摘要:

238 J.C.S. DaltonInteractions and Reactions in Restricted Polar Media. Binding ofCyanide Ion to Hemin in Surfactant-solubilized Methanol in BenzeneBy Willie Hinze and Janos H. Fendler,’ Department of Chemistry, Texas A & M University, College Station,One molecule of monomeric hemin is solubilized by 21 00 f 400 molecules of uncharged surfactant lgepal CO-530in benzene containing 0.2% (v/v) methanol. The substrate-surfactant binding constant in this system is (3.5 f 0.5)x lo4 I mol-I. The environment of hemin in this restricted methanol pool is less polar than in bulk methanol.The interaction of cyanide ion with hemin, localized in the polar cavity of surfactant aggregates in benzene, hasbeen treated in terms of a two-step process. Rate (k,, k-,, k,, k-.J and equilibrium ( K ) constants have beendetermined.Values for K, k-,, k,/k-,, and k, in 0 . 7 0 ~ lgepal CO-530 in benzene, containing 1 .O% (v/v) methanolare 960-times greater, 200-fold smaller, 1 O-fold greater, and identical, respectively, to those in bulk methanol.The significance of these results are discussed and compared to those available in aqueous surfactant systems.Texas 77843, U.S.A.IMPORTANCE of hemoglobins in biochemical processes 1-4has prompted the kinetic investigations of reactions in-volving hemin in aqueous and non-aqueous ~olutions.~-~~Although these studies have provided a wealth ofinformation, electrostatic, hydrophobic, and othermicroscopic medium effects of hemoproteins in theirnative environments could not be assessed. Dimeriz-ation of hemin as well as solvent co-ordination to thecentral iron atom introduced additional dissimilaritiesin the chemistry of hemin and hemoproteins. In orderto minimize these problems Simplicio and his co-workers 5-7 have studied the interactions in and cyanidebinding with aqueous micelle solubilized hemin.Inthese systems the hydrophobic interaction between themicelle and hemin apparently overcomes the hemin-hemin interactions. Kinetic and thermodynamic datacould, therefore, be obtained for the binding of cyanideion to hemin rnon~mers.~*~ Ligand substitution occurred1 J. E. Falk, Cow@. Biochem., 1963, 9, 3.2 J. N. Phillips, Comp. Biochem., 1963, 9, 34.3 G. S. Marks, ‘ Heme and Chlorophyll, Chemical, Biochemicaland Medical Aspects,’ Van Nostranjl, London, 1969.Hemoglobin and Myoglobinin their Reactions with Ligands,’ North Holland Co., Amsterdam,1971.E.Antonini and M. Brunori,5 J. Simplicio, Biochemistry, 1972, 11, 2524.J. Simplicio, Biochemistry, 1972, 11, 2529.7 J. Sirnplicio and k’. Schwenzer, Biochemistry, 1973, 12, 1923.E. B. Fleischer, S. Jacobs, and L. Mestichelli, J . Amer.Chern. Soc., 1968, 90, 3527.through the penetration of cyanide ion across the* membrane-like sheath imposed about the hemin at themicelle-water interface.’ 5An alternative approach is to bring both the heminmonomer and cyanide ion, along with a limited amountof protic solvent, into a benzene solution by a suitablesurfactant. We have reported that surfactants in non-polar solvents form relatively small aggregates, termedas reversed micelles, which provide unique media forinvestigating interactions and reactions.ll Possibilitiesof solubilizing controlled amounts of water or methanolin the polar cavities of these aggregates as well as bindingsubstrates fairly rigidly accounts, among other factors,for the uniqueness of the system and allows its utilizationas a model for interactions at enzyme-active sites andmembrane processes. Indeed we have reported thatrate constants for the mutarotation of 2,3,4,6-tetra-methyl-a-D-glucose l2 and those for the decompositionof Meisenheimer complexes l3 in the polar cavities of9 N.B. Angerman, B. B. Hasinoff, H. B. Dunford. and R. B.Jordan, Canad. J . Chem., 1969, 47, 3217.10 B.B. Hasinoff, N. B. Dunford, and D. G. Howe, Canad. J .Chem., 1969, 47, 3225.l1 E. J. Fendler, S. A. Chang, J. H. Fendler, R. T. Medary,0. A. El Seoud, and V. A. Woods in, ‘ Reaction Kinetics inMicelles,’ ed. E. H. Cordes, Plenum Press, New York, 1973, p. 127.l2 J. H. Fendler, E. J. Fendler, R. T. Medary, andV. A. Woods,J . Amer. Chem. Soc., 1972, 94, 7288.l3 J . H. Fendler, E. J . Fendler, and S. A. Chang, J. Amer.Chewz. Soc., 