Langmuir 1996,11, 1038-1040
1038
Notes PhenazineDihydrophenazine Redox Couple as an Inoffensive Catalytic Probe Discerns Premicellar Aggregation in Dilute Aqueous Solutions of Triton X-100
Ri
Ri
@jj
NI
R a o M. Uppu‘
NI
Rz
Rz
AP+
AP’
Division of Biophysics, National Institute of Nutrition, Indian Council of Medical Research, Jamai-Osmania, P.O., Hyderabad-500 007, India Received November 4, 1994
Lindman and Wennerstroml have once commented on the need for “a potentially chemically reactive species in the solution that can diffuse to the micelle where the reaction may be catalyzed” to understand the various dynamical processes going on in a micellar solution. A recent claim that 5-alkylphenazines (Figure 1)exhibit a n altered mechanism of redox coupling between NAD(P)H and tetrazolium salts in the presence of micelle-forming amphiphiles suggests the possibility of a molecular probe that fits into the above d e s c r i p t i ~ n .5-Alkylphenazines ~~~ as a class of electron-transfer catalysts have been extensively used in the spectrophotometric assays of dehydrogenases for coupling the production of NAD(P)H to the reduction of tetrazolium salts to deeply colored f ~ r m a z a n s . ~This - ~ nonenzymatic chemical coupling by phenazines has also been the basis for the activity staining of dehydrogenases after polyacrylamide gel electrophores ~ sthe , ~visualization of NAD(P)H-producinghistochemical reactions,8and the estimation of nicotinamide adenine dinucleotides in tissue extracts by enzymatic r e c y ~ l i n g . ~ J ~ The balance of evidence indicates that in these reactions NAD(P)H first reduces the 5-alkylphenazinium cation (AP+)to 5,10-dihydro-5-alkylphenazine(APH) (Figure 2).11-14 Under aerobic conditions, the dihydrophenazine thus formed undergoes autoxidation, allowing the univalent reduction of 0 2 to the superoxide anion radical (OZ’-) which, in turn, reduces tetrazolium salts by successive one-e- s t e p ~ . ~ J In - ’ the ~ presence of detergent micelles, however, the reduced phenazines, which are sparingly
RZ
APH
Figure 1. Structures of 5-alkylphenazinium cations (AP+) at various reduction and protonation states. R1 = H and R2 = CH3 for 5-metylphenazinium cation (MP+),M P , MPH+, and MPH; R1 = H and Rz = C2H5 for 5-ethylphenazinium cation (EP+),E P , E P H + ,and EPH; and R1= OCH3 and Rz = CH3 for 1-methoxy-5-methylphenaziniumcation (MMP+), MMP’, MMPH+, and MMPH.
Figure 2. A proposed scheme of reactions leading t o the formation of superoxide a n i o n radical during the redox cycling of 5-alkylphenazinium alkyl sulfate (AF’+C,H~,+ISO~-).~ AP+-
NAD (P) H
APH
202
,20;-
(aqueous)
‘I’
t Address for correspondence: The Biodynamics Institute, 711 Choppin Hall, Louisiana State University, Baton Rouge, LA 70803-
APH
(micellar)
1800. (1)Lindman, B.; Wennerstrom, H. Topics Curr. Chem. 1980,87, 1. (2) Rao, U. M. Free Radical Biol. Med. 1989, 7, 513. (3) Experimental evidence has been presented that indicates the formation of a high-affinity, charge-transfer type complex between the micelle-bound dihydrophenazine and the tetrazolium salt: Rao, U. M. Free Radical Biol. Med. 1989, 7, 491. (4) Nachlas, M. M.; Margulies, S. I.; Goldberg, J. D.; Siligman, A. M. Anal. Biochem. 1960,I, 317. (5) Babson, A. L.; Phillips, G. E. A. Clin. C h i n . Acta 1965,12, 210. (6) Michal, G.; Mollering, H.; Siedel, J. In Methods of Enzymatic Analysis; Bergmeyer, H. U., Bergmeyer, J., Grassl, M., Eds.; Verlag Chemie: Basel, 1983; Vol. 1,pp 197-232. (7) Gabriel, 0. Methods Enzymol. 1971,22, 578. (8) Pearse,A. G. E. Histochemistry Theoretical andApplied;Churchill Livingstone: London, 1972; Vol. 2, pp 880-961. (9) Nisselbaum, J. S.; Green, S. Anal. Biochem. 1969,27, 212. (10)Narayanareddy, K.; Belavady, B. Biochem. Med. 1984,32,404. (11) Nishikimi, M.; Rao, N. A,; Yagi, K. Biochem. Biophys. Res. Commun. 1972,46, 849. (12) Ponti, V.; Dianzani, M. U.; Cheeseman, K.; Slater, T. F. Chem.Biol.Interact. 1978,23, 281. (13)Halaka, F. G.; Babcock, G. T.; Dye, J. L. J . Biol.Chem. 1982, 257, 1458. (14) Picker, S. D.; Fridovich, I. Arch. Biochem. Biophys. 1984,228, 155.
