BY GEORGE KARREMAN, HELMUT MIJELLER, AND ALBERT SZENT-GP~RGYI INSTITUTE FOR MUSCLE RESICARCH, MARINE BIOLO(:ICAL LABORATORY, WOO%3 HOLE, MASSACHUSETTS ~~~~~r~~~a~ed farce .I 1, 1967 Introduction.-The lifetime of electronic singlet excitation in molecules is too short to allow the utilization of these excitational energies in biological systems.' Triplet excitations last longer, but the probabilities of their occurrence are small. Circumstances which increase the probability of these transitions may thus have biological importance. It has been shown' that dyes and certain other substances which are excited to singlets by ultraviolet light, in their dilute aq~~eous solution, go into triplets if the solu.tion is frozen. This transition may be due to changes taking place in the solvent or in the solute, or in both. It can be assumed that freezing causes crystallization of the solvent and with it a consequent increased local concentration of the solute. Increasing the concentration af certain dye- stuffs in aqueous solution may lead to reversible molecular aggregations in the form of dimers and polymers, which, on ex~itat`~o~~, may tend to go into the triplet state. That this is the case for acridine orange has been demonstrated in particular by the work of Zanlier.2 Acridine orange is especially suited for the study of these relations, since it shows a green fluorescence under ultraviolet light in singlet, and a red phosphorescence in triplet, excitation. Accordingly, a dilute aqueous solution (1O-3-1.O-J M) shows a vivid green color, which turns gradually into red as the coll~ent,ratioll of the dye is increased and polymers are formed. The nature of the bond between two molecules of the acridine orange dimer can be decided by the behavior in organic solvents like alcohol or acetone, which will dissociate complexes held together by dispersion forces,3 while they leave electro- polar bonds unaffected. Accordingly, addition of alcohol or acetone to a concen- trated aqueous solution of aeridine orange causes a change in color from red to green, as observed under the ultra~~iolet lamp, dispersion forces being respolls~ble for the bond holding the dimers together. As is being shown in this laboratory, ATP forms a red phosphorescent complex with acridine orange, as does adenine, indicating that it is the purine part of the molecule which dim.erizes with the dye. Thus the dye can also form complexes with molecules of a different substance. Dimers formed by two equal molecules are, in this laboratory, called "homodimers," while dimers formed by two different molecules are called "heterodimers." If the psoas muscle of the rabbit, extracted with glycerol,4 is washed out with water, then suspended in a 10p3 M acridine orange solution, and placed, after a while, on filter paper, it is found to show an intense red color if illuminated wit,h a high-pressure mercury lamp, the visible light of which is eliminated by an appropri- ate filter. The dye seeping out into the underlying filter paper is green. This indicates that the dye has been bound by a muscle constituent in the form of a complex, a heterodimer, which goes into the triplet state on excitation. A drop of alcohol placed on the muscle fiber changes the red color into ,green,, showing that dispersion forces underlay the formation of the complex. ( "' 373 374 3~~~~EMI~T~Y: KARREMAN ET AL, PROC. N. A. s. It has been suggested6 that ATP energetiaes the muscle by converting the bond energy of its high-energy phosphate into an excitation energy on its purine ring, then transmitting this excitation energy to the contractile protein, How this transmission from the purine to the protein takes place is left open. The experi- ments with acridine orange suggest the possibility that, like the dye, the ATP molecules also form a complex with a ~onstituer~t of muscle, probably a group on the protein-a complex capable of triplet, excitation. This excitation of the ATP- protein dimer could represent the transmission of the energy of the nu~leotide on to the protein. The formation of complexes between ATP and arridine orange, on the one hand, and the formation of complexes between acridine orange and muscle, and between ATP and muscle, on the other hand, also suggest that dye and nucleotide might, be bound by the same atomic groups of the ~~~l~tl,a(It,ile matter. If ATP and the dye react with the same atomic groups, then t,hc dye should inhibit the contraction induced by ATP, and this inhibition should be a competitive one. Experiments.