1973, 95, 33731975 239alkylammonium carboxylate surfactant aggregates inbenzene are several orders of magnitude greater thanthose in pure benzene or in pure water. Aquation ofthe tris(oxa.lato)chromate(m) anion in the polar cavitiesof associated alkylammonium carboxylates proceededsix million-fold faster than that in pure water.l4~l6Even more significant is the observed specificity.Aquations of tris(oxalato)cobaltate or ethylenediamine-bis(oxalato)chromate(m) are only modestly affected bysurfactants in benzene.16 It emerges that both theobserved magnitude and specificity of catalyses in thesesystems significantly exceed those generally observedin aqueous micellar soluti0ns.l7-~ It seemed to usworthwhile, therefore, to examine if relatively largemolecules such as metal porphyrins could be solubilizedin non-polar solvents by surfactants and if the rates oftheir reactions are affected in the restricted polarenvironments provided by this media.Availability ofdata on the interaction of hemin with cyanide ion inaqueous micellar systems 6-7 dictated this reaction to bethe subject of our initial investigations.EXPERIMENTALHemin, ferriprotoporphyrin IX (Eastman), was used asreceived.Polyoxyethylene( 6) nonylphenol, Igepal CO-530,was used as received from GAF Corporation. Sodiumdi(2-ethylhexyl) sulphosuccinate, Aerosol-OT (Aldrich) ,was dried prior to use. Reagent grade benzene wasdistilled from sodium and stored over Linde 5A molecularsieve. Stock solutions of hemin (generally 10-3~) wereusually made up under a stream of nitrogen in dry 0.05~-methanolic sodium methoxide and stored in the refrigerator.These solutions were stable for a t least two weeks, asshown by spectrophotometry. Individual solutions forspectral and kinetic determinations were prepared byinjecting appropriate volumes of the methanolic heminstock solutions into benzene solutions of the surfactants.Final concentrations of hemin, methoxide ion, and methanolranged between (5-8) x 10-6~, (1-5) x 1 0 - 4 ~ , and0.2-5-0% (v/v), respectively.Water concentrations, (9-10) 10-4~, in these solutions were carefully controlled andmonitored by gas-liquid-partition chromatography usinga Porapak Q column. Cyanide stock solutions wereprepared by dissolving dry sodium cyanide (Baker Reagentgrade) immediately prior to use in methanol and standard-ized by a spectroscopic method.21 All other chemicalsused were the best available reagent grade.Isolation of Dicyanoferri'rotoporphyrin Complex.-Hemin(0.100 g, 0.153 mmol) and sodium cyanide (0.186 g, 3.79mmol) were shaken vigorously with water (1.0 ml) untilcoloured crystals appeared.After 15 min excess of acetone(5.0 ml) was added and the solvent was evaporated in zlacuo.The solid was washed with benzene three times. Theprocess was repeated subsequent to the addition of sodiumcyanide. The isolated brownish crystals were driedin vacuo.l4 C. J. O'Connor, E. J. Fendler, and J. H. Fendler, J . Amer.Chem. Soc., 1973, 95, 600.l6 C. J. O'Connor, E. J. Fendler, and J. H. Fendler, J . Amer.Chem. SOC., 1974, 96, 370.l6 C. J. O'Connor, E. J. Fendler, and J. H. Fendler, J.C.S.Dalton, 1974, 626.l7 E. H. Cordes and R. B. Dunlap, Accounts Chem. Res., 1969,2, 239.l* E. H. Cordes and C. Gitler, Progr. Bio-org. Chem., 1973, 2, 1.Solubilities of the isolated dicyanoporphyrin complexhave been determined by measuring concentrations ofsaturated solutions a t 25.0 "C spectrophotornetrically.Excess of solid complex was added to a series of solutionscontaining different amounts of Igepal CO-530 in benzene-methanol 99 : 1% (v/v). These solutions were vigorouslyshaken for 2 h, allowed to stand overnight, and thencentrifuged.Diluted aliquots were used to determineabsorbances a t 402 nm using appropriate blank solutions.Concentrations of the saturated solutions were thencalculated using €402 ,,,,, = 8.0 x lo4 1 mold' cm-l.Solubilities of sodium cyanide were determined by amodified spectroscopic method of Humphrey 22 on aliquotsof saturated solutions in benzene both in the absence andpresence of Igepal CO-530.A small volume (0.5 ml) ofthe cyanide solution was added to 2-0 ml of methanol.Addition of an excess of solid Hg,Cl, to