RZ
APH t
Ht
\ i
Tetrazolium salt
Formazan
Figure 3. Schematic representation of the altered redox coupling between NAD(P)H and tetrazolium salts by 5-alkylphenazinium alkyl sulfate in the presence of a micelle-forming am~hiphile.~
soluble in aqueous solutions,15J6rapidly migrate into the micellar pseudophase where tetrazolium salts are reduced in preference to 0 2 (Figure 3).3 Exclusion of 0 2 reaction in the micellar pseudophase has been claimed in earlier reports of both electron spin resonance probes of micellar viscosity17and in redox studied8 as well as in photoprocesses such as fluorescence q u e n ~ h i n g . ~ ~ ~ ~ ~ The results presented in Figure 4 demonstrate that the (15) Zaugg, W. S. J . Biol. Chem. 1964,239, 3964. (16)NAD(P)H, tetrazolium salts, and the oxidized phenazines are all highly soluble in water. In fact, the partition coefficients for NAD(P)Hand tetrazolium salts between the aqueous and the micellar phases are shown to be close to 1,suggesting an apparent lack of increase in their concentration within the micellar volume: Uppu, R. M. J . Inorg. .. Biochem. 1995 (in press). (17) McIntire, G. L.; Chiappardi, D. M.; Casselberry, R. L.; Blount, H. N. J . Phys. Chem. 1982,86,2632.
0743-7463/95/2411-1038$09.00/00 1995 A m e r i c a n Chemical Society
Langmuir, Vol. 11, No. 3, 1995 1039
Notes
0.02
0.01
0.03
0.04
Triton X - 100
(Yo, w/v) Figure 4. Effect of Triton X-100on the redox coupling between NADH and NBT by a catalytically low concentration of MP+. The reaction mixture in a final volume of 2.5 mL contained 0.05M sodium phosphate buffer, pH 6.0,50 yM NBT, 85 yM NADH, L O p M MP+,0.2 m M EDTA, and 0-0.04% (w/v)Triton X-100:( 0 )no additional effector and (A) plus SOD (40 units/ mL). The reaction was initiated with the addition of MP+and the appearance of NBT-monoformazan was monitored at 560 nm using a Gilford 250 spectrophotometer equipped with a thermoregulator (25 f 1 "C). Control assays were done by omitting MP+ from the reaction mixture. 02'--independent reduction of tetrazolium salts in the NAD(P)WAP+/Ozsystem is also possible a t concentrations of Triton X-100 much lower than its critical micelle concentration (cmc). In this particular assay, using NADH as a donor of electrons, the reduction of nitroblue tetrazolium (NBT) was studied a t pH 6.0 in the presence of a catalytically low concentration of 5-methylphenazinium methyl sulfate (MPfCH3S04-; 1.0 pM) and varying but low concentrations of Triton X-100 (0.001-0.04%, w/v). Ethylenediaminetetraacetic acid (0.2 mM) was included in the assay to minimize the effects of putative divalent cation impurities. An acid pH was chosen to ensure a rapid spontaneous dismutation of 0 2 ' - , which involves H+ ( 0 2 ' - + OZ'--HZOZ 0 2 ) . As expected, in the absence of TritonX-100, the reduction ofNBT was negligibly small. Addition ofTriton X-100 resulted in a progressive increase in the reduction of NBT until the concentration of the detergent approached the cmc and then tended to plateau off (Figure 4). When MP+ was omitted from the assay, the detergent either alone or in combination with NADH did not bring about any measurable reduction of the tetrazolium salt. Thus, the observed enhancements in the tetrazolium reduction are not consequent on a direct reduction by the detergent or some putative contaminant present in it. Also, since Triton X-100 NADH does not reduce NBT, the contribution of the direct catalytic action of detergent micelles, like the one reported earlier,16r21,22 appears to be negligibly small in these assays. The absence of catalysis by detergent micelles probably is due to the fact that the concentration of tetrazolium salt (50 pM) employed in these assays was much smaller than that used elsewhere.16J1,2zTaken together, these observations suggest that Triton X-100 exerts its action by altering the mechanism of chemical coupling by MP+ as discussed earlier,3 but presumably involving the premicellar aggregate~.~~
+
+
(18)Worsfold, M.; Marshall, M. J.; Ellis, E. B. Anal. Biochem. 1977, 79,152. (19) Turro, N. J.; Aikawa, M.; Yekta, A. Chem. Phys. Lett. 1979,473. (20)Turro, N. J.;Cox, G. S.; Li, X. Photochem. Photobiol. 1983,149. (21)Rao, U.M. Biochem. Int. 1982,5,585. (22)Rao, U.M. Biochem. Biophys. Res. Commun. 1989,159,1330. (23)The formation of small aggregates prior to micellization is often referred to as premicellar aggregation.
