Spectral analysis of interactions between proteins and dye ligands

✍ Scribed by J. Hubble; A.G. Mayes; R. Eisenthal

• Publisher: Elsevier Science
• Year: 1993
• Tongue: English
• Weight: 926 KB
• Volume: 279
• Category: Article
• ISSN: 0003-2670
• DOI: 10.1016/0003-2670(93)85080-4

✦ Synopsis

Interactions between the triaxine dye Cibacron Blue F3GA and the protein lysozyme have been investigated by monitoring the characteristic spectral shift which accompanies the binding phenomenon. This technique, which is both quick and convenient, allows determination of apparent dissociation constants, and in the case of supported dyes, the fraction of total dye available for protein binding. In this paper, this approach has been used to compare experimental results obtained with both free and dextran coupled dye allowing the effects of dye conjugation to be quantified in the absence of diffusional limitations seen with insoluble supports. In addition to equilibrium studies, changes in absorbance have also be used to follow binding kinetics using a "stopped flow" system. Results obtained indicate that dextran-supported dye molecules over a range of dye/dextran ratios show a constant ration of dye availability, but show a decrease. in dissociation constant with increased loading. Kinetic studies show the feasibility of applying a standard saturation model to the dye-protein interaction where free dye is used. However, in the case of dextran-supported dyes a model which takes account of dye stacking is required to describe adequately the results obtained.