this solutionresulted in a disproportionation reaction to form Hg(CN),.Subsequent addition of solid NaI results in the formationof [HgI4I2-, which absorbs a t 300 nm (E = 1.2 x lo4 1 mol-lcm-l). Calibration was obtained on standard cyanidesolutions in Igepal CO-530 in benzene.Spectrophotometric determinations were carried outusing a Cary 11 8-C spectrophotometer whose cell compart-ment was thermostatted to 24.8 f 0.1 "C. Kinetic datawere obtained on a Cary 118-C, or a Beckman KintracVII spectrophotometer and on the Durrum model 110stopped-flow T-jump system. Temperatures for the kineticruns were maintained a t 24.8 0.1 "C by water circulation.Critical micelle concentrations were determined by thedye method 23 using Bromophenol Blue.RESULTS AND TREATMENT OF DATAHemin is completely insoluble in benzene.However,stable solutions of (6-8) x 10-6M-hemin are obtained byinjecting small volumes of methanolic stock solutions, in thepresence of sodium methoxide, into benzene. Absorptionspectra of these solutions [i.e. 6-2 x 10-6M-hemin, 1.0 x10-4~-sodi~methoxide in benzene in the presence of0 . 0 2 ~ 0 (v/v) methanol] indicate hemin to be in aggregatedform.24 Successive addition of Igepal CO-530 to thissolution results in an increase of absorption, parallel with aslight bathochromic shift in the Soret band, up to a maxi-mum after which there is no further change (Figure 1).The molar absorptivity of hemin a t 402 nm in benzene inthe presence of 0-6O~-Igepal CO-530, 8.1 X lo4 1 mo1-l cm-l,is in good agreement with that reported for hemin mono-mers in aqueous micellar sodium dodecyl sulphate (8.2 x104 1 mol-1 cm-1) , hexadecyltrimethylanimonium bromide(7.0 x 104 1 mol-l cm-l), andTriton X-100 (6.2 x 1 0 4 ~ 1 mol-lcm-l).Furthermore the molar absorptivity of hemin in0-60~-Igepal CO-530 in benzene was found to be unaffectedby changes in the hemin [in the (2-20) x 10-6~-range] orin the methanol [in the 0.02-4.0% (v/v) range] concen-trations. Changes of the absorbances of hemin at 402 nmas functions of Igepal CO-530 concentrations are alsoE. J. Fendler and J. H. Fendler, Adv. Phys.Ovg. Chem., 1970,8, 271.2o J. H. Fendler and E. J. Fendler, ' Catalysis in Micellar andMacromolecular Systems,' Academic Press, London, 1975.I1 W. Hinze and R. E. Humphrey, Analyt. Chem., 1973,45, 385.I2 R. E. Humphrey, unpublished results, 1974.29 P. Mukerjee and K. Mysels, ' Critical Micelle Concentrationsof Aqueous Surfactants,' NSRDS-NBS 36, Washington, D.C.(1971).a4 A . C. Maehly and A. Akeson, Acla Clzem. Scand., 1958, 12,1259240 J.C.S. Daltonillustrated in Figure 1. Drawing parallels to the two limbsof the obtained curve result in an intersection at 6.5 x10-2M-Igepal CO-530. This value can be taken as thecritical micelle concentration of the surfactant in benzeneand i t agrees well with that determined independently bythe dye [(4.5-5.6) x 10-2~1] and IH r ~ .m . r . ~ ~ [(3*5-5.5) x 10-2~] methocls.IQ, O-LO-2 1- .'^ **.. j ,.1* I .I I I I I 350 370 390 L10 L30 450A I n mFIGURE 1 Absorption spectra of 6.2 x 10-sM-hemin, containing1.0 x 10-4~-sodium methoxide and 0.02% (v/v) methanol in(1) benzene; (2) O.O2~-Igepal CO-530 in benzene; (3) 0 . 1 0 ~ -Igepal CO-530 in benzene; (4) 0*32~-Igepal CO-530 in benzene;(5) 0.50ni-Igepal CO-530 in benzene; (6) 1.2~-Igepal CO-530 inbenzene; (7) neat methanol. Insert shows a plot of absorb-ance of 6.2 x lO-%¶-hemin, containing 1.0 x 10-4~-sodiummethoxide and 0.02% (v/v) methanol, in benzene at 402 nm us.Igepal CO-530 concentrationAddition of Aerosol-OT to solutions of 6.2 x 10-s~f-hemin in benzene, containing 1.0 x l o - 4 ~ sodium methoxideand 0.2% (v/v) methanol, resulted in the development of anew band a t 407 nm with a shoulder at 378 nm.As theconcentration of Aerosol-OT increased this shoulderdeveloped into an absorption maxima. The developmentof the absorption maxima at 378 and 407 nm coincidedwith the aggregation of Aerosol-OT as established from a+ [H !]