n s t a n i s is described in the p a r a g r a p h on spectropk, o t o m e t r i e e x p e r i m e n t s . E x p t s . I a n d 2 were done in z955 and s957 using, a]~ eari3~ cell ',vil:]~ a. t i m e resolution of several seconds. Therefore, the kinetic d a t a from ~:]iese '.:xper[rnems are n o t included. The record of expt. 5 is shown in Fig. 3. Methyl h y d r o g e n i)ercxide v:a~ h-~jected twice. The biphasJc t r a n s i e n t s a c c o m p a n y i n g ti:e injections (I) ;~re d~e i> j a r r i n g of the i n s t r u m e n t when tl~e stopcock was turned. The stopcock was ~urned off (S) with less disturbance. The s t e a d y - s t a t e reaction m i x t u r e in the cell dr.,ring thence 5~iections corresponds to o.so sec after the beginning of the reaction. Essentially r,o defleeiion has occurred a t tl~is early stage. During the second injection period, i-]-.,eflow is stopped a n d the m a g n e t i c changes a c c o m p a n y i n g the r e m a i n d e r of the --eactio~7 aJe % i i o w e d
F !
flow beqins [njectSon of MeOOH
S
[,~jeclion stopped
O
flow off
]~'ig-. 3. ]-iecord of e x p t . 5.
The m o l a r p a r a m a g n e t i c s u s c e p t i b i l i t y of the c o m p o u n d % t r e e d in {he ,~eac~io.~ with m e t h y l h y d r o g e n p e r o x i d e is a s s u m e d to be i n d e p e n d e n t of the p H because i-hc~ a b s o r p t i o n s p e c t r u m is p H i n d e p e n d e n t L The s u s c e p t i b i l i t y is calculated frorr, Zeomponnd = Z u n r e a e l e d - ! /~Z
where A Z - - zoaAm~,~a~/[ferrimyogiobin] is the m e a s u r e d change i~ m o l a r susceptibility. The u n r e a c t e d ferrimyoglobin exists in two forms, a brown neutral %rn ~, with a molar p a r a m a g n e t i c s u s c e p t i b i l i t y at 2o ° of Z~e = s3,98o ' zo -~ emu and a red alkaline form with a s u s c e p t i b i l i t y of XFeOK = 11,33o" IO -° emu ta. (These values have beer~ revised according to th.e calculation of GRIrI~ITHa). The m o l a r s u s c e p t i b i l i t y of the u n r e a c t e d ferrimyoglobin solution 5s Z,~nreaeted = X~.Fe -}- ( ! - - - X)ZFeOI_ {
where the mole fraction x of the n e u t r a l form is given b y log
X 1 ---X
-- p K ,--- p I ]
The t e m p e r a t u r e d e p e n d e n c e of the p K has been d e t e r m i n e d ~4. After correcting t h e values for 2compound [O 20 ° b y the Curie law, the m e a n value i~5 calcu]at,~d to be 3300 • zo -6 emu w i t h a s t a n d a r d error of 50o . zo --Gemil. ]~';'oc~is~. 13iophys. Ac#~,
40 0:96o) 3;Y-3~9
MAGNETO-KINETICS
OF A FERRIMYOGLOBIN
REACTION
317
At the high methyl hydrogen peroxide concentration used in the experiments, there was the possibility of a second reaction between the compound and unreacted MeOOH to form another compound, resulting in a mixture of compounds. This possibility was examined by recording the change in the absorption spectrum of ferrimyoglobin upon addition of MeOOH to make molar ratios of 1.5, 3.0, 4-5, and 7.5. The reactions were permitted to go to completion, and then the differential absorption spectrum was recorded from 500 m/z to 650 m/x. Confirming the observation of GEORGE AND IRVINE7, the data show that a molar ratio somewhat greater than 3 : I is required for full formation of the compound at this pH. Within the accuracy of the spectrophotometer, the change in the absorption spectrum is the same for molar ratios of 4.5 and 7.5. At the molar ratios of 1.5 and 3.0 the change in the absorption spectrum is simply less intense. Therefore, only one spectrophotometrically identifiable compound is formed in this range of relative concentrations. Spectrophotometric data were obtained on the kinetics of formation of the compound under the conditions of the magnetic expts. 3, 4 and 5. For the spectrophotometric studies, a split-beam recording spectrophotometer operated with a 0-67 % rise time of I sec was used 15. The time course of the reaction, which was followed at 550 m~, was found not to obey second-order kinetics even when the effective excess oxidant was taken smaller than the nominal excess ratio, as might be suggested by the high molar ratio required for complete reaction. In the absence of full knowledge of the actual reaction mechanism, one can still define a rate constant as the rate of formation of compound per unit concentration of ferrimyoglobin and per unit concentration of oxidant. Since the concentrations are known only at the beginning of the reaction, ,and the initial 1. 5 or 2 sec are obscured by a mixing artefact, it was necessary to extrapolate the time course of the observed compound concentration back to the initial point. For this purpose of extrapolation, some empirical descriptions of our data were tried. With a power-law dependence of the form y = m 0 [ 1 - - ( 1 +t/T)-l/q] where y is the concentration of compound at the time t and m o is the initial ferrimyoglobin concentration, it was possible to adjust q and T to obtain an excellent fit. The second-order rate constant, k = IMe OOHI-1 (too - - y)-t dy/dt, is then given by k ~
