Abstract. The emission spectra of polar aromatic molecules in rigid, polar solution are shown to depend on the exciting wavelength. Occurrence of the phenomenon depends on both the excited-state lifetime of the chromophore and the degree of rigidity of the medium. The results are interpreted in terms of a model which stresses the contribution of micro-environmental heterogeneity to electronic absorption and emission spectra.Shifts in chromophore electronic absorption and emission spectra as a function of the polarity and polarizability of the solvent are well known and have been interpreted by Bayliss and McRae in terms of permanent and induced dipolar interactions between a molecule in its ground and excited states and the solvent.' The interactions have been qualitatively treated as occurring between the chromophore and a general molecular environment. For aromatic amino acid residues in proteins2-4 and for aromatic hydrocarbons trapped in polycrystalline matrices' a considerable body of evidence exists that indicates that chromophores of the same species can have different electronic energies as a consequence of the existence of several distinct local environments. Even in a homogeneous phase, solute molecules can be expected to occupy a variety of solvation sites at any given time, generating an array of electronic transition energies whose summation comprises the absorption or emission spectrum of the sample as a whole. Such variation in solute-solvent local interactions is thought to be a significant source of the broadening of the absorption and emission spectra of a chromophore in a condensed phase.In the present article we provide evidence for the contribution of microenvironmental heterogeneity to solution spectra through our observation of an exciting-wavelength dependence for fluorescence and phosphorescence spectra of aromatic molecules in dilute rigid solution. In exploring the consequences of this environmental heterogeneity we show that it provides a rationalization for the failure of fluorescence concentration depolarization at the red edge of the solute absorption band7-9-the so-called "Weber red edge effect." 10 The Model. No attempt is made in this paper to present a quantitative model for the contribution of environmental heterogeneity to electronic spectra. We only emphasize three qualitative assumptions: (1) that electronic energies of chromophores in solution are a function of geometry-dependent solute-solvent interactions, (2) that at any instant in time there is an ensemble of interac-1116

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The disappearance of the exciting-wavelength dependence of the phosphorescence spectra of polar, aromatic chromophores in supercooled glycol-water mixtures is utilized to monitor the kinetics of solvent reorientation. Reorientation times in the nanosecond to second range are obtained for (3:2 v I v) glycerol-water and (1:1 vlv) ethylene glycol-water at I40-240 'K. The results suggest that the process is one involving a cluster of solvent molecules and in which the chromophore plays a relatively passive role. Steady-state data and direct measurements of phosphorescence shifts as a function of time indicate that the solvent reorientation process is nonexponential in nature. The decay function derived from the temperature dependence of the steady state data is consistent with the decays observed directly as a function of time. Interpretation of this nonexponential decay in terms of a summation of rate processes leads to a distribution dominated by two reorientation rate constants. The relative contributions of the slow and faster reorientation rate constants in addition to their activation parameters differ for the glycerol-water and ethylene glycol-water mixtures.

Heavy atoms, such as bromine or iodine, perturb the excited-state properties of aromatic chromophores through a spin-orbital coupling mechanism. In the present work the use of specifically directed spinorbital probes to study subtle structural relationships in biopolymers is described. Heavy atoms are introduced into defined sites in biochemical systems and the emission spectrum of a ligand or intrinsic chromophore is monitored for perturbation by the bound heavy atom. This technique is illustrated by a study of acridine dye binding to the copolymer poly(dA-BrdU). The results are interpreted in terms of an "externally" bound dye fraction whose emission is perturbed by the heavy atom in the polymer and an intercalated dye component unperturbed by bromine.The perturbation of electronic excited-state properties of aromatic molecules through a spin-orbital coupling mechanism, mediated by solvent molecules possessing certain heavy atoms as constituents (the so-called "external heavy-atom effect"), was initially observed by Kasha (1). Since that time the influence of solvents containing heavy atoms on the spectroscopic properties of simple molecules has been studied in some detail (2-4). Experimentally, the most accessible manifestations of the external heavy-atom effect are decreases in fluorescence efficiency and phosphorescence lifetime, and an increase in the phosphorescence efficiency of the aromatic molecule. The first of these, fluorescence quenching, has been exploited in biochemical preparations by several investigators who have observed partial fluorescence quenching due to the exposure of a fraction of the emitting molecules to a solvent containing halide ion. This solvent perturbation approach has been used to estimate the number of tryptophyl residues exposed to solvent in proteins (5, 6), and it is capable of distinguishing free proflavin from the dye complexed with DNA (7). Perturbation of thymine phosphorescence by mercuric ion in stoichiometric mercury-polynucleotide complexes has also been reported (8).In The copolymers consisting of alternating adenine and 5-bromouracil deoxyribonucleotides, poly(dA-BrdU), and alternating adenine and thymine deoxyribonucleotides, poly(dAdT), were purchased from General Biochemicals. Poly(dABrdU) was also prepared in this laboratory in a synthesis primed by poly(dA-dT) after the procedure of Riley and Paul (22), with Escherichia coli DNA polymerase generously provided by Dr. David Denhardt, Dept. of Biochemistry, McGill University. Proflavin was a K and K product. Acridine orange was obtained from Eastman. All other materials were of reagent grade. Samples consisted of 0.2-0.3 mM poly(dABrdU) in 0.01 M cacodylic buffer (pH 7.5) diluted 1:1 with an ethylene glycol or glycerol solution of dye. Ionic strengths were adjusted with sodium chloride. Spectra and lifetimes were recorded at 77°K on an instrument already described (23). RESULTSThe changes in phosphorescence that characterize an external heavy-atom perturbation are exemplified by the influence of ethyl b...

A DNA-dependent ATPase formed after T4 phage infection is purified to apparent homogeneity. The molecular weight of the purified enzyme is SO 000 when determined by glycerol gradient centrifugation and by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The enzyme at an earlier stage in purification (prior to DEAE-cellulose chromatography) exists as a complex with a molecular weight of 100000. However, molecular weight determinations by Sephadex gel chromatography give considerably decreased molecular weights for the complex and for the enzyme after DEAEcellulose chromatography. The enzyme is stimulated to varying degrees by a variety of singlestranded polydeoxyribonucleotides or by single-stranded DNA, but no chemical change in the polynucleotide has been detected as a result of the enzyme action.In a preliminary report we described the finding of a DNA-dependent ATPase from extracts of Escherichia coli infected with bacteriophage T4 [l]. This enzyme catalyzes the hydrolysis of ATP to ADP and inorganic phosphate in the presence of heat-denatured DNA without detectable nuclease activity. A number of DNA-dependent ATPases have subsequently been found in E. coli [2 -51 and in eukaryotic systems [6,7]. However, it is not clear how ATP hydrolysis is coupled to an overall enzymatic reaction or how it participates in various functions such as DNA synthesis [3, .This communication described the purification of a T4 phage-induced DNA-dependent ATPase, as well as new studies on the interaction of the enzyme with DNA. These studies were made to illuminate the nature of the DNA requirement and its functional significance. However, the function of the enzyme in vivo remains inexplicable in terms of its known catalytic properties. Therefore we have considered the possibility that the enzymatic reaction as studied represents a partial reaction for which the 'complete' reaction is only evident with a larger physical complex or that the enzyme might participate in a coupled reaction in a more complex medium. We have not succeeded in finding this hypothetical 'complete' reac-