/>I - [OMe-]/h% __tFIGURE 2 Changes of absorbance, a t 383 nm, for 7.0 x lo-"-hemin in benzene-methanol [99-6 : 0.4% (v/v)] containing0*65~-Igepal CO-530. as functions of hydrogen-ion concentra-tions. Data on the dotted line were taken immediately aftermixing while those on the solid line obtained 3 h subsequent tomixing. The logarithm of absorbance differences for the samedata arc plotted against the hydrogen-ion concentration in theinsertplot analogous t o that illustrated in the insert of Figure 1.Formation of an additional peak at 378 nm is taken toindicate the replacement of solvent ligands of the heminby Aerosol-OT molecules.Due to this complication therest of the present investigations were limited to usingIgepal CO-530.Changing the stoicheiometric acidity of the IgepalCO-530 surfactant solubilized hemin in benzene, by theaddition of appropriate amounts of sodium methoxide andnitric acid, results in significant absorbance changes.Such changes at 383 nm give the usual titration curve(Figure 2). On plotting absorbance differences logarithmic-ally against the logarithm of stoicheiometric hydrogen ionconcentration a straight line is obtained (see insert inFigure 2) from which an apparent pK, value of 4.4 f 0.3 iscalculated for the equilibrium :Hemin(Ohle,MeOH).(S) + H+ __The slope of this straight line, 0.8, substantiates a one toone interaction between the proton and the Igepal CO-530solubilized hemin.Since subsequent investigations of theinteraction of cyanide ion with hemin were carried out inthe presence of sodium methoxide (>€GO x 10-%I) theunprotonated species is the reacting substrate, There is aslow change of absorbance over the entire acidity range(Figure 2). At base concentrations greater than 10-3~1it is due to dimer formation and a t the lower base concen-tration it is accountable in terms of auto-oxidation of thevinyl side-chain of hemin.26 In agreement with thissuggestion is the observed decrease of the decay rate from(4-8) x sP1 to (8-12) x s-l on careful exclusionof the oxygen by nitrogen purging.The time scale ofsubsequent kinetic investigations are considerably faster,this slow absorbance change can, therefore, be neglected.Solubility of sodium cyanide in benzene is 2-5 x 10-4~~.This value is in fair agreement with that extrapolatedfrom known solubilities of NaCN in water and methanolusing a correlation between solubilities and solvent polarityparameter, 2. Addition of Igepal CO-530 increases itssolubility substantially (Table 1). A linear relationship isTABLE 1Solubilities of NaCN in Igepal CO-530 in benzene aHemin(MeOH),+.(S) (1)[Igepal C0-530]/~ Solubility of NaCN/hi0~000 (2.5 f 1) x 10-40.030 (3.6 + 2) x 10-40.060 (8.8 & 2) x lo-*0.090 (0.3 i 2) x 10-40.126 (13 5) x 10-4(85 30) x 10-40.633 (69 * 30) x 10-41.260 (186 * 10) x 10-41.580 (250 & 20) x 10-40.3160.945 (138 -JI 15) xa At ambicnt temperature ; solubility of sodium cyanide inH,O is 8.04~ a t 25.4 "C and that in MeOH is 1 .2 4 ~ a t 25.1 "C.obtained on plotting the left-hand side of equation (2) us.V ( C D - ccxc): l91 - a N 1( 3 ) - = - .ci K (c, - ccxc)where ci is the solubility of NaCX in given Igepal CO-530concentrations relative to that in benzene, K is the bindingz6 J. H. Fendler and P.-S. Sheih, J . Phys. Chew., in the press.26 S. B. Brown, P. Jones, and A. Suggett, Trans.Favaday SOC.,1968, 64, 9861975constant between the surfactant and NaCN, c, is thestoicheiometric Igepal CO-530 concentration and CCMOis the critical micelle concentration. The slope of thisline gives 1.42 for N/K and assuming an aggregation numberof 5 for Igepal CO-530 (ref. 25) a value of 3.5 f 1 1 mol-l isestimated for K .Addition of increasing amounts of cyanide ion to thesurfactant solubilized hemin, or that in pure methanol,results in increase of absorbaiices at 432.5 and 424 nm atthe expense of absorptions at 402 nm and 398 nm forIgepal CO-530 and methanol, respectively. Figure 3illustrates typical spectral changes for the interaction ofcyanide ion with liemin in neat and Igepal CO-530 solu-bilized methanol. These data suggest the equilibriumformation of a new species :liemin(ONe,r\leOH)-(S) + 2(CX-) _ _ ,Good relationships have been obtained under the pseudo-first-order conditions (i.e.