1/qTao
where a 0 is the initial concentration of MeOOH. q determines the shape of the reaction curve, and will therefore depend upon the conditions of the reaction. For the spectrophotometrically observed reactions, the shape factor q is found to be the following function of the excess ratio ao/mo, q - - g.3 (aO/~O) -3/2
The time courses of the magnetic susceptibilities fit the same empirical power function when q was calculated from the above formula and only T was adjusted. Thus there is no indication of different shape between the magnetic and spectrophotometric kinetic curves. However, the magnetic data do not allow of an accurate shape analysis. The rate constants for the magnetic curves are calculated in the same way as the spectrophotometric constants and are significantly smaller at lower temperatures. Biochim. Biophys. Acta, 4 ° (196o) 3 1 3 - 3 1 9
3I~
A. S. BRILL, A. EHRENBERG, H. DEN HARTO(;,
This is shown in Fig. 4 which is an Arrhenius p]ot of the r a t e o:m:_-tani~. SO:a.igbt lines through the m a g n e d e and s p e c t r o p h o t o r n e t r i c points were ]Stted bTF ,:]~e med~od of ]east squares. P'ig. 4 Arr]~enius p]o£ of s-~agJ,etic ~J'-,d Spoctrophotometric ~:atc dai:>;,
+i
AR~i
,_Q@@
~
Ci:x
~-"7[!"i~
]"ig. 5. a: {he productitm arid disappea:'a~ce c:,f free radicals; b: convelTsJoP of ferJ:J~]])rogie)bi~-J to compound; c: observed cLmnge i~, nmg;~etic susceptibili~3 .= a ~ 1.
DISCUSSION The existence of differences between t h e rates of change of optical densit~ and mag-netic s u s c e p t i b i l i t y with t i m e is consistent with the l i b e r a t i o n of a free radical whe~ ferrimyoglobin a n d m e t h y l hydrogen p e r o x i d e react. This mechar~iam is one of two discussed b y GEORG]: AND II~.7]INE for the reaction between ferrh~yogJobJ~ ;~nd h y d r o g e n p e r o x i d e ~°. I t is interesting to note d m t the l a t t e r reaction proceed>~ a b o n i 5 t i m e s more slow]y b u t has the same activatior, energy of ~4,ooo cg,] as c a i c u l a t e d from t h e s p e c t r o p h o t o m e t r i c d a t a . The magnetJca]]y d e t e r m i n e d ra~e consian~s would not be e x p e c t e d to obey an Arrhenhls relation if two or more ma~.u~etic p~:oce.~÷ses c o n t r i b u t e to the obser--ed kinetic curve. The effeci of l h e p r o d u e t i o c and d i s a p p e a > ance of free radical u p o n the t i m e course of the m a g n e t i c su:~cep!fi)ilil 7 i:-: s h o > ~ s c h e m a t i c a l l y in Fig. 5- Ti~e o b s e r v e d change proceeds at a slower ra#c t h a n ti~e r a t e of conversion of ferrimyoglobin to c o m p o u n d alone. S p e c t r o p h o t o m e t r i c a i ] y O1qi37 this conversion is foi]owed (except possibly for a small effect dtle to the d e g r a d a t i o n of m y o g l o b i n ;---see below). Because of the factor of eight between ti~e m o l a r n:,ag~eiic s u s c e p t i b i l i t y change in the myoglobin a n d the much. smaller molar susceptibiii*y of free radicals, it is difficult to account for t h e differences Jn the spectrophotonnetri .... cally a n d magnetome~rically d e t e r m i n e d rates on this basis alone. H o w e v e : , there 5f; a n o t h e r observation which bears u p o n the problem. P y r i d i n e h e m o c h r o m o g e n d e t e r m i n a t i o n s of heroin concentration before and after the e x p e r i m e n t s show t h a t as much as Ix % of the heroin of l h e myoglobh~ is d e s t r o y e d during t h e reaction. The n a t u r e of the b r e a k d o w n p r o d u c t s has not y e t been i n v e s t i g a t e d so ! h a t their c o n t r i b u t i o n s to the m a g n e t i c s u s c e p t i b i l i t y and to the O.D. are n o t q u a n t i t a t i v e ] y k n o w n b u t are ahnost certainly ob:;ervabie. I t is v e r y likely t h a t the r a t e at which t h e d e g r a d a t i o n p r o d u c t s are formed has an influence u p o n the s p e c t r o p h o t o m e t r i c a n d m a g n e t o m e t r i e kinetic curves. £?iocJihT*. ]?io/)hy.v. .I ~h~, .4o (J 9~'o) 3 ; 3-3 ] 9
