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BBA 46499 OXI DATI ON- REDUCTI ON POTENTI AL OF THE F ERRO- F ERRI CYANI DE SYSTEM I N BUFFER SOLUTI ONS JAMES E. O'REILLY Department of Chemistry, University of lllinois at Urbana-Champaign, Urbana, Ill. 61801 (U.S.A.) (Recived October 2nd, 1972) SUMMARY The r edox pot ent i al (Eo' ) of t he pot assi um f er r ocyani de- pot assi um ferri cyani de oxi dat i on- r educt i on pot ent i al buffer was measured in f our pH buffer solutions, acetate, Tris, phosphat e, and borat e, under specified condi t i ons of pH, sol ut i on composi t i on, and t emperat ure. The pot ent i al s r epor t ed shoul d be accurate, on t he hydr ogen el ect rode scale, t o within about 2 mV and precise t o within at least + 0.3 mV. Al t hough t he pot ent i al of t he f er r o- f er r i cyani de coupl e is sensitive t o fairly small changes in experi ment al condi t i ons, several met hods are discussed by which this pot ent i al can be est i mat ed with a reasonabl e degree of accuracy ( about 2- 10 mV) in sol ut i ons whi ch differ somewhat f r om the part i cul ar solutions r epor t ed here. At t ent i on is called t o t he need f or great er care in specifying experi ment al condi t i ons in the det er mi nat i on of r edox pot ent i al s of vari ous biological species. INTRODUCTION The f er r o- f er r i cyani de r edox coupl e is a widely used oxi dat i on- r educt i on pot ent i al buffer f or t he det er mi nat i on of r edox pot ent i al s of vari ous biological systems. It has been used, f or example, in the st udy of mammal i an cyt ochr ome c (refs 1 and 2), vari ous bact eri al and algal cyt ochromes of the c-type t-5, cyt ochr ome f (ref. 6) and ot her biological compounds. Some of the advant ages of t he f e r r o- ferri cyani de system is t hat it is relatively easy t o prepare, its pot ent i al is quite stable with time, and its use allows the empl oyment of relatively simple spect r ophot omet r i c met hods, as opposed t o t he r at her t edi ous pot ent i omet r i c t i t rat i on met hods pi oneered by Michaelis, Clark, and ot hers in t he 1920's and 1930's. In the course of an investigation concerni ng the r edox potentials of cert ai n bact eri al cyt ochr omes of the e-type, the purpose of which was t o det ermi ne i f it woul d be possible to correl at e known differences in st ruct ure with r at her small variations in r edox potential, it qui ckl y became evi dent t hat a good deal of ambi gui t y exists in the knowledge of the actual r edox pot ent i al of the f er r o- f er r i cyani de couple. Al t hough a good many workers have investigated the r edox pot ent i al of t he f er r o- f er r i cyani de system 7-9, most investigators in the bi ochemi cal field make refer- ence t o the met hod of Davenpor t and Hill 6 which assumes an E o' f or t he f e r r o- ferri cyani de system of +0. 43 V at 30 C- - t aken f r om the dat a of Cl ark et al. 8. Un- 510 J. E. O'REILLY f or t unat el y the oxi dat i on pot ent i al of this system is very dependent on the t ot al ionic strength as well as the individual solution component s 7,~ increasing f r om about +0. 36 V at zero ionic strength to +0. 45 V at about 1 M ionic strength 7. Cl ark ~ hi msel f has strongly caut i oned against the uncritical use of the value of 0.43 V. It is t he pur pose of the present paper, therefore, to present experi ment al values f or the pot ent i al of the f er r o- f er r i cyani de system in f our commonl y used buffers at specified pH values, as well as to offer a rat i onal met hod f or estimating the pot ent i al in buffer solutions of slightly different pH and ionic strength. EXPERIMENTAL Chemi cal s All solutions were pr epar ed with doubl e distilled water. Pot assi um ferri cyani de [K3Fe(CN)6] and pot assi um ferrocyani de di hydrat e [ K4Fe( CN) 6. 