On the use of self-quenching fluorophores in the study of membrane fusion kinetics. The effect of slow probe redistribution.

In glycoprotein-mediated pH-induced fusion of virus to animal cells, the mixing of materials between membranes or between cytoplasmic spaces occurs after the virus-cell complex has gone through a number of activation reactions. The monitoring of the fluorescence changes measured in a fusing system using self-quenching probes could reflect not only the kinetics of activation, but also the redistribution reaction of probes. For instance, time delay seen in the onset of fluorescence changes after triggering the fusion reaction (S.J. Morris, D.P. Sarkar, J.M. White and R. Blumenthal, J. Biol. Chem. (1989) 3972), could be due to rate-limiting probe redistribution kinetics. In this paper we examined in detail the effect of probe redistribution rates on fusion kinetics. Simulations were performed using a very simple model with two fusion-activation steps and an exponential probe redistribution kinetics. We conclude that if the rates of probe redistribution are faster than or equal to those of viral glycoprotein activation, the kinetics of the fusion reaction are not significantly affected.