MAGNETO-KINETICS
OF A F E R R I M Y O G L O B I N
REACTION
319
The value obtained for the molar susceptibility of the compound does not contain contributions from free radicals since these will have reacted to form stable diamagnetic products by the end of a magnetic run. However, the breakdown products will contribute to the extent that their molar susceptibilities differ from that of the compound. The amount, and possibly the types oi breakdown product as well, depend upon the experimental conditions, and this variability m a y account for part of the spread in the values obtained for the molar susceptibility of the compound. The mean value of 3300" lO -6 emu at 20 ° is that for a complex of unit spin (two unpaired electrons). a spin which is theoretically possible for the d-shell configuration of GEORGE'S postulated quadrivalent iron. It must be realized that for the small temperature range over which the magnetic experiments were made, the data are not sufficiently precise to test whether or not there is the Curie law dependence appropriate to a complex of unit spin. The molar susceptibility does not differ from the values obtained earlier b y THEORELL AND EHRENBERG for both the hydrogen peroxide and methyl hydrogen peroxide reactions and supports the contention of GEORGE AND IRVINE that the same compound is formed in both reactions. It is advisable now to extend the range of temperature and to investigate the breakdown products so that their effects upon the data can be evaluated. I t would also be most useful to obtain data on the time course of the oxidant concentration. Until single large crystals of the compound are available, it is not likely that paramagnetic resonance will be of further help in elaborating the configuration of the d-shell. ACKNOWLEDGEMENTS
The authors wish to thank F. A. MULLER for his assistance in analysing the data, and B. CHANCE, P. GEORGE Aand D. H. IRVlNE for stimulating and helpful discussions. This investigation was supported in its early stages by a Predoctoral Research Fellowship to A. S. B~ILL from the Division of Research Grants of the United States Public Health Service.
REFERENCES 1 t3. CHANCE,Rev. Sci. Instr., 22 (1951) 619. 2 A . S. BRILL, H . DEN HARTOG AND V. LEGALLAIS, Rev. Sei. Instr., 29 (1958) 3 8 3 . 3 H . THEORELL AND A. EHRENBERG, Arch. Bioehem. Biophys., 41 (1952) 4 4 2 . 4 H . THEORELL AND A. EHRENBERG, Arkiv Fysik, 3 (1951) 299. 5 j . S. GRIFFITH, Biochim. Biophys. Acta, 28 (1958) 439. 6 p . GEORGE AND ]). H . IRVlNE, Biochem. J., 52 (1952) 5 1 1 . 7 p . GEORGE AND D. H . IRVlNE, Biochem. J., 55 (1953) 2 3 0 . s p . GEOEGE AND D. H . IRVlNE, Biochem. J., 58 (1954) 188. 9 p . GEORGE AND D. H . IRVlNE, Biochem. J., 6o (1955) 596. 10 p . GEORGE AND D . H . IRVlNE, J. Colloid Sci., I I (1956) 3 2 7 . 11 j . F . GIBSON, D . J . E . INGEAM AND P. NICHOLLS, Nature, 181 (1958) 1 3 9 8 . 12 K . G . PAUL, H . THEORELL AND ~x. ,~kKESON, Acta Chem. Scand., 7 (1953) 1 2 8 4 . 13 H . THEORELL AND A . EHRENBERG, Acta Chem. Scan&, 5 (1951) 823. 14 p . GEORGE AND G. HANANIA, Bioehem. J., 52 (1952) 5 1 7 . IS C. C. YANG AND V. LEGALLAIS, Rev. Sci. Instr., 25 (1954) 8Ol.
Biochim. Biophys. Acta, 4 ° (196o) 3 1 3 - 3 1 9
GLUCOSE METABOLISM BY BRAIN
6 H. KEWlTZ AND I. B. WILSON, Arch. Biochem. Biophys., 60 (1956) 261. 7 E. SCHOFFI~NIELS, I. B. WILSON AND D. •ACHMANSOHN, Biochim. Biophys. Acla, 27 (1958) 629. s I. ]3. WILSON, Biochim. Biophys. Acta, 27 (1958) 196. 9 j. j . GHOSH AND J. H. QUASTEL, Nature, 174 (1954) 28. 10 E . ANNAU, I. BANGA, B. G6zsY, ST. HUSZ~K, K. LAKI, B. STRAUB AND A. SZENT-GYORGYI, Z. physiol. Chem., Hoppe Seyler's, 236 (1935) I. 11 R. A. PETERS, Brit. Med. Bull., 9 (1953) 116. 12 C. MARTIUS AND D. NITZ-LITZOW, Biochim. Biophys. Acta, 12 (1953) 134. 13 A. R. SCHULZ AND H. Goss, Biochim. Biophys. Acta, 20 (1956) 443. i4 F. C. G. HOSKIN, Can. J. Biochem. and Physiol., 34 (1956) 743. 15 R. V. COXON AND R. J. ROBINSON, Proc. Roy. Soc. (London), B, 145 (1956) 232. 16 L. H. COHEN AND ~V. t{. NOELL,Federation Proc., 18 (1959) 28. 17 j. BERNSOHN, I. NAMAJUSKA AND L. S. G. COCHRANE, Arch. Biochem. Biophys., 62 (1956) 274. 18 D. NACHMANSOHN,Chemical and Molecular Basis o/Nerve Activity, Academic Press, New York, 1959.