0.04
0.08
0.12
0.16
Triton X - 100 (Yo, w/v)
Figure 5. Effect of urea on the NADH-dependenl IP+mediated reduction of NBT in the Dresence of varv low concentrationsof Triton X-100.The iiitial velocity ofreduction of NBT was determined in aerobic mixtures consisting of 0.05 M sodium phosphate buffer (pH 6.0),50yM NBT, 85yM NADH, 0.2 mM EDTA, 0.001-0.16% (w/v)Triton X-100,1 p M MP+, and 0-4 M urea: (0)no urea, (m) 1 M urea, (0) 2 M urea, and (0)4M urea. Other assay conditions are as mentioned in Figure 4. The inset is a secondary plot of the concentrationof Triton X-100at BP versus the concentrationof urea employed in the assay. The velocity curves obtained by plotting the magnitude of tetrazolium reduction against the concentration of Triton X-100 were subjected to break point (BPI analysis as described by Sitaramam and S a m ~ a The . ~ ~concentration of Triton X-100 a t the BP was found to be 0.0180.02% (w/v) (Figure 4). Similar results were obtained when iodonitrotetrazolium was used in place of NBT and when 5-ethylphenazinium ethyl sulfate (0.5 pM) or 1-methoxy-5-methylphenazinium methyl sulfatez5(0.25 pM) replaced MP+in the above reaction (data not shown). Since the concentration of Triton X-100 a t the BP is a close approximate of its cmcz6and since the concentration of solubilized dihydrophenazine (APH) is very low (51.0 pM), the latter being a necessary condition resulting from the limited availability of AP+ (0.25-1.0 pM), it is reasonable to propose that, a t low concentrations of the detergent, certain premicellar aggregates serve as limiting structures for the expression of redox coupling by AP+.At the cmc and above, where an extensive association of monomers commences, leading to the formation of large aggregates (i.e., micelles), the catalytic efficiency of AP+ is expected to tend toward saturation (Figure 4). While a direct experimental verification of the involvement of premicellar aggregates is conceivably difficult to obtain, it is of interest to note that superoxide dismutase (40 units/ mL) did not inhibit the reduction ofNBT a t concentrations of Triton X-100 well below the cmc (Figure 4). Furthermore, inclusion of urea a t a final concentration of from 1 to 4 M resulted in a progressive increase in the concentration of Triton X-100 a t the BP (Figure 5), much in the same way as it alters the cmc of the detergent.27 Urea might alter the degree and the nature of interaction (presumably of hydrophobic type) of reactants with both premicellar aggregates and micelles, since the magnitude of the reaction under the plateau conditions was also dropped significantly (Figure 5).z1 (24)Sitaramam,V.; Sarma, M.K. J. Proc. NatLAcad. Sci. USA 1981, 78, 3441. (25)1-Methoxy-5-methylphenaziniummethyl sulfate is a photochemically stable substitute for MP+ in the activity staining of dehydrogenases (Nakamura, S.;Arimura, K.; Ogawa, K.; Yagi, T. Clin. Chim.Acta 1980,101,321). ItwasagiftfromProf.T.Yagi,Department of Chemistry, Shizuoka University, Shizuoka, Japan. (26)Helenius, A.;McCaslin, D.R.; Fries, E.; Tanford, C. Methods Enzymol. 1978,56, 734 and the references cited therein. (27)Gratzer, W. B.; Beaven, G. H. J . Phys. Chem. 1969,73, 2270.