--A thin bundle of glycerol-extracted psoas muscle fibers,5 approx- imately 0.25 mm. in diameter, was cut in two in the middle. One half was allowed to contract in. an ATP solution and served as a control for the other half, which contracted in a bath containing an identical ATP concentration but also containing acridine orange. This met,hod eliminated errors due to variations among di~ere~~t fiber bundles. Contraction (shortening) was plotted against time. Acridine orange concentrations from lo-* to lo--" M were studied. Hy varying the ATP ~on~eI~trat'ior1, one set of experiments was performed for each acridine orange concentration employed. Since all experiments showed a uniform pat'tern, only a few representative examples will be des(~ribed here. Figure 1 illustrates the inhibitory effect of lo-" M acridine orange on fibers con- tracting in a 2, 4, 8-, and lo-~nillimolar ATP solution. It can be seen that with 2 mM AT1 the contraction is almost completely inhibited. Rising ATP concen- trations diminish this inhibitory effect, and with 1 G mM ATP, inhibition is minimal. Similar experiments performed with lower acridine orange concentrations (10--3, lc .6o `7;: 0 .40 t O 0 Ooo .20 i_ -L-L-I 0 0 0 * 8 8 0 0 0 0 0 0 e 0 8 O 0 moo O 0 0 @e 0 0 * Ooo mins. 0 5 IO 15 20 5 IO 15 20 5 IO 15 20 5 IO I5 20 0) W cl 4 FIG. I.-Inhibition of isotonic contraction by 100~ M acridine orange. IEffect of rising ATP concentrations: a, 2 mM ATP; 6, 4 ml% ATP; c, 8 mM ATP; d, 16 mM ATP. Pull circles: fiber contracted in presence of IO+! M acridine orange; open circles: fiber contracted in absence of acridine orange, 1,: rest length; 1,: contracted length. VOL. 43, 1957 B~~~~~~~~TRY: KARREMAN ET AL. 375 5 X lO+, lO+ I@) showed only quantitative differences: a lesser degree of in- hibition, which is further diminished with rising ATP concentration. The inhibitory effect of a certain acridine orange concentration in the contraction bath was increased by incubating the fiber with the dye prior to contraction. Even short incubations of one minute and less were effective; longer incubations produced more pronounced inhibition. Rising ATP concentrations in the con- traction bath diminished this additional inhibition. The pattern illustrated in Figure 1 was observed in all experiments. IVjth acridine orange concentrations as low as 10m4 &I, in a contraction bath with 2 mM ATP, inhibitory effects were no longer shown. Even 20 minutes' il~cubatjo~l of the fiber in this dye con(~e~tration failed to produce an ir~hibitory effect for ATP concentrations of 2 mM and more (see Ifig. 2, a). However, in the weaker and slower contractions obtained with 0.2 mM ATP, the inhibitory effect of IO-* M acridine orange was again apparent (Pig. 2, b), mins. 0 5 IO 15 20 0 5 to I5 20 4 b) FIG. Z,--`Effect of 10d4 M acrid&e orange on isotonic contraction: a, with 2 mM ATP; b, with 0.2 mM ATP. Full circles: fiber con- tracting in IO+ 1cf acridine orange after 20 minutes' in~uba.tion; open circles: control fiber in absence of acridine orange. I, and I, as in Fig. 1. These opposite effects of ATP and acridine orange on the rate of shortening and degree of maximal contraction have been observed in all experiments. They seemed to suggest a competitive inhibition, i.e. a competitive adsorption of dye and ATP. Such a process would be analogous to the competitive adsorption of a sub- strate on its enzyme in the presence of a competitive jr~h~bitor and should show characteristic quantitative relations. The formal and qua~ltitative analysis of our experimental data should clarify this question and is presented below. The reversibility of the acridine orange action could also be demonstrated by contracting acridine orange-incubated fibers in an ATP solution free of dyestuff. Here the rate of shortening was slowed, but finally the same maximal contraction was developed as by the nonincubated control fibers, Figure 3 illustrates two examples of these experiments. Maximal contraction was reached more rapidly with increasing ATP and more slowly with increasing dye eon~entrat,ion or pro- 376 UiOCIHBMiS1'JiY: KAJhU'MAN JU' AL. I'lwc. N. A. s. longation of the time of incubation. This excludes a denaturation of the contractile proteins by the dyestuff under the experimental conditions employed. 1.00 .80 .60 , r lc 0 II .40- .O 00 oh* 20 - I---L mins 0 5 IO 15 20 a) --l----L- O 5 IO 15 20 30 b) FIG. 3.---Fibers incubated in acridine orange prior to contraction. The rate of shortening is decreased, while the degree of maximal contraction is not impaired. a, 2 minutes' incubation in 10-a M acri- dine orange;. b, 2 minutes' incubation in lop2 M acridine orange. Pull circles: incubated fiber; open circles: nonincubat,ed control fiber. ir and I, as in Fig. I. Discussion.-In the case of competitive inhibition of a reaction, as, for example, that of an enzyme E with a substrate S and that of the same enzyme with an inhibitor I, ES and EI denote the enzyme-substrate and the enzyme-inhibitor complexes, respectively. The velocity ZJ of the enzymic reaction is proportional to the con- centration of ES. As is well known, if the reciprocal of the velocity of the enzymk reaction is plotted against the reciprocal of the substrate concentration, we obtain, in the case of competitive inhibition, a straight line which cuts the ordinate (l/v) axis at a point which is independent of the inhibitor concentration, I. However, the slope is dependent on 1. Consequently, the straight lines for different values of 1 cut the (l/v)-axis at the same point, whereas the slope is larger, the larger the value of I. In the present case of muscle a constituent X corresponds to E, ATP to S, acridine orange (AO) to I. Thus X + ATP ~=f X(ATF'), X + A0 -d X(A0). If we call K the equilibrium constant of the reaction