Bioehim. Biophys. Acta, 4 ° (196o) 3o9-313
A M A G N E T O - K I N E T I C S T U D Y OF T H E R E A C T I O N B E T W E E N FERRIMYOGLOBIN
AND METHYL HYDROGEN
A R T H U R S. B R I L L * , A N D E R S E H R E N B E R G * * AND H E N D R I K
PEROXIDE DEN HARTOG***
E. R. Johnson Foundation Jor ~Iedical Physics, University o/Pennsylvania, Philadelphia, Pa. (U.S.A.) (Received August 26th, 1959)
SUMMARY
I. The reaction between ferrimyoglobin and methyl hydrogen peroxide has been studied with a new instrument for measuring rapid changes in the magnetic susceptibility of dilute aqueous solutions of proteins. 2. A comparison of the magnetically and spectrophotometrically obtained rate data shows differences, increasing toward lower temperatures, which can be explained in part by the production of free radicals. 3. The molar susceptibility at 20°C of the myoglobin compound formed is 330o" IO-6 emu with a standard error of 5oo • IO-6 emu.
INTRODUCTION
The reactions of pertinent to the They have been years. The recent
the hemoproteins with peroxides are of interest in themselves and elucidation of the mechanisms of peroxidase and catalase action. studied spectrophotometrically and magnetometrically for m a n y development of rapid spectrophotometric techniques 1 has permitted
* Present address: D e p a r t m e n t of Engineering Physics, Rockefeller Hall, Cornell University, Ithaca, N e w York, U.S.A. ** Present address: Biochemical D e p a r t m e n t , Medical Nobel Institute, Stockholm, Sweden. *** P r e s e n t address: N a t u u r k u n d i g L a b o r a t o r i u m , University of A m s t e r d a m , A m s t e r d a m , The Netherlands.
Biochim. Biophys. Acta, 4 ° (196o) 313-319
~I 4
A. ~:~. BIe,I L L , A- E H R E N B E R C ,
N. D E N
HARTOC
observation o f the kinetics of formation and decay of labile intermediate:.: as wel~ as the formation of stable compounds, and hence permitted the dct:<;minatio~ of th~ absorption spectra of t]~e short-Iived compounds. C~ANCF~ suggested ~)?at ~-: ]Zaukinebalance type magnetic susceptorneter be combined with a flow xystcm to achieve great time resolution J~; magnetic susceptibility measurements, so ki~;~t t]:~c maguetic m o m e n t of such labile intermediates could be determined. A most sensitive :magnetic susceptometer of this nature has been. developed a~d is described e]eswi~ercL Fig. x shows the performance of this instrument. Fig. ~ depicts a. flow experimer~t ,vhere t}~e magnetic susceptibility of the r e a d i o n mixture in half-cell ~ (whic]J is corL~tant i~> time for the constant flow rates ,Q5~ and. Qs) is compared with the m~_,gnetic susceptibility of the unreacted solution i~ half-cell z. \~:hile flow is in prog';e% ~.~teady-state reaction mixtures are observed with a time resolution of o.o5 sec or ]ee..s, co,' respo~?dJng to the time spent b y the mixture in the neighborhood of the mag~et. V;:he~ f%w i; stopped, the magnetic changes in the reaction mixture, now stat%~~ary :i~ the cell, are followed with a time resolution corresponding to t]~e e]ectr,micaU? adj~st:able response of the magnet~ In the experiments to be described, the respo~sc v.:'as ~omewhaf faster than in f:Jg. xA; for the fastest reaction tl~e u n d a m p e d angular fy, queney was ~ rad sec -~ and tl~e ratio of damping to criticaJ damping was 0.55i
i u Ion
A ~ : 5 5 X 1 0 - f l e.m.u.
Ai=15.5po Fig.
'
/.mogee!
half ceil I, conjoining i - - - ! , o f f .ei 2, Canto rdng. main so,u,ion ~_~--. reocf ~n m xt .... i
, secondary so~utmn ~
Qs, flow ro,t~ cf set:o-'ds '., .~.or~fio,-
I . Response of inst:rument t o a. era'rent
step in the directive force circuit (]eft), a,nd to a nickel-chloride conce~]i:ra,tJonstep (right) *.