2HzO ] were each recrystallized twice f r om wat er; the ferri cyani de was air dried and stored over anhydr ous CaCI2, and the ferrocyani de was qui ckl y air dried and st ored in a tightly sealed bottle. Analysis of the pot assi um ferrocyani de by per manganat e t i t rat i on 11 indicated a puri t y of 99. 7+0. 2%. St ock solutions of ferro- and ferri cyani de were pr epar ed fresh daily and prot ect ed f r om light. Qui nhydr one was obt ai ned f r om East man and recrystallized twice f r om wat er at 70 C as r ecommended by Bates 12. All buffer solutions were pr epar ed f r om analytical reagent grade chemicals wi t hout f ur t her purification. The following buffer systems were used: pH 4.20 and 4.75, acetate ( CH3 COOH+Na OH) ; pH 7.00 and 8.00, Tris (t ri shydroxymet hyl - ami no) met hane+ HC1); pH 7.00 and 7.80, phosphat e ( KH2PO 4 + NaOH) ; pH 9.00 and 10.00, bor at e (H3BO, ~+NaOH). The pH values of all solutions were checked with a pH met er and were within 0.03 unit of t he nomi nal value. Ioni c strengths (I) were calculated using the st andard expression I = 1/2 SCI Zi 2, where Ci is the concent r at i on of a part i cul ar ionic species and Zi is t he charge of t hat species, and assuming no associ at i on of charged species. The following pK a values were used in the cal cul at i on of ionic strength values: acetic acid, 4.757; di hydrogen phosphat e ion, 6.865; Tris ion, 8.080; bori c acid, 9.194. It shoul d be not ed t hat part i cul arl y in solutions of high ionic strength and buffer concent rat i ons, where i on pai ri ng and ot her ionic-association phenomena become increasingly i mport ant , the last t wo digits in the pK, values are most cert ai nl y not significant, but were ret ai ned simply f or cal cul at i on purposes. Ni t rogen, used t o deoxygenat e test solutions and bl anket t hem duri ng pot ent i al measurement s, was purified and presat urat ed with wat er vapor by bubbl i ng successively t hr ough t wo acidic vanadous solutions 13, sat urat ed calcium hydroxi de, and distilled water. Ref er ence el ect rode All pot ent i al s were measured vs a qui nhydr one reference el ect rode in 0.01 M HC1, 0.09 M KC1, and correct ed t o pot ent i al s vs the nor mal hydr ogen electrode. All pot ent i al s quot ed in this paper are in V vs the nor mal hydr ogen electrode. The best val ue f or the st andard pot ent i al of the qui nhydr one el ect rode is +0. 69976 V at 25 C (ref. 12). To obt ai n the pot ent i al of the par t i cul ar reference el ect rode used FERRO-FERRICYANIDE REDOX POTENTIAL 511 0.00020 V is subt r act ed f r om the f or mer val ue due t o the "sal t er r or " of the el ect rol yt e sol ut i on used x4, and t hen 0.1241 t V is subt r act ed since the p H of the sol ut i on is 2.098 (a Nat i onal Bureau of St andar ds secondar y p H st andar d) 15. Therefore, + 0.57545 V is the pot ent i al of the par t i cul ar reference el ect rode used. E q u i p m e n t All pot ent i al s were measur ed wi t h a Leeds and Nor t hr up 8687 Vol t Pot ent i o- met er. Test sol ut i ons were cont ai ned in a wat er-j acket ed t hr ee- compar t ment cell~6: one end compar t ment cont ai ned the f er r o- f er r i cyani de test sol ut i on, the ot her cont ai ned the qui nhydr one reference electrode, and the mi ddl e compar t ment con- t ai ned an appr opr i at e sal t sol ut i on (usually 0.1 M KC1) which al l owed electrical cont act bet ween t he reference- and t est -el ect rode compar t ment s wi t hout appr eci abl e mi xi ng and cont ami nat i on of the t wo solutions. Temper at ur e of the entire cell was mai nt ai ned at 25.00 + 0.02 C, unless ot herwi se stated. El