[CN-] > [hemin],) on plottingthe left-hand side of equation (4) against 1/[CN-]2Khemin(CN),*(S) + Oh/le-/MeOH (3)[hemin] 1 , 1 1 -- ----r A E K E [CN-Izwhere [hemin],, and [CX-] are the originalconcentration of these reactants, A is(4)stoicheiometricthe observedO00Dk/d0/I \ I 360 380 LOO 420 LLO 450hlnmFIGURE 3 Absorption spectra of 6.0 x lO-%-hemin, containing1.0 x 10-4~-sodium methoxide: (1) in neat methanol; (2) in2.8 x 10-3~-NaCN in methanol; (3) in 0*70~-Igepal CO-530 inbenzene; (4) in 0*70~-Igepal CO-530 and 7-2 x 10-4~-NaCNin benzene.Data for the interaction of hemin with sodiumcyanide in the O.70~-Igepal CO-530 benzene system at 432.5 nmare plotted according to equation (5) in the insertabsorbance due to the dicyano-complex, a t the appropriatewavelength, and K and E are the equilibrium constant andmolar extinction coefficient for hemin(CN),*(S). Values of16.4 and 2.28 x lo4 1 niol-l and 56 000 and 60 000 1 mol-lcm-1 have been obtained for K and E in methanol andIgepal CO-530 in benzene, respectively.Alternatively equation (5) can be used where AIln, A ,1 -- -- A,, - A14 - A , IqCN-Inand A , are absorbances d u e to hemin in the absenceof cyanide ion, that in the presence of different amountsof cyanide ion, and that for the hemin(CN),*(S) complex,respectively. A plot of the logarithm of the left-hand sideof equation ( 6 ) us. tlie logarithm of the reciprocal cyanideion concentration (illustrated for the Igepal C0-530-benzene system in tlie insert of Figure 3) gives straightlines with slopes of 2-2 f 0.2 and 1-75 f 0.05, for theinteraction of hemin with cyanide ion in benzene in thepresence of Igepal CO-530 and in neat methanol, re-spectively.These values substantiate the validity ofequation- (3) with respect to theTABLE 2Interaction of hemin with1 04[cN-] 1111 k4ls-l5.00 0.087010.00 0.062020.00 0.1 1040.00 0.24050.00 0.38065.00 0.56097.50 1.24 (0.98)150.0 3-42 (2.18)195.0 4-00308.0 6.82 (6.56)In methanol Qinvolvenient of twocyanide ion600.0 18.28 (18.28)O.TO&r-Igepal CO-530 in benzene c0-479 d0.980 0.001 541.467 d 0.001641.904 d 0.005962.376 d 0.00957.128 0.03615.0 8 0-117 (0.122) f30-0 0.27655.0 0.66060.0 0.874 (0.904) f100.0 1.70120.0 2.87 (2.73) f0.70 hi-Igepal CO-530 in benzene31.0 0.34140.0 0.46250.0 0.585100 1.661 GO 2-90310 6.300-7O~-Igepal CO-630 in benzene0.76 0*001;551.52 0.006901 - 9.98 0.009203.04 0-01443.80 0.02535.00 0.04161.36~-Igcpal CO-530 in benzene1.95 0.004253-90 0.006665.85 0.0 1 6 17.80 0.01989.75 0.026!111.7 0.042321.0 0.14045.0 0-34590.0 0.715120 1.08141 1.35280 3.15Abs.0.0180.0800.1790.2790.3100.3150-3210.3170.32 10.0400.0980.1750.2300.2600.3100.3100.3 150.3250.325In pure methanol, containing 1.0 x 10-4hf-NaOMe.[Hemin] = 6.5 x 10% a t 24.0 "C.Followed a t 424 nm(build-up of the dicyano-complex) unless otherwise stated.b Follou~ed a t 398 nm (decay of parent). c Solution contains1% methanol (vlv), and (8.6-20) x 10-5ni-NaOMe. [Hemin]= 6.7 x 1 0 - 6 ~ ~ . Absorbances determined at 432.6 nm (build-up of dicyano-complex) unless otherwise stated. d 24.6 "C.e 24.8 "C. /Followed decay oE parent at 402 nm. g 6 %MeOH, 1 x 10-4~-OMe. [Hemin] = 5.0 x 1 0 - 5 ~ at 25.0 "C.hSolution contains 0.15:; methanol (v/v), 1.0 x 10-4~-NaOMe and 5.0 x 10-7~r-hcmin. Followed a t 433.5 nm at24-8 "C242 J.C.S. Daltonmolecules of cyanide ion per one molecule of hemin. Valuesof K (15 and 2.4 x 104 1 mol-1) calculated from equation(5) agree, of course, with those obtained using equation (4)(16.4 and 2.3 x lo4 1 mol-1).Depending on the concentration of cyanide ion, theattainment of equilibrium (3) could be followed either byconventional or stopped-flow spectroscopic methods. Thedata are given in Table 2.Equilibrium formation of thehemin(CN),*(S) complex occur in two steps:hemin(OMe,MeOH)*(S) + CN- klk-1hemin(OMe,CN)*(S) + MeOH (6)kak-2hemin(OMe,CN).