]?~g, ?-, Schematic
of ion, e x p e r i m e n t .
The red compound %treed in the reaction between horse %rrimyoglobi~ and m e t h y l hydrogen peroxide was studied b y THEORELL AXD EUReN]~ER(Y~spcc!.rophoto-metrically, using a F,eckman apparatus, and magnetometriea]ly, usJ~g ti~ei_r special G o u y - t y p e balal~ce 4. Because of the instability of the compound at the ferrin~yoglobi> concentration of 65o ~3.f required for the G o u y susceptometer, some of the compound had reverted to ferrimyoglobiD during the relative]y long time required to make a measurement with this instrument. Correcting for the a m o u n t e4 ferrJmyoglobin present on the basis of spectrophotometric measurements, they obtail~ed %r the molar paramagnetic susceptibility of the compound a tentative value of 3ooo-~o ~ emu. T h e y also measured the mo]ar paramagnetic susceptibility ~)f the compound formed in the reaction between ferrimyoglobin and t I 2 0 2, ohtaining 35o0 , zo -'; enlu. Recent calculations of (;RIFfZ'rH'~ show t h a t a paramagnetic correction !.o the diamagnetism of the comparison hemoproteins should be made. Accordingly, these values are revised to 330o and 38oo • zo -a ernu. • F r o m this F i g u r e i t can be seen t]m.t t]~e p o o r t i m e resolution of t h e origins] flo-,v system
(see Fig. z4 of reff) ha.s bee]] remedied by the introductioJ~ of a. ceil of im]?~:ovc.ddesi..,}m
MAGNETO-KINETICS
OF A YERRIMYOGLOBIN REACTION
315
In an extensive series of experiments, GEORGE AND IRVINE6-1° have studied the reactions of ferrimyoglobin with hydrogen peroxide, methyl and ethyl hydrogen peroxides, and other strong oxidizing agents. They have shown that all the myoglobin compounds formed have the same absorption spectrum and state of oxidation, one equivalent higher than ferrimyoglobin. They have suggested that there is only one compound and that it contains iron in the quadrivalent state. Additionally they have shown that when the peroxides, which have two oxidizing equivalents, react with ferrimyoglobin, a transient oxidizing entity is produced which they suggest is a free radical. More recently G I B S O N , I N G R A M AND N I C H O L L S 11, utilizing electron spin resonance, have demonstrated the appearance of a free radical in the frozen reaction mixture of ferrimyoglobin and hydrogen peroxide. Their evidence indicates that the free radical is a product of a reaction of the transient oxidant rather than the peroxide, and is most likely situated in the globin where it is stabilized at the low temperature (9o°K) required for the experiments. Additionally, in experiments with the magnetic field set for a g-value of 6, they find a quenching of the five unpaired electron signal of ferrimyoglobin when the peroxide compound is formed, which implies a small magnetic moment for the compound. E X P E R I M E N T A L METHODS AND ANALYSIS OF DATA
The ferrimyoglobin, methyl hydrogen peroxide reaction has now been studied with the new rapid susceptometer. For reasons of temperature control, tile flow scheme of Fig. 2 was used in these experiments so that the unreacted ferrimyoglobin was in one half-cell, the reaction mixture in the second half-cell, and the difference in the magnetic susceptibilities was measured. Table I is a compilation of the experimental conditions and the data obtained. The ferrimyoglobin concentrations were measured TABLE
I
EXPERIMENTAL CONDITIONS AND DATA OBTAINED Expt.
[ F e r r i m y o g l o b i n Z × 106, M IMe O O H I × lO 6, 216r pH* T e m p e r a t u r e °C z]• X IO l l , e m u (volume magnetic suscept, change) h ( r a t e c o n s t a n t ) , sec -1 M -1
•
2
3
4
5
45-3
18.5
25.2
26.9
23.6
230o
21o
24 °
200
19o
8.36 25 --46.1 ( m e a n of 4) --
8.23
8.25
26 --21.1
21 --2o. 5 ( m e a n of 2)
--
37 °
8.29 13.9 -21o
8.28 25.2 --27. 9 lO3O
* I n a l l e x p e r i m e n t s o . I o 2"V/borate b u f f e r w a s used.
in terms of heroin by the pyridine hemochromogen method 12. Some heroin is destroyed in this reaction (see DISCUSSION). The concentrations given are for unreacted ferrimyoglobin solutions. The methyl hydrogen peroxide was kindly supplied by A. C. MAEHLY*. The hydrogen peroxide content, determined by the pertitanic acid complex * T h e a u t h o r s a r e p l e a s e d t o t h a n k DR. MAEHLY for h i s g e n e r o u s h e l p . Biochim.
Biophys.