ect rodes were shi ny pl at i num flags, about 1 c m 2 in area. P r o c e d u r e s At t he st art of measur ement s, qui nhydr one was added to the 0.01 M HC1, 0.09 M KC1 in one end compar t ment of the wat er - j acket ed cell, the pl at i num-fl ag el ect rode inserted, and the sol ut i on deoxygenat ed wi t h purified ni t rogen f or at least 15 mi n. The el ect rol yt e sol ut i on was added t o t he mi ddl e compar t ment ; and t he par t i cul ar f er r o- f er r i cyani de test sol ut i on to the ot her end compar t ment , the el ect rode i nsert ed, and the sol ut i on deoxygenat ed. Car e was t aken to keep the sol ut i on levels equal in the t hree compar t ment s t o avoi d gross sol ut i on t ransfer. Pot ent i al s of the i ndi vi dual sol ut i ons were measur ed at least t hree different t i mes on separ at e days; i ndi vi dual measur ement s usual l y agreed within 0.2 mV. Several measur ement s were made on equi mol ar mi xt ures of f er r o- f er r i cyani de wi t hout buffer. The values obt ai ned agreed with t hose r epor t ed by Kol t hof f and Tomsi cek 7 t o wi t hi n about 2 mV; however, 1.36 mV of this is a syst emat i c difference due t o t hei r use of 2.075 as the p H of the 0.01 M HC1, 0.09 M KC1 sol ut i on, r at her t han the cur r ent l y accept ed N. B. S. val ue of 2.098. Thr oughout this paper the f er r o- f er r i cyani de syst em studied is an equi mol ar 10 mM pot assi um f er r ocyani de and pot assi um ferri cyani de ( i . e . 5 mM of each species), o f ionic st rengt h 0.080 M. RESULTS The exper i ment al results f or the oxi dat i on pot ent i al of equi mol ar (10 mM) f er r o- f er r i cyani de in several c ommon buffer syst ems is present ed in Tabl e I. I t can be seen t hat the redox pot ent i al vari es f r om about 0.40 V t o al most 0.51 V as t he concent r at i on of the buffer syst em is i ncreased f r om 0.01 t o 0.90 M. Al so pr ovi ded, f or compar i s on pur poses, is a value f or E 0' - Ek, where E L is the val ue f or t he pot ent i al of an equi mol ar f er r o- f er r i cyani de sol ut i on whose t ot al concent r at i on is such t hat t he ionic st rengt h of the sol ut i on is equal to the t ot al ionic st rengt h of the f e r r o- ferri cyani de buffer syst em studied*. I t can be seen t hat the measur ed pot ent i al s of * The potentials reported by Kolthoff and Tomsicek 7 were plotted on a large sheet of graph paper vs 1, and the potential at any given ionic strength was then determined from the resultant E v s I ~ plot. 512 J . E. O' REI LLY TABLE I OXI DATI ON POTENTIALS AT 25 C FOR EQUI MOLAR (10 mM) POTASSI UM FERRO- FERRI CYANI DE AS A FUNCTI ON OF BUFFER C O N C E N T R A T I O N The ionic strength, I, of the solution is the total ionic strength, i.e. the calculated ionic strength of the buffer system plus 0.080 M due to the ferro-ferri cyani de. Ek refers to the potential reported by Kolthoff and Tomsicek7 for a solution containing only equimolar potassium ferro- and ferri- cyanide at a total concentration so as to have an ionic strength equal to that reported in the I column. Buffer concn ( M) Acetate ( p H 4.20) Tris ( p H 7.00) Phosphate ( p H 7.00) Borate ( p H 9.00) 1 Eo" E o ' - E k 1 Eo" Eo' - - Ek 1 Eo' Eo' - - Ek 1 Eo' Eo' - - Ek ( M ) ( V) ( mV) ( M ) ( V) ( mV) ( M ) (V) ( mV) ( M ) ( V) ( mV) 0.90 0.50 0.20 0.10 0.05 0.02 0.01 0.525 0.4280 3.2 0.954 0.5012 54.9 1. 42 0.4559 - 8 . 8 0.656 0.4310 - 2 . 0 0.434 0.4224 4.2 0.736 0.4798 42.6 1.08 0.4460 - 5 . 2 0.524 0.4246 - 0 . 2 0.353 0.4157 4.7 0.514 0.4522 28.0 0.716 0.4332 - 2 . 9 0.398 0.4160 1.0 0.319 0.4120 4.1 0.415 0.4356 19.0 0.544 0.4247 - 1 . 4 0.345 0.4103 0.0 0.301 0.4098 3.6 0.355 0.4232 12.2 0.434 0.4182 0.1 0.315 0.4075 - 0 . 