(S) f CN-hemin(CN),*(S) + -0Me (7)and equations (6) and (7) are related to the associationconstant [equation (3) J by relation (8). Assuming that thesteady-state approximation holds (i.e. d[hemin(OMe,CN)*-(S)]/dt = 0) the observed rate constant for the decayof the absorption due to the disappearance of hemin-(OMe,MeOH)*(S) or that for the increase of absorbance due tothe formation of hemin(CN),*(S), k4, is given by relation (9) :(9)Equation (9) a t high cyanide concentration simplifies to :k4 = k,[CN-] ( 10)= k-2 (11)while at low cyanide ion concentration it approximates to:The data obtained (Table 2) approximate well equation(9) and taking limiting slopes at high and low cyanide ionconcentration (as illustrated in Figure 4) values for k , andk-, are obtained and given in Table 3.Alternatively thedata can be treated according to equation (l2).7 Good-- [CN-] K-,[OMe] 1k4 k,k2[CN-] + h, -linear relationships have been obtained on plotting the1 04[CN-]/~FIGURE 4 Plot of the observed rate constants, k4, for the inter-action of hemin with cyanide ion us.stoicheiometric cyanide ionconcentration. Data are plotted according t o equation (12)in the insert. (Q) I n neat methanol; (m) in benzene in thepresence of 0*70~-Igepal CO-530 and 1.0% (v/v) methanol;(0) in benzene in the presence of 0.70hl-Igepal CO-530 and 5.0%(v/v) methanol; (0) in benzene in the presence of 1 . 3 5 ~ -Igepal CO-530 and 5.0% (v/v) methanolleft-hand side of equation (12) vs. l/[CN-] (see insert inFigure 4), from the intercepts and slopes of which k , andk-,/k,k, are obtained. These values are also given inTable 3.TABLE 3Kinetic and thermodynamic parameters for the interaction of hemin with cyanide ionk l l k l l k2lk-5Conditions 1 mol-l s-lo 1 mo1-1 s-1 b COMel k-,ls-l k,,/s-l a K/1 m o F * K/1 mol-lfMeOH, 1.0 x 10-4MeONa, 365 & 30 385 f 10 (3.6 & 0-6)10-3 (6 f 2)10-, (4 f 0.6)10-2 26.0 f 1.0 16.4 & 0.5O-'lO~-IgepalCO-530in 388 f 20 350 f 60 (3.6 & 0.6)10-2 (3 i.l)IOw4 (7.1 f 0.6)10-4 (2-6 f 0.8)104 (2.3 & 0*5)1024 "Cbenzene, 1.0% MeOHMeONa, 244 "Cbenzene, 0.15% MeOHMeONa, 24.8 "Cbenzene, 6% MeOHMeONa, 25.0 "Cbenzene, 5.0% MeOHMeONa, 26.0 "C(v/v), 1-0 x 10-431-0*70~-Igepal CO-630 in 170 f 30 (3.8 f 0.3)10-2 (8 3)10-4 (1-2 f 0*3)10-3 (1.2 4 0*5)104(VIV), 1.0 x 1 0 - 4 ~ -(v/v), 1.0 x 10-4~-(+), 1.0 x 10-4~-0*7O~-Igepal CO-530in 210 & 20 250 3 30 (1.9 f 0-7)10-2 (2.0 -J= 0.6)10-3 (2.4 -j= 0*3)1031-36hf-Igepal CO-630 in 80 f 5 106 f 20 (5.5 f 0*4)10-2 (2.1 f 0*4)10-3 (2.8 & 0*6)10-3 (2.0 & 0*2)1034% CTAB in H,O2% NaLS in H,O g 3400&- 185 276 x 5.7 x 10-8 16.4 14.4 -J= 43% Triton X-100 610 f 60 1.26 x 2-16 x lo2 1.72 x lo311200 f 2000 (2.66 f 0*2)10-2 (8.8 _t 1-8)104 (5-9 f 1*2)1040 Using equation (10).b Using equation (12). c Using equation (11). From the decay of isolated hemin(CN),-(S). Usingequation (8) and multiplying by [OMe]. f Using equation (4) and multiplying by [OMe]. Taken from ref. 71975Rate constants for the decomposition of hemin(CN) %*(S),K, were obtained independently by dissolving the isolatedcomplex (see Experimental section) in the appropriatesolvent system and following either the decay of absorbancedue to this species or, alternatively, the appearance of theparent peak due to hemin(OMe,MeOH)*(S) as a function oftime.Values of k-, obtained by this method agree wellwith those determined by using equation (11) (Table 3).Values for k-2, using the isolated complex, have also beenobtained in benzene at different concentrations of surfactantand methanol. Table 4 summarizes the data. At constantmethanol concentration increasing the surfactant concen-tration results in an increase in k+ Similarly, at constantsurfactant concentration the rate is enhanced by increasingmethanol concentration. This latter dependency allowsthe calculation of the second-order rate constant for thedecomposition of hemin(CN) z*(S) with respect to methanol.4 LOLL38 -0 89 M-Igepal CC-SSO0 247 wMeCH In benzeneIn 0 7 0 ~ - l g e p o l CO L32430 :- 428- BU'CH-E G26-2 4 2 4 -422- 4-530,l 99v-MeOH in benzenein 2% HaLS in H2O0H20I60 65 70 75 80 85 90 95Z values-FIGURE 6 CorreIation between the absorption maxima of theSoret band of the isolated hemin(CN), complex and solventpolarity parameter 2The obtained value in 0.89M-Igepal CO-530 in benzene,1-02 x 10-3 1 mol-1 s-l, agrees well with 1.6 x 1 mol-1 s-lcalculated for the same reaction in neat methanol (bydividing 24.7). The absorption maximum of the Soretband of the isolated dicyanohemin complex is highlysolvent sensitive.A good relationship is, in fact, obtainedon plotting its absorption maxima against the microscopicsolvent polarity parameter, 2 (Figure 5). Such correlationallows the estimation of the microscopic environment of thehemin