A c t a , 4 ° (196o) 313 319
316
5 . s. B R I L L , ,4, E H R E N B E R G ,
H. D E X
HARTOG
m e t h o d , was 6 %. A correction was m a d e for the O.D. of this impm;ity, and ,:he c o n c e n t r a t i o n of the m e t h y l h y d r o g e n p e r o x i d e was m e a s u r e d spectrop~:o!:ometricaily at 230 m/* using an e x t i n c t i o n coefficient of 32 M - ~ cm q , Ti>: cl:m~ge :in n~ag~etic s u s c e p t i b i l i t y o b t a i n e d in e x p e r i m e n t 4 is considered !ess reliable tha,,~ o~ber d a t a and has not been inc!uded. The changes shown for expts. I and 3 aie the ~-~ea;~ -,;ah~e~ from identical reactions a n d c a r r y weights of 4. a n d 2 respectively. The rneth.od of o b t a i n i n g the r a t e c o n s t a n i s is described in the p a r a g r a p h on spectropk, o t o m e t r i e e x p e r i m e n t s . E x p t s . I a n d 2 were done in z955 and s957 using, a]~ eari3~ cell ',vil:]~ a. t i m e resolution of several seconds. Therefore, the kinetic d a t a from ~:]iese '.:xper[rnems are n o t included. The record of expt. 5 is shown in Fig. 3. Methyl h y d r o g e n i)ercxide v:a~ h-~jected twice. The biphasJc t r a n s i e n t s a c c o m p a n y i n g ti:e injections (I) ;~re d~e i> j a r r i n g of the i n s t r u m e n t when tl~e stopcock was turned. The stopcock was ~urned off (S) with less disturbance. The s t e a d y - s t a t e reaction m i x t u r e in the cell dr.,ring thence 5~iections corresponds to o.so sec after the beginning of the reaction. Essentially r,o defleeiion has occurred a t tl~is early stage. During the second injection period, i-]-.,eflow is stopped a n d the m a g n e t i c changes a c c o m p a n y i n g the r e m a i n d e r of the --eactio~7 aJe % i i o w e d
F !
flow beqins [njectSon of MeOOH
S
[,~jeclion stopped
O
flow off
]~'ig-. 3. ]-iecord of e x p t . 5.
The m o l a r p a r a m a g n e t i c s u s c e p t i b i l i t y of the c o m p o u n d % t r e e d in {he ,~eac~io.~ with m e t h y l h y d r o g e n p e r o x i d e is a s s u m e d to be i n d e p e n d e n t of the p H because i-hc~ a b s o r p t i o n s p e c t r u m is p H i n d e p e n d e n t L The s u s c e p t i b i l i t y is calculated frorr, Zeomponnd = Z u n r e a e l e d - ! /~Z
where A Z - - zoaAm~,~a~/[ferrimyogiobin] is the m e a s u r e d change i~ m o l a r susceptibility. The u n r e a c t e d ferrimyoglobin exists in two forms, a brown neutral %rn ~, with a molar p a r a m a g n e t i c s u s c e p t i b i l i t y at 2o ° of Z~e = s3,98o ' zo -~ emu and a red alkaline form with a s u s c e p t i b i l i t y of XFeOK = 11,33o" IO -° emu ta. (These values have beer~ revised according to th.e calculation of GRIrI~ITHa). The m o l a r s u s c e p t i b i l i t y of the u n r e a c t e d ferrimyoglobin solution 5s Z,~nreaeted = X~.Fe -}- ( ! - - - X)ZFeOI_ {
where the mole fraction x of the n e u t r a l form is given b y log
X 1 ---X
-- p K ,--- p I ]
The t e m p e r a t u r e d e p e n d e n c e of the p K has been d e t e r m i n e d ~4. After correcting t h e values for 2compound [O 20 ° b y the Curie law, the m e a n value i~5 calcu]at,~d to be 3300 • zo -6 emu w i t h a s t a n d a r d error of 50o . zo --Gemil. ]~';'oc~is~. 13iophys. Ac#~,
40 0:96o) 3;Y-3~9
MAGNETO-KINETICS
OF A FERRIMYOGLOBIN
REACTION
317
At the high methyl hydrogen peroxide concentration used in the experiments, there was the possibility of a second reaction between the compound and unreacted MeOOH to form another compound, resulting in a mixture of compounds. This possibility was examined by recording the change in the absorption spectrum of ferrimyoglobin upon addition of MeOOH to make molar ratios of 1.5, 3.0, 4-5, and 7.5. The reactions were permitted to go to completion, and then the differential absorption spectrum was recorded from 500 m/z to 650 m/x. Confirming the observation of GEORGE AND IRVINE7, the data show that a molar ratio somewhat greater than 3 : I is required for full formation of the compound at this pH. Within the accuracy of the spectrophotometer, the change in the absorption spectrum is the same for molar ratios of 4.5 and 7.5. At the molar ratios of 1.5 and 3.0 the change in the absorption spectrum is simply less intense. Therefore, only one spectrophotometrically identifiable compound is formed in this range of relative concentrations. Spectrophotometric data were obtained on the kinetics of formation of the compound under the conditions of the magnetic expts. 