FLUORESCENCE QUENCHING DATA INTERPRETATION IN BIOLOGICAL-SYSTEMS - THE USE OF MICROSCOPIC MODELS FOR DATA-ANALYSIS AND INTERPRETATION OF COMPLEX-SYSTEMS

In micro-heterogeneous media (e.g. membranes, micelles and colloidal s ystems), the fluorescence decay in the absence of quencher is usually intrinsically complex, e.g. due to the existence of several sub-popula tions with different microenvironments. In this case it is impossible to analyze data in detail (accounting for transient effects) and simpl er formalisms are needed. The objective of the present work is to pres ent and discuss such simpler formalisms. The goal is to achieve simple data analysis and meaningful, clear data interpretation in complex sy stems using microscopic models that consider several sub-populations o f chromophores. Two points are dealt with in detail. (i) It is shown t hat the approximation of the transient effects by the quenching sphere -of-action model is not always possible. The quenching sphere-of-actio n concept can be regarded as a valuable tool, although crude, only in a limited range of experimental conditions, namely time resolution. (i i) The Stern-Volmer equation usually used for data analysis is only va lid for a limited range of small and moderate equilibrium association constants, K-a, although this is frequently overlooked in the literatu re. Self-consistency criteria are presented for the proposed methods. The well-known downward curvature due to a fraction of fluorophores wh ich is not accessible to the quencher is only a limiting case from a s et of possible situations which result in deviations to linearity. A s ystematic classification of the different types of quenching is presen ted. (C) 1998 Elsevier Science B.V. All rights reserved.