1 0.290 0.4074 2.3 0.314 0.4127 5.2 0.351 0.4106 - 0 . 3 0.296 0.4056 - 0 . 1 0.287 0.4065 1.6 0.299 0.4084 2.4 0.319 0.4075 - 0 . 4 0.290 0.4043 - 0 . 8 t he f e r r o - f e r r i c y a n i d e buf f er s ys t ems agr ee r e a s o n a b l y wel l wi t h t hos e r e p o r t e d f or t he p u r e f e r r o - f e r r i c y a n i d e s ol ut i ons , p a r t i c u l a r l y at l ow buf f er c o n c e n t r a t i o n s . The s o me wh a t l a r ge r pos i t i ve di f f er ences i n Tr i s buf f er a r e p r o b a b l y due t o a hi ghe r degr ee o f a s s o c i a t i o n o f t he Tr i s c a t i o n [ ( HOCH2 ) 3 CNH 3 +] wi t h t he f e r r o c y a n i d e a n i o n t h a n is t he cas e wi t h K + or Na +. Du e t o t he n a t u r e o f t he p r o c e d u r e s us ed i n t hi s s t udy, t he r ef er ence e l e c t r ode , et c. i t is e s t i ma t e d t ha t t he p o t e n t i a l s r e p o r t e d i n Ta bl e I a nd el s ewher e i n t hi s p a p e r a r e a c c u r a t e t o wi t hi n a b o u t 2 mV, wi t h r e s pe c t t o t he n o r ma l h y d r o g e n e l e c t r ode . Becaus e o f t he excel l ent r e p r o d u c i b i l i t y o f t he r es ul t s , a n i n d i v i d u a l p o t e n t i a l is e s t i ma t e d t o be pr eci s e wi t hi n at l eas t _+ 0. 3 mV. Al t h o u g h t he r e dox p o t e n t i a l s s hown i n Ta bl e I f or f o u r p a r t i c u l a r buf f er s ys t ems s h o u l d be us ef ul t o ot he r wor ke r s , i t wo u l d be hi ghl y de s i r a bl e t o be a bl e t o p r e d i c t t he r e d o x p o t e n t i a l o f t he f e r r o - f e r r i c y a n i d e s ys t em u n d e r a wi de va r i e t y o f e x p e r i me n t a l c ondi t i ons . To wa r d t hi s end, f u r t h e r me a s u r e me n t s wer e ma d e on t he f e r r o - f e r r i c y a n i d e s ys t em i n t he s a me f o u r buf f er s at di f f er ent p H val ues . Ta bl e I I pr e s e nt s t hes e da t a , as wel l as a c o mp a r i s o n wi t h t he val ues r e p o r t e d b y Ko l t h o f f a n d To ms i c e k 7 (ER) a n d wi t h t hos e o b t a i n e d wi t h t he " s t a n d a r d " buf f er s i n Ta b l e I. As c a n be seen, a me a s u r e d E o' va l ue i s u s u a l l y f a i r l y cl os e (i . e. wi t hi n a b o u t 3 mV) t o t he o t h e r t wo p o t e n t i a l val ues. Si nce, as wi l l be di s cus s ed l at er , bes t r es ul t s a r e a c hi e ve d whe n t he c ont r i - b u t i o n o f t he buf f er t o t he t o t a l i oni c s t r e ngt h is f a i r l y s mal l , onl y 0. 05 a n d 0. 10 M buf f er s ys t ems wer e s t udi ed. Va r i o u s t ype s o f " c o r r e c t i o n " p r o c e d u r e s wer e t r i e d i n a n a t t e mp t t o c o r r e l a t e me a s u r e d Eo' val ues wi t h Ek or Eo' ( Ta bl e I ) , b y t r yi ng t o a c c o u n t f or c ha nge s i n p o t e n t i a l due t o t he ( known) di f f er ences i n i oni c s t r engt hs . Al t h o u g h s ever al s uch p r o c e d u r e s wer e t r i ed, n o n e of f er ed a ny s i gni f i cant a d v a n t a g e ove r a n e s t i ma t i o n b a s e d on E k val ues or E o' val ues f r o m Ta b l e I. FERRO-FERRICYANIDE REDOX POTENTIAL 513 TABLE II OXIDATION POTENTIALS AT 25 C FOR EQUIMOLAR (10 mM) POTASSIUM FERRO- FERRICYANIDE IN SEVERAL BUFFER SYSTEMS Ek and I have the same meaning as in Table I. E0' (Table I) is the potential of the ferro-ferricyanide couple in the "standard" acetate, Tris, phosphate, or borate buffer at 0.05 or 0.10 M concentration taken from Table I. Buffer Buffer 1 (M ) Eo" (V) Ek (V) Eo'(Tablel) conch (M) (V) Acetate (pH 4.75) 0.05 0.324 0. 4103 0.4084 0. 4098 0.10 0. 443 0.4142 0. 4189 0.4120 Tris (pH8.00) 0.05 0. 321 0.4200 0. 4081 0.4232 0.10 0.354 0. 4311 0. 4112 0.4356 Phosphate (pH7.80) 0.05 0. 468 0.4192 0. 4208 0. 4182 0.10 0.599 0. 