Competitive Binding of Atp and Acridine Orange by Muscle.

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 aqueous solution, go into triplets if the solution 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 of certain dyestuffs in aqueous solution may lead to reversible molecular aggregations in the form of dimers and polymers, which, on excitation, 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 Zanker.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 (10-3-10-4 M) shows a vivid green color, which turns gradually into red as th'e concentration 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 electropolar bonds unaffected. Accordingly, addition of alcohol or acetone to a concentrated aqueous solution of acridine orange causes a change in color from red to green, as observed under the ultraviolet lamp, dispersion forces being responsible 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 dimerizes 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 10O3 M acridine orange solution, and placed, after a while, on filter paper, it is found to show an intense red color if illuminated with a high-pressure mercury lamp, the visible light of which is eliminated by an appropriate 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.