Kinetics of Fluorescence Fading of Acridine Orange‐heparin Complexes in Solution

Abstract

The irreversible photochemical fading of fluorescence of acridine orange-heparin complexes indicates that fading involves the reaction of a "reactive" excited bound dye dimer with one in the ground state. A kinetic parameter, r", is derived, which has a constant value over a considerable range of conditions, and which is directly related to rate constants for photophysical and photochemical processes. This parameter appears to be characteristic for heparin and may serve to identify it in the presence of other glycosaminoglycans.

Structural basis for the anticoagulant activity of heparin. 2. Relationship of anticoagulant activity to the thermodynamics and fluorescence fading kinetics of acridine orange-heparin complexes

“…Evaluations of the fading parameter /-" (Menter et al, 1978) were carried out at [AO]0 = 8 µ for the heparin samples and at 10 µ for the dermatan sulfate sample. The polymer-binding site to dye ratio was 6.6 in all cases.…”

“…The polymer-binding site to dye ratio was 6.6 in all cases. These concentrations ensured that the photochemical fading, and not AO-AMPS complex formation, was rate determining [see Menter et al (1978)]. Thus, the fading constant, r", is independent of AO binding.…”

“…Firstly, among the various GAGs, differences in the value of the cooperative binding constant, ATq, primarily reflect differences in the electrostatic properties of the polymer (Menter et al, 1977(Menter et al, , 1979a. Secondly, the photochemical fading of AO-GAG fluorescence under continuous irradiation with exciting light involves a second-order photooxidation of AO in which two adjacently bound AO molecules react in the rate-limiting step (Menter et al, 1978(Menter et al, , 1979b. Because of the involvement of two dye molecules in the slow step, the rate of fluorescence fading (r") depends critically on the geometry and conformation of the binding sites.…”

Photochemistry of Heparin‐acridine Orange Complexes in Solution: Photochemical Changes Occurring in the Dye and Polymer on Fluorescence Fading

“…Preparation of solutions, treatment of glassware, measurement of absorption and corrected emission spectra. as well as fluorescence fading measurements, have been previously described (Menter et a[., 1978a). Heparin concentrations are based on uronic acid assays (Bitter and Muir, 1962).…”

“…Since the fading parameter r" is sensitive to polymer structure and conformation (West rt al., 1976;Menter et al, 1978a), photochemically-induced conformational and structural changes in the polymer Volume Eluted (ml) Figure 4. Gel filtration profiles on Biogel P-30 (0.6 x 70 cm column) of heparin recovered from photolysis.…”