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Biophys J, November 2002, p. 2652-2666, Vol. 83, No. 5

Measuring pKa of Activation and pKi of Inactivation for Influenza Hemagglutinin from Kinetics of Membrane Fusion of Virions and of HA Expressing Cells

Aditya Mittal,* Tong Shangguan,dagger and Joe Bentz*

 *Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania 19104 USA; and  dagger Elan Corporation, 1 Research Way, Princeton, New Jersey 08540 USA

The data for the pH dependence of lipid mixing between influenza virus (A/PR/8/34 strain) and fluorescently labeled liposomes containing gangliosides has been analyzed using a comprehensive mass action kinetic model for hemaglutinin (HA)-mediated fusion. Quantitative results obtained about the architecture of HA-mediated membrane fusion site from this analysis are in agreement with the previously reported results from analyses of data for HA-expressing cells fusing with various target membranes. Of the eight or more HAs forming a fusogenic aggregate, only two have to undergo the "essential" conformational change needed to initiate fusion. The mass action kinetic model has been extended to allow the analysis of the pKa for HA activation and pKi for HA inactivation. Inactivation and activation of HA following protonation were investigated for various experimental systems involving different strains of HA (A/PR/8/34, X:31, A/Japan). We find that the pKa for the final protonation site on each monomer of the trimer molecule is 5.6 to 5.7, irrespective of the strain. We also find that the pKi for the PR/8 strain is 4.8 to 4.9. The inactivation rate constants for HA, measured from experiments done with PR/8 virions fusing with liposomes and X:31 HA-expressing cells fusing with red blood cells, were both found to be of the order of 10-4 s-1. This number appears to be the minimal rate for HA's essential conformational change at low HA surface density. At high HA surface densities, we find evidence for cooperativity in the conformational change, as suggested by other studies.

Biophys J, November 2002, p. 2652-2666, Vol. 83, No. 5
© 2002 by the Biophysical Society   0006-3495/02/11/2652/15  $2.00


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