3, 4 and 5. For the spectrophotometric studies, a split-beam recording spectrophotometer operated with a 0-67 % rise time of I sec was used 15. The time course of the reaction, which was followed at 550 m~, was found not to obey second-order kinetics even when the effective excess oxidant was taken smaller than the nominal excess ratio, as might be suggested by the high molar ratio required for complete reaction. In the absence of full knowledge of the actual reaction mechanism, one can still define a rate constant as the rate of formation of compound per unit concentration of ferrimyoglobin and per unit concentration of oxidant. Since the concentrations are known only at the beginning of the reaction, ,and the initial 1. 5 or 2 sec are obscured by a mixing artefact, it was necessary to extrapolate the time course of the observed compound concentration back to the initial point. For this purpose of extrapolation, some empirical descriptions of our data were tried. With a power-law dependence of the form y = m 0 [ 1 - - ( 1 +t/T)-l/q] where y is the concentration of compound at the time t and m o is the initial ferrimyoglobin concentration, it was possible to adjust q and T to obtain an excellent fit. The second-order rate constant, k = IMe OOHI-1 (too - - y)-t dy/dt, is then given by k ~
1/qTao
where a 0 is the initial concentration of MeOOH. q determines the shape of the reaction curve, and will therefore depend upon the conditions of the reaction. For the spectrophotometrically observed reactions, the shape factor q is found to be the following function of the excess ratio ao/mo, q - - g.3 (aO/~O) -3/2
The time courses of the magnetic susceptibilities fit the same empirical power function when q was calculated from the above formula and only T was adjusted. Thus there is no indication of different shape between the magnetic and spectrophotometric kinetic curves. However, the magnetic data do not allow of an accurate shape analysis. The rate constants for the magnetic curves are calculated in the same way as the spectrophotometric constants and are significantly smaller at lower temperatures. Biochim. Biophys. Acta, 4 ° (196o) 3 1 3 - 3 1 9
3I~
A. S. BRILL, A. EHRENBERG, H. DEN HARTO(;,
This is shown in Fig. 4 which is an Arrhenius p]ot of the r a t e o:m:_-tani~. SO:a.igbt lines through the m a g n e d e and s p e c t r o p h o t o r n e t r i c points were ]Stted bTF ,:]~e med~od of ]east squares. P'ig. 4 Arr]~enius p]o£ of s-~agJ,etic ~J'-,d Spoctrophotometric ~:atc dai:>;,
+i
AR~i
,_Q@@
~
Ci:x
~-"7[!"i~
]"ig. 5. a: {he productitm arid disappea:'a~ce c:,f free radicals; b: convelTsJoP of ferJ:J~]])rogie)bi~-J to compound; c: observed cLmnge i~, nmg;~etic susceptibili~3 .= a ~ 1.
DISCUSSION The existence of differences between t h e rates of change of optical densit~ and mag-netic s u s c e p t i b i l i t y with t i m e is consistent with the l i b e r a t i o n of a free radical whe~ ferrimyoglobin a n d m e t h y l hydrogen p e r o x i d e react. This mechar~iam is one of two discussed b y GEORG]: AND II~.7]INE for the reaction between ferrh~yogJobJ~ ;~nd h y d r o g e n p e r o x i d e ~°. I t is interesting to note d m t the l a t t e r reaction proceed>~ a b o n i 5 t i m e s more slow]y b u t has the same activatior, energy of ~4,ooo cg,] as c a i c u l a t e d from t h e s p e c t r o p h o t o m e t r i c d a t a . The magnetJca]]y d e t e r m i n e d ra~e consian~s would not be e x p e c t e d to obey an Arrhenhls relation if two or more ma~.u~etic p~:oce.~÷ses c o n t r i b u t e to the obser--ed kinetic curve. The effeci of l h e p r o d u e t i o c and d i s a p p e a > ance of free radical u p o n the t i m e course of the m a g n e t i c su:~cep!fi)ilil 7 i:-: s h o > ~ s c h e m a t i c a l l y in Fig. 5- Ti~e o b s e r v e d change proceeds at a slower ra#c t h a n ti~e r a t e of conversion of ferrimyoglobin to c o m p o u n d alone. S p e c t r o p h o t o m e t r i c a i ] y O1qi37 this conversion is foi]owed (except possibly for a small effect dtle to the d e g r a d a t i o n of m y o g l o b i n ;---see below). Because of the factor of eight between ti~e m o l a r n:,ag~eiic s u s c e p t i b i l i t y change in the