4263 0.4297 0.4247 Borate (pHI0.00) 0.05 0. 351 0.4106 0. 4109 0. 4075 0.10 0.408 0. 4151 0.4160 0. 4103 TABLE III OXIDATION POTENTIAL FOR EQUIMOLAR (10 mM) POTASSIUM FERRO-FERRI- CYANIDE AS A FUNCTION OF TEMPERATURE E (reference electrode) is the value of the quinhydrone 0.01 M HCI, 0.09 M KC1 reference electrode used at the temperature indicated. This was calculated using the E0 values compiled by Bates 12 and assuming that the "salt error" remained constant at -0. 20 mV and the pH constant at 2.098. The experimental Eo" values fit the equation E0 ' = 0.4032-2.52' 10 -3 ( T- 25) - 6 54" 10 -6 ( T- 25) 2 to within 0.2 mV. T (C) Eo" (V) E (reference electrode) (V) 20 0.4157 0.5812 25 0.4031 0.5754 30 0.3907 0.5697 35 0.3773 0.5639 40 0.3641 0.5581 Since it woul d be of i nt erest t o know the pot ent i al of the ferro-ferri cyani de coupl e as a f unct i on of t emperat ure, for example, many redox pot ent i al measur ement s of bi ol ogi cal compounds are made at 30 C, the dat a present ed i n Tabl e III for equi- mol ar (10 mM) f er r o- f er r i cyani de were obt ai ned. To a first appr oxi mat i on, this redox coupl e has a t emperat ure coefficient of - 2. 52. 10- a V/ C; this t emperat ure coefficient can now be used, in conj unct i on wi t h the dat a present ed i n Tabl es I and II, to est i mat e the pot ent i al of the f er r o- f er r i cyani de redox buffer at t emperat ures different f r om 25 C. For example, the pot ent i al i n 0.05 M phosphat e (pH 7.00) at 30 C shoul d be 0.4182 V- 5 C(0.00252 V/ C ) =0. 4056 V. 514 J. E. O' REILLY DISCUSSION In general, the r edox pot ent i al of the f er r o- f er r i cyani de coupl e in aqueous buffer sol ut i ons is least affected by vari ous exper i ment al var i abl es when t he buffer syst em is at a l ow concent rat i on, hence, low ionic st rengt h. I t is t her ef or e r ecom- mended t hat the f er r o- f er r i cyani de oxi dat i on- r educt i on buffer be used in l ow con- cent r at i on ( <0. 05 M) buffer systems. For an i nvest i gat or who wishes to use the f er r o- f er r i cyani de syst em in the st udy of redox pot ent i al s of some bi ol ogi cal compound, t he par t i cul ar Eo' selected under a given set of exper i ment al condi t i ons must , of necessity, depend on t he degree of preci si on and accuracy desired. I f pot ent i al s are to be det er mi ned t o an accur acy bet t er t han about 1 mV, it is necessary t hat the oxi dat i on pot ent i al of each par t i cul ar sol ut i on be measur ed in some appr opr i at e fashi on, since it has been shown t hat the E 0' of this system, cal cul at ed using the Ner nst equat i on, depends on the r at i o of f er r ocyani de :ferricyanide, even when al l owance is made f or t he difference in ionic st rengt h 7. For exampl e, when the rat i o is 1:10, the cal cul at ed E o' is a little hi gher ( about 1-3 mV) t han t hat f or the equi mol ar sol ut i on; conversel y, when the rat i o is 10: 1, the cal cul at ed E0' is a few mV lower. It is obvi ousl y i mpract i cal , however, to measur e the redox pot ent i al of each and every sol ut i on. I f the i nvest i gat or is satisfied in knowi ng t he pot ent i al wi t hi n a few mV, t hat is, about + 3 - 1 0 mV, the Eo' values in Tabl es I and I I can be used even i f t he ferro- cyani de: ferri cyani de rat i o differs f r om 1 : I, as is usually t he case, as l ong as the buffer concent r at i on is fairly low ( <0. 05 M). About the same degree of " e r r or " or uncer t ai nt y can be expect ed in Eo' values est i mat ed f r om these t abl es f or sol ut i ons t hat differ onl y a small a mount f r om t hose listed. For exampl e, t he E 0' in 0.02 M acet at e buffer ( pH 4.50) shoul d not differ significantly f r om t hat f or 0.02 M acet at e ( pH 4.20). Fur t her mor e, as l ong as the buffer concent r at i on is l ow ( <0. 