complex in the surfactant-solubilized methanol(Figure 6).DISCUSSIONMicelles and surfactant aggregates interrupt hemin-hemin interactions both in aqueous5-7 and in benzenesolutions.It is instructive to compare the magnitudeand site of solubilization of hemin as well as its re-activity in aqueous and non-aqueous surfactant systems.In aqueous anionic and uncharged micelles, hemin ispredominantly solubilized at the micelle-water inter-face.5 Indeed, deep substrate penetration into themicellar core in aqueous solutions rarely occurs.1*Conversely, in benzene hemin is predominantly localizedin the methanol pool which is surrounded by surfactant27 F. M. Fowkes in ' Solvent Properties of Surfactant Solu-tions,' ed. K. Shinoda, Marcel Dekker, New York, 1967, p.65.molecules. Positions of the absorption maxima of thedicyanohemin complex in the different surfactantsystems substantiate this postulate. Using the linearcorrelation obtained between the absorption maxima ofthe Soret band of hemin(CN),*(S) in solvents of differentpolarities 710s. the solvent polarity parameter 2 (Figure 5 ) ,the apparent environment of hemin(CN), complex inaqueous sodium dodecyl sulphate and Triton-X7 iswater like (ie. the extrapolated 2 values are 92.5 and886). Cationic micellar hexadecyltrimethylammoniumbromide intercalated hemin is in an apparently less-polarenvironment than it is in the other aqueous surfactants.Manifestation of the most pronounced micellar effects inaqueous cationic surfactants can, therefore, be rational-ized in terms of deeper substrate penetration.Theextrapolated 2 value for hemin(CN),*(S) in benzene inthe Igepal CO-530 system depends on the concentration(ie. the size) of the methanol pool. The apparentpolarity of hemin solubilized in benzene by 0 . 8 9 ~ -Igepal CO-530 in the presence of 0.24na-MeOH is less thanacetone. Increasing the concentration of solubilizedmethanol or decreasing the concentration of IgepalCO-530 result in an increase of the microscopic polarityof the hemin molecule (Figure 5). Changing the con-centration of the polar solvent can, therefore, bringabout changes in the effective polarities of hemin.Information on the number of Igepal CO-530 moleculesutilized for the solubilization of one hemin moleculehave been obtained from solubility measurements (seeExperimental section).The concentration of micelles,[MI, is given by :(13) CD - COMC CM1 = NSince hemin is completely insoluble in dry benzene,assuming a 1 : 1 interaction between hemin and thesurfactant, its solubility in given surfactant solutionsrepresents the concentration of micelles (Le. hemin isbeing used to titrate the micelles). A good straight linehas been obtained on plotting hemin solubilities, that ismicelle concentrations, against (cD - cmc) (equation(13)]. From the slope of this line a value of 2100 & 400is calculated for N . Although the assumptions involvedin this calculation may not entirely be valid, the numberof surfactant molecules needed to solubilize a heminmolecule is considerably greater than the range ofaggregation numbers quoted for reverse micelles in non-polar solvents27 Evidently such a large molecule ashemin is solubilized in non-polar solvents by beingwrapped around surf actant molecules of some thickness,forming grossly enlarged aggregates.We have en-countered similarly large dodecyl ammonium propionateaggregates in the solubilization of vitamin B12a.28Since no information is available on the shape of sur-factant aggregates containing large porphyrin moleculesin benzene, we are somewhat reluctant to call them' reversed micelles ' in the accepted sense of termin01og-y.~'Lack of data on the size of aqueous micelles in the28 J. H. Fendler, F.A. Nome, and H. Van Woert, J . Amer.Chem. SOG., 1974, 96, 6745J.C.S. Daltonpresence of hemin does not allow comparisons betweenthe extent aqueous and non-aqueous aggregates enlargeupon solubilizing hemin.From the decomposition of hemin(CN),*(S) in benzenesolutions containing different amounts of Igepal CO-530TABLE 4Rate constants for the decomposition of isolatedhemin(CN),*(S) complex in benzene at 24.8 "C[IgepalcO-530]/~ [MeOH]/hr lO3k_,/s1 lo3 k-,/s-l0.890 0.0 0.810.890 0.247 1.030.890 0.494 1.30 1.310-890 0.988 1-800.890 1-98 2.830.175 0.247 0.28 0.320-350 0.247 0 0.40 0.420.700 0.247 C 0.72 0.741-43 0.247 C 0-84 0.850.70 1.235 2.101.35 1.235 2.80Obtained by following the decay of absorbance due t ohemin(CN),.