The binding of proflavine to poly(cc-L-glutamic acid) a t p H 7.5 has been investigated by means of absorbance measurements a t 444 nm. Equilibrium properties were registered as a function of the total dye concentration and the polymer to dye ratio. The results agreed very well with the predictions of a basic model theory of binding to a linear lattice where cooperative interaction is restricted to nearest neighbor binding sites. The parameters g (number of binding sites per amino acid residue), K (cooperative binding constant), and q (factor measuring the strength of cooperativity) could be determined for various concentrations of added KC1 electrolyte. While g of approx. 1.1 and q of approx. 200 were found to be little affected by increasing the ionic strength, a considerable decrease of K was observed. The latter is interpreted as the effect of competitive inhibition due to non-cooperative binding of potassium ions (Kz = 160 M-I).Extrapolation to zero electrolyte concentration yields K -+ KO = 8 x lo4 M-I. It is concluded that the binding between the ligands and the polymer is of essentially electrostatic nature. Cooperative interaction, on the other hand, must be attributed to the stacking tendency of neighboring bound dye molecules.

The binding of acridine orange to poly(α‐l‐glutamic acid) at pH 7.5 has been investigated by means of absorbance measurements at 492 nm. The equilibrium as well as chemical relaxation behavior is interpreted in the light of a basic model theory of cooperative binding. Binding and cooperativity are found to be stronger than in the previously studied case of proflavine. In principle, the static properties are analogous in both cases. The only distinct contrast is given by the fact that the degree of cooperativity is considerably affected by ionic strength and polymer to dye ratio. This is attributed to the effect of unfavorable electrostatic interactions which tend to prevent long aggregates of bound dye. Temperature jump measurements resulted in relaxation curves which agreed very well with the predictions of the theory. Mean relaxation times of cooperative binding could be determined and evaluated in terms of an effective rate constant for recombination of free dye with bound aggregates of dye. The effect of varying the concentration of added KCl can be attributed to blocking of reaction sites by bound K+ ions. Extrapolation to zero KCl concentration yields a diffusion controlled rate constant for cooperative binding of monomer dye (kRo is approx. 1.5 × 109 M–1 sec–1). The life time of terminal elements of bound aggregates of dye turns out to be about 400 μsec.

~-Glyceraldehyde-3-phosphate dehydrogenase has been reacted with spin labels, which were designed to elucidate the geometry of the active site of the enzyme. Three series of spin labels were used, which represented either substrate and coenzyme analogs or irreversible inhibitors, the separation of the active site directing part from the nitroxide radical being readily adjusted through the length of the intervening methylene chain.The effect of this variation on the electron paramagnetic resonance spectrum of the labeled protein as well as the effect of coenzyme binding on these spectra have been investigated. All spectra are complex and represent the superposition of a t least three different types of spectra. These three types of spectra are correlated to nitroxide radicals in three different types of motion: (a) very fast rotation in a fluid region, (b) medium fast rotation of a hindered radical and (c) slow rotation of an almost immobilized radical. The spin labels differ only in the relative population in the three states related to the three types of motion. Also addition of the coenzyme NAD only alters this relative population. A spin labeled coenzyme analog, which has the nitroxide in the region of the sugar-nicotinamide bond, is bound to the protein and displays an electron paramagnetic resonance spectrum typical for an immobilized radical. The geometry of the active of ~-glyceraldehyde-3-phosphate dehydrogenase is complex and cannot be described as a simple cleft, but instead as a system of narrow and wider spaces, which is changed by the binding of the coenzyme.The spin labeling technique has been introduced by McConnell [l]. It has been applied to a wide spectrum of problems and has been especially successful in biochemical and biological applications. Reviews on spin labels have appeared [Z, 31. Spin labels have been used in biochemistry to detect conformational changes of haemoglobin [3,4], aspartate transcarbamylase [5] as well as to study the activity [6] and the geometry of the active site [7] of ol-chymotrypsin. Spin labels were used in this work in an attempt to determine conformational and geometrical properties of the tetrameric enzyme D-glyceraldehyde-3-phosphate dehydrogenase. ~-Glyceraldehyde