of X and ATP, and K' that of the reaction of acridine orange and X, we have VOL. 43, 1957 BIOCHEMISTRY: KARREMAN ET AL. 377 and coc3 K, C4 > M in which Co represents the concentration of the free X, C1 that of free ATP, Cz that of X(ATP), Cs that of AO, and Cd that of X(A0). Furthermore, calling X0 the total concentration of X, which is equal to the sum of that of t.he free X and that of the bound X in X(ATP) as well as in X(AO), we also have cc + c2 + Cd = x,,. Simple algebraic manipulation gives, from equation (l), and substitution of equation (4) into equation (2) leads to K CzG "`2+-c. 1 (5) Introduction of equations (4) and (5) into equation (3) yields, after some simple rearrangements, c2 ( K c + 1 + g, $ = x0, 1 1 > from which we obtain X0 " = y--i K/C1 + (K/K') (C,/C,j ' 01 Cl " = cl + K(1 + C3/K') xo* (f-9 Since, experimentally, the length of the muscle fiber was studied as function of time, the natural and most simple assumption which can be made here is that the shortening in the final (equilibrium) state is proportional to the concentration, C2, of the X(ATP) complex: I?, - I, = cd& (7) in which Z, is the rest length, I, the length of the muscle fiber in the final (equilibrium) contracted state, and cr a proportionality constant. From equation (6) we see that the concentration Cz is a (hyperbolically) increasing function of C1 with saturation value X0. Therefore, assumption (7) leads to 1, - L, = &X0, (8) 378 BIOCHEMISTRY: KARREMAN ET AL. PROC. N. h. s. in which I,, is the length of the maximally contracted fiber in its final (equilibrium) state, as occurs with a (infinitely) large concentration of ATP. Elimination of CY from equations (7) and (8) yields I, - lnzc x0 1, - I, = c:! ' or 1 1 X" ___ = -~- -, 1, - I, 1, - L c`2 Simple rearrangement from equation (6) leads to x0 -- = c, Introduction of expression (10) into equation (9) gives (9) (10) from which we see that the intercept of the plot of the reciprocal of the shortening, l/(1, - LJ, versus that of the ATP concentration, l/C,, should yield a straight line with an intercept equal to the reciprocal of the maximal shortening, l/(L - lmc), which is independent of the inhibitor concentralion Ctj, and a slope [1/(L7 - Lmc)]- K(1 + G/K') h h w ic is a linear function of the inhibitor concentration Ca, as might have been expected from what has been said before about the competitive inhibition in general and assumption (7). Without inhibitor, equation (11) leads, with Cs = 0, to (12) The plot of the value of l/(lr - 1,) versus l/C, for the experiment without acridine orange indeed yields a straight line, as shown in Figure 4, fitted by the least-squares method. From the intercept we obtain l/(& - I,,) = 1.14, leading to l,, = 0.124 (lr is taken as unity), so that the maximal contraction with a very large concentration of ATP is 87.6 per cent. For the slope we find 7.37 X lo-", from which we obtain with the above-mentioned value of l/(& - Z,,) the value 6.5 X low4 for K. Similarly, the plot of l/(& - L) versus l/C,, for an experiment containing acridine orange in the concentration lo-m2 M, yields a straight line, as shown in Figure 4. Fcr this line we find, by the method of least squares, the value 1.11 for the intercept I/(1, - I&, , h h w ic value is the same within the experimental ac- curacy as that found in the experiment without dye. The latter result proves that, there is competitive inhibition of ATP and acridine orange for the same site of muscle constituent X. Also from the slope 5.28 X 1O-3 of the last straight line, from the above values of J/(1, - I,,) and K, and from the concentration Ca = lop2 M of acridinc orange, we find the value 15 X 10B4 for K', which shows that the dye is bound by the same site as ATP but is bound somewhat more than twice as strongly. VOL. 43, 1957 BIOCHEMISTRY: KARREMAN El' AL. 0 too 200 1 300 400 500 -5 FIQ. 4.-Reciprocal value of the final shortening plotted against the reciprocal value of the molar concentration of ATP. Open circles: in presence of 10 2 M acridinr: orange. fiber in absence of acridina orange; f&l circles: fiber lc and I,. as in Fig. 1. Summary.--,4cridille orange and ATP form complexes with the same muscle constituents. The formation of the ATP complex is an essential step in the energetization of muscle. Acridine orange inhibits contraction, and this inhibition has the properties of a competitive absorption. Dye and ATP molecules compete for the same site. * This research was sponsored by the National Institutes of Health, Grant No. H2042(R), the Commonwealth Fund, the American Heart Association, Inc., the Muscular Dystrophy Associa- t.ions, the Association for the Aid of Crippled Children, the National Science Foundation, and the IJnited Cerebral Palsy Foundation, l A. Szent-Gyorgyi, Bioenergetics (New York: Academic Press, Inc., 1957). 2 V. Zanker, 2. physik. Chem., 199, 225, 1952. 3 E. Rabinowitch and 11. F. Fpstein, J. Am. Chem. Sot., 63, 69-78, 1941. 4 A. Szent-Gyorgyi, Biol. Bull., 96, 140, 1949. 6 A. Szent-Gyorgyi, Henry Ford Hospital Symposium.. Enzymes, Units of Biological Structure and Function, ed. Oliver H. Gable (New York: Academic Press, Inc., 1955).