(S).Obtained by following the increase ofabsorbance due t o hemin(OMe,i\IeOH).(S). C In the presenceof 1 x ~ o - ~ M - M ~ o N ~ .(Table 4) the magnitude of substrate-surfactant bindingconstant, K , can be estimated by using equation (14) : l9where kt2 and k_", are the observed first-order rateconstants at given Igepal CO-530 concentrations andin the micellar phase. A plot of the left-hand side ofequation (14) against l / ( c D - cCMC) gives a good straightline from which a value of 0.06 is calculated for N / K .Taking a value of 2100 -+ 400 for N , as obtained fromsolubility measurements [equation (14)], K is estimatedto be (3.5 & 0-5) x lo4 1 mol-l. Hemin is apparentlyappreciably bound to Igepal CO-530 in the benzene-methanol 99.8 & 0.2 (v/v) solvent system.Rate and equilibrium constants for the formation ofhemin(CN),=(S) complex are strongly medium dependent(Table 3).The observed differences among the differ-ently charged aqueous micelles have been rationalizedprimarily on electrostatic grounds.' Dimer formationprecludes, of course, the availability of data in water.Effects of Igepal CO-530 in benzene containing smallvolumes of methanol can be related, however, to that inbulk methanol. It is seen that equilibrium constantsin benzene in the presence of 0.7OwIgepal CO-530 areup to 960-fold greater than that in methanol (Table 3).The magnitude of this enhancement decreases withincreasing volumes of methanol in the Igepal CO-530system. Increasing the size of the methanol pool willnot only decrease the beneficial effect of substrate-surfactant interactions but will likely alter the effectiveactivity of methanol.Analogous and even more pro-nounced effects have been observed in ligand exchangereactions at vitamin B12, in surfactant solubilized waterin benzene.%Effects of surfactant aggregates on the equilibriumconstant is a composite of the individual rate constants[see equation (S)]. Of these, values for k,, k-2, andk2/k-, are available (Table 3). Effects on the rateconstant for the decomposition of hemin(CN),*(S), k-,,are the most straightforward to interpret since it re-presents a unimolecular process. In aqueous micellarNaLS the value for k-, is identical, within experimentalerrors, to that in methanol. Conversely, values for k-,are factors of up to 200-fold smaller in surfactant-solubilized methanol than in methanol (Table 3). Thisrate retardation is likely to be the consequence of en-hanced ground-state stability of hemin(CN),*(S) in therestricted methanol pool. Surprisingly a 10-fold re-duction of the methanol pool (to 0.15% v/v methanol)does not affect further the inhibition of k-, (Table 3).Rate-constant ratios for k2/k-, are enhanced up to afactor of 10 in the benzene-solubilized methanol withrespect to bulk methanol. Enhancements of the rateconstants for cyanide addition to hemin, k,, in aqueousmicellar systems (Table 3) have been largely rationalizedon electrostatic consideration^.^ Absence of surfactanteffects in benzene is necessarily a composite of heminstabilization, and the meagre surfactant cyanide ionassociation.CONCLUSIONThe present results demonstrate the utility of sur-factants to solubilize such a large molecule as hemin inpools of protic solvents in bulk benzene. In manyrespects this system bears analogy to hydrophilicpockets in native proteins and membranes. Theobserved structural and reactivity differences of heminin the bulk solvent and in the cavities of surfactantaggregates question the validity of extrapolatinginformation from dilute aqueous solutions to complexbiochemical systems in a straightforward manner. Weare extending these investigations to other reactionsinvolving hemins as well as other porphyrins.Support of this work by the Robert A. Welch Foulidationand by the National Science Foundation is gratefullyacknowledged.[4/1393 Received, 10th July, 1974

ISSN:1477-9226    

DOI:10.1039/DT9750000238

出版商:RSC    

年代:1975

数据来源: RSC