Quantitative Analysis of Oxonol V Fluorescence in Submitochondrial Particles” JEFFREY c. FREED MAN,"^ TERRI s. NOVAK,~ HARVEY S. PENEFSKY! AND WILFRED D. STEINdpe bDeparttnents of Physiology and dBiochernishy and Molecular Biology State Universiq of New York Health Science Center Syracuse, New York 13210 eSilberman Institute ofLife Sciences Hebrew University Jerusalem, Israel 91904 In recent studies of the Na/K-ATPase, Lauger and colleagues1,* reported that certain fluorescent styryl dyes that had been thought to measure transmembrane voltage actually measure changes in the intramembranous electrostatic potential due to alterations in the charge state of the transport protein during the pumping cycle. We reexamined the changes in oxonol V fluorescence that occur on energization of beef heart submitochondrial particles3; our quantitative analysis raises the possibility that the fluorescence of this dye may likewise monitor electrostatic charging rather than transmembrane voltage. To obtain characteristic curves that constrain the construction of models for the mechanism of dye response, the concentration of submitochondrial particles was varied at constant [dye] (FIG.1) both before and after adding 10 mM succinate to energize the membranes. The top two panels in FIGURE 1 depict the decreases in fluorescence that occur on energization with succinate at 1 JLMoxonol V. At 20 nM Oxonol V, the dye fluorescence increases upon energization (bottom panel). With a model that assumes that (1) a partition coefficient, y, describes the equilibrium of dye between the membrane and the external aqueous phase, (2) the total fluorescence, FT, is the sum of that due to free dye, Ff, and bound dye, Fb, and (3) the molar fluorescence of bound dye, fb, differs from that of free dye, ff, and decreases exponentially with increasing bound dye with a quenching constant k, the derived Eadie-Hofstee type equation for ( F - F,)/c, is: (F - F,)/c, = p(fge-kP - ff)

where p = y c ~ / ( 1+ ycm), cT and c, are the known total [dye] and [membrane], respectively, F, is dye fluorescence in the absence of membranes, F is fluorescence at a given c,, and is the molar fluorescence of bound dye at infinite dilution. The pattern of results for energized submitochondrial particles could not be fitted by Equation 1. Consequently, the partition quench model was modified to include an additional fraction of membrane-associated dye, represented by a second partition “This work was supported by a grant-in-aid from the American Heart Association (J.C.F.) and USPHS grant GM21737 (H.S.P.). ‘Address for correspondence: Dr. Jeffrey C. Freedman, Department of Physiology, SUNY Health Science Center, 750 East A d a m Street, Syracuse, h T 13210. 493

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PROTEIN (pg/ml) FIGURE 1. Fluorescence of Oxonol V before (hollow circles) and after @[led cireles) and the percentage of change in fluorescence (squares) on energization with succinate at varied concentration of submitochondrialparticles. A series of cuvettes, each containing a different membrane protein concentration, was prepared and the fluorescence was recorded at 23°C before and after the addition of 10 mM succinate. (Top two panels) Oxonol V 1 p M , lines are theoretical with the parameters given in the caption of FIGURE2. (Bottompanel) Oxonol V 20 nM, line is theoretical according to Equation 1 with parameters similar to those at 1 p M dye (see text).

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coefficient. The modified expression describing this two partition/quench model is as follows: whereg = yl + y2, andp’ now equalsgcT/(l + gc,,,). The pattern of fluorescence in nonenergized membranes (FIG.2, upper curve) is well described by Equation 1, whereas the triphasic pattern of fluorescence after energization with succinate (lower curve) or with ATP (not shown) is consistent with Equation 2. We examined alternative models (equations not shown) with the following results: a model with one class of dye binding sites gives a plot with 2; a model with one curvature opposite to that found in the lower curve of FIGURE partition coefficient plus a class of parallel binding sites mathematically fits the data 2, but with a physically unreasonable binding constant; a model based on in FIGURE voltage-dependent redistribution of dye between internal and external compartments and with membrane binding predicts increases rather than decreases in fluorescence on energi~ation.~ The derived parameters indicate that the effect of energization is threefold: (1) the partition coefficient increases by 20 times; ( 2 ) the quenching constant k also

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FIGURE 2. Eadie-Hofstee plots before and after energization with succinate at 1 pM Oxonol V. To a series of cuvettes, each containing a different concentration of submitochondrial particles up to 2.4 mg/ml was added 1 pM Oxonol V, and the fluorescence was noted before (hoNow symbols) and after @/Zed symbols) the addition of 10 m M succinate (upper panel). Triangles and squares are from two different experiments. Solid lines are theoretical fits according to Equation 1 (before energization) or Equation 2 (after energization). Without succinate,fF was 885 and k was 3.95; from the curve fits, y was 50 and and fel were 6.7. lo3. With succinate, fF was 855; from the curve fits, k was 87, yl was 261, y2 was 1.2. lo3, a n d f i and fsl were 6.5 . lo3.

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increases by 20 times; and (3) a second partitioning fraction is evident in the triphasic curve at high ratios of dye to membrane (or low values of F - F,). The same derived increases in the partition coefficient and the quenching constant describe the effects of energization for concentrationsof Oxonol V ranging from 20-3 pM. The marked increase in the quenching constant k could arise from dynamic quenching due to a local concentration of anionic Oxonol V around a site of accumulated positive charge. REFERENCES

1. BUHLER,R.,W.S ~ R M E H.-J. R , APELL& P. GUGER. 1991. J. Membr. Biol. 121: 141-161. 2. STURMER, W.,R. BUHLER,H.-J. &ELL & P. LAUGER. 1991. J. Membr. Biol. 121: 163-176. 3. SMITH,J. C., P. Russ, B. S. COOPERMAN & B. CHANCE. 1976. Biochemistry 15:5094-5105. 4. APELL,H.4. & B. BERSCH. 1987. Biochim.Biophys. Acta 903: 48M94.