myoglobin a n d the much. smaller molar susceptibiii*y of free radicals, it is difficult to account for t h e differences Jn the spectrophotonnetri .... cally a n d magnetome~rically d e t e r m i n e d rates on this basis alone. H o w e v e : , there 5f; a n o t h e r observation which bears u p o n the problem. P y r i d i n e h e m o c h r o m o g e n d e t e r m i n a t i o n s of heroin concentration before and after the e x p e r i m e n t s show t h a t as much as Ix % of the heroin of l h e myoglobh~ is d e s t r o y e d during t h e reaction. The n a t u r e of the b r e a k d o w n p r o d u c t s has not y e t been i n v e s t i g a t e d so ! h a t their c o n t r i b u t i o n s to the m a g n e t i c s u s c e p t i b i l i t y and to the O.D. are n o t q u a n t i t a t i v e ] y k n o w n b u t are ahnost certainly ob:;ervabie. I t is v e r y likely t h a t the r a t e at which t h e d e g r a d a t i o n p r o d u c t s are formed has an influence u p o n the s p e c t r o p h o t o m e t r i c a n d m a g n e t o m e t r i e kinetic curves. £?iocJihT*. ]?io/)hy.v. .I ~h~, .4o (J 9~'o) 3 ; 3-3 ] 9
MAGNETO-KINETICS
OF A F E R R I M Y O G L O B I N
REACTION
319
The value obtained for the molar susceptibility of the compound does not contain contributions from free radicals since these will have reacted to form stable diamagnetic products by the end of a magnetic run. However, the breakdown products will contribute to the extent that their molar susceptibilities differ from that of the compound. The amount, and possibly the types oi breakdown product as well, depend upon the experimental conditions, and this variability m a y account for part of the spread in the values obtained for the molar susceptibility of the compound. The mean value of 3300" lO -6 emu at 20 ° is that for a complex of unit spin (two unpaired electrons). a spin which is theoretically possible for the d-shell configuration of GEORGE'S postulated quadrivalent iron. It must be realized that for the small temperature range over which the magnetic experiments were made, the data are not sufficiently precise to test whether or not there is the Curie law dependence appropriate to a complex of unit spin. The molar susceptibility does not differ from the values obtained earlier b y THEORELL AND EHRENBERG for both the hydrogen peroxide and methyl hydrogen peroxide reactions and supports the contention of GEORGE AND IRVINE that the same compound is formed in both reactions. It is advisable now to extend the range of temperature and to investigate the breakdown products so that their effects upon the data can be evaluated. I t would also be most useful to obtain data on the time course of the oxidant concentration. Until single large crystals of the compound are available, it is not likely that paramagnetic resonance will be of further help in elaborating the configuration of the d-shell. ACKNOWLEDGEMENTS
The authors wish to thank F. A. MULLER for his assistance in analysing the data, and B. CHANCE, P. GEORGE Aand D. H. IRVlNE for stimulating and helpful discussions. This investigation was supported in its early stages by a Predoctoral Research Fellowship to A. S. B~ILL from the Division of Research Grants of the United States Public Health Service.
REFERENCES 1 t3. CHANCE,Rev. Sci. Instr., 22 (1951) 619. 2 A . S. BRILL, H . DEN HARTOG AND V. LEGALLAIS, Rev. Sei. Instr., 29 (1958) 3 8 3 . 3 H . THEORELL AND A. EHRENBERG, Arch. Bioehem. Biophys., 41 (1952) 4 4 2 . 4 H . THEORELL AND A. EHRENBERG, Arkiv Fysik, 3 (1951) 299. 5 j . S. GRIFFITH, Biochim. Biophys. Acta, 28 (1958) 439. 6 p . GEORGE AND ]). H . IRVlNE, Biochem. J., 52 (1952) 5 1 1 . 7 p . GEORGE AND D. H . IRVlNE, Biochem. J., 55 (1953) 2 3 0 . s p . GEOEGE AND D. H . IRVlNE, Biochem. J., 58 (1954) 188. 9 p . GEORGE AND D. H . IRVlNE, Biochem. J., 6o (1955) 596. 10 p . GEORGE AND D . H . IRVlNE, J. Colloid Sci., I I (1956) 3 2 7 . 11 j . F . GIBSON, D . J . E . INGEAM AND P. NICHOLLS, Nature, 181 (1958) 1 3 9 8 . 12 K . G . PAUL, H . THEORELL AND ~x. ,~kKESON, Acta Chem. Scand., 7 (1953) 1 2 8 4 . 13 H . THEORELL AND A . EHRENBERG, Acta Chem. Scan&, 5 (1951) 823. 14 p . GEORGE AND G. HANANIA, Bioehem. J., 52 (1952) 5 1 7 . IS C. C. YANG AND V. LEGALLAIS, Rev. Sci. Instr., 25 (1954) 8Ol.
Biochim. Biophys. Acta, 4 ° (196o) 3 1 3 - 3 1 9