05 M), the dat a of Kol t hof f and Tomsi cek 7 can be used to obt ai n a r easonabl e est i mat e of the E 0' f or f er r o- f er r i cyani de by cal cul at i ng the t ot al ionic st rengt h of t he sol ut i on. I f it is desi rabl e to use higher concent r at i ons of f er r o- f er r i cyani de, e.g. 20 or 50 mM, the dat a of Kol t hof f and Tomsi cek 7 shoul d be j ust as useful. Al t ernat i vel y a small correct i on f act or can be appl i ed to the pot ent i al values in Tabl es I and I I due to the i ncreased ionic st rengt h. Finally, t he i nvest i gat or can now experi ment al l y det ermi ne, wi t h rel at i ve ease, t he redox pot ent i al of f er r o- f er r i cyani de under a par t i cul ar set of exper i ment al condi t i ons by using one of the accurat el y det er mi ned syst ems in Tabl es I and I I as a "r ef er ence el ect r ode" and measur i ng the redox pot ent i al of t he unknown syst em wi t h a pot ent i omet er or ot her suitable high i mpedance device. For exampl e, the redox pot ent i al of f er r o- f er r i cyani de in 0.04 M a mmoni a buffer ( pH 9.27) coul d be measur ed rel at i ve t o t hat of t he 0.05 M Tri s buffer ( pH 8.00) listed in Tabl e I I . Thi s el i mi nat es t he need f or pr epar i ng and checki ng a different reference el ect rode. The i nvest i gat or is caut i oned agai nst the uncriticai use of t he val ue of 0.43 V f or t he E o' of f er r o- f er r i cyani de at 30 C. Fr om i nspect i on of Tabl e I and a consi der- at i on of the t emper at ur e dependence of the pot ent i al , it woul d a ppe a r t hat this val ue is even moder at el y accurat e onl y at fai rl y hi gh buffer concent r at i ons (0. 1-0. 9 M) or hi gh f er r o- f er r i cyani de concent r at i ons ( about 0.08 M) , at about 0.02 M buffer and 0.01 M f er r o- f er r i cyani de, this val ue mus t be t oo hi gh by s ome 30 mV or so. FERRO- FERRI CYANI DE REDOX POTENTI AL 515 Al t h o u g h i t ma y s eem t o s ome t h a t t hi s p a p e r is c o n c e r n e d wi t h a p p a r e n t l y s ma l l v a r i a t i o n s i n p o t e n t i a l , i t s h o u l d be n o t e d t h a t a l t h o u g h t he r e d o x p o t e n t i a l s o f v a r i o u s b a c t e r i a l c y t o c h r o me s c2, such as t hos e f r o m Rhodospirillum rubrum, Rho- dopseudomonas capsulata, or Rhodopseudomonas palustris, a r e h i g h e r t h a n t ha t o f ma mma l i a n c y t o c h r o me c by onl y a b o u t 50- 80 mV at p H 7 ( r ef s 1 a n d 3), t hes e di f f er ences i n p o t e n t i a l r ef l ect a p p r e c i a b l e di f f er ences i n bi ol ogi c a l f unc t i on. Pa r t i c u- l a r l y wi t h r e g a r d t o a t t e mp t s t o r el at e c he mi c a l s t r uc t ur e t o bi ol ogi c a l f unc t i on (i.e. r e dox pot e nt i a l ) , a gr e a t de a l o f car e a n d a t t e n t i o n mu s t be p a i d t o t he effect s o f f ai r l y s ma l l di f f er ences i n e x p e r i me n t a l c o n d i t i o n s on t he Eo' o f t he f e r r o - f e r r i c y a n i d e o x i d a t i o n r e d u c t i o n p o t e n t i a l buf f er . ACKNOWLEDGEMENTS Th e a u t h o r wo u l d l i ke t o expr es s hi s g r a t i t u d e t o Dr Ka r l M. Du s o f t he De p a r t me n t o f Bi o c h e mi s t r y f or ma n y he l pf ul di s c us s i ons a n d t o t he Es s o Co mp a n y f or f i nanci al as s i s t ance. REFERENCES 1 Vernon, L. P. and Kamen, M. D. (1954) J. Biol. Chem. 211,643-662 2 1-[orio, T. and Ohkawa, J. (1968) J. Biochem. Tokyo 64, 393-404 3 Kamen, M. D. and Vernon, L. P. (1955) Biochim. Biophys. Acta 17, 10-22 4 Ni shi mura, M. (1959) J. Biochem. 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