1040 Langmuir, Vol. 11, No. 3, 1995
Notes
The concept of premicellar aggregation is consistent with the postulate that aqueous solutions of amphiphiles must contain finite concentrations of species of all degrees of aggregation, however narrow there distribution may be.28129 In fact, several lines of evidence have been presented in the literature in support of the formation of premicellar aggregates in dilute aqueous solutions of ionic amphiphiles, based on the measurement of such solution properties as c ~ n d u c t i v i t y ,molar ~~ volume,31 osmotic c ~ e f f i c i e n t ,transport ~ ~ , ~ ~ number,33h y d r o l y s i ~and , ~ ~amphiphile partitioning between aqueous and organic phases.35 In most ofthese studies a doubly charged dimer is considered to be the predominant form ofthe premicellar aggregate. Analyses of kinetic data also suggest the formation of small concentrations of oligomer in these solutions.28 However, it is often very difficult to obtain experimental evidence for aggregates smaller than micelles, particularly for nonionic amphiphiles with low critical micelle concentrations. At 25 "C, a Triton X-100 micelle has an average aggregation number of 140.26,36This high aggregation number suggests the micellization to be a strongly cooperative process. In view of this, and also in view of the fact that commercial preparations of Triton X-100 are somewhat molecularly inhomogeneous with regard to the length of ethylene oxide chains,37 it was thought that premicellar aggregates, if formed, would be predominant over a narrow range of concentration around its cmc.
Contrary to this, the results presented here indicate that premicellar aggregation is possible a t concentrations of the detergent 20-50-fold lower than its cmc (Figures 4 and 5). The suppression of the reactivity of dihydrophenazines toward 0 2 lends further support to the existence of premicellar aggregates (Figure 4). A demonstration of premicellar aggregates without perturbing the system, such as the one reported in this system, becomes significant in view of the growing interest in the electron-transfer reactions catalyzed by ionomolecular aggregates (these are induced premicellar aggregates formed between ionic amphiphiles and anionic or cationic dyes) as a promising means of conservation of solar energy p r o c e s ~ e s . ~Similarly, ~ - ~ ~ there are claims of enhanced water solubility of certain extremely waterinsoluble organic solutes in the premicellar region of nonionic amphiphiles3' that may provide newer strategies for environmental decontamination of sparingly soluble solutes. It is hoped that a detailed study of the cyclic oxidationheduction reactions of phenazines in various systems may add to our understanding of the physicochemical characteristics of the premicellar aggregates formed and their involvement in catalysis.
(28)Aniansson, E. A. G.; Wall, S. N.; Almgren, M.; H o f h a n n , H.; Kielmann, I.;Ulbricht, W.; Zana, R.; Lang, J.; Tondre, C.J . Phys. Chem. 1976,80, 905. (29)Vold, M. J. J . Colloid Interface Sci. 1986,116,129. (30)Mukerjee, P.(1958)J . Phys. Chem. 1958,62,1404. (31)Franks, F.; Quickenden, M. J.; Ravenhill, J . R.; Smith, H. T. J. Phys. Chem. 1968,72,2668. (32)Walton, H.F.; Hiebert, E. N.; Sholtes, E. H. J.Colloid Sci. 1946, 1, 385. (33)Mukerjee, P.J . Phys. Chem. 1968,62,1397. (34)Lucassen, J. J . Phys. Chem. 1966,70,1824. (35)Goodman, D. W. J.A m . Chem. Sac. 1958,80,3887. (36)Robson, R. J.; Dennis, E. A. J . Phys. Chem. 1977,81, 1075. and (37)Kile, D. E.;Chiou, C. T.Enuiron. Sci. Technol. 1989,23,832 the references cited therein.
LA940872C
Acknowledgment. I thank late Dr. S. G. Srikantia, Dr. B. Sivakumar, and Dr. William A. Pryor, for helpful discussions, and Dr. V. Reddy, Director, National Institute of Nutrition, for supporting this study.
(38)Yamagishi, A. J . Colloid Interface Sci. 1981,86, 468. (39)Sato, H.; Kawasaki, M.; Kasatani, K.; Ban, T. Chem. Lett. 1982, p. 1139. (40)Atherton, S. J.; Dymond, C. M. G. J . Phys. Chem. 1989,93, 6809. (41)Kusumoto, Y.; Watanabe, J.; Kurawaki, J. Chem. Express 1989, 4 , 153. (42)Jiang, Y.; Wu, S. Huazua Xuebao 1990,48, 447. (43)Neumann, M.G.; Gehlen, M. H. J . Colloid Interface Sci. 1990, 135,209. (44)Barber, D. C.;Freitag-Beeston, R. A. J . Phys. Chem. 1991,95, 4074. (45)Niu, S.;Gopidas, K. R.; Turro, N. J . Langmuir 1992,8, 1271.
