l-Anilino-naphthalene 8-sulfonate (ANS) has been used as a probe for the hydrophobic patches on the surface of human IgG. The binding of ANS to human IgG, its Fab and Fe fragments, and heataggregated IgG was measured using equilibrium dialysis. 3.83:. 0.6 ANS binding sites on IgG were detected and the dissociation constant for the interaction at 25°C was 720 i160 uM. There are 0.77 i 0.02 binding sites for ANS on each Fab region and 2.1 i 0.3 sites on the Fc region of IgG at 25°C. The dissociation constants for the interactions are 12 _+_ 2 uM and 440 i 80 uM respectively. At 4°C, 1.5 3; 0.2 ANS binding sites on IgG were detected with a dissociation constant of 210 j; 70 uM, and the Fab region has 0.61i0.02 binding sites with a dissociation constant of 93 i 4 uM. At 4°C, ANS binding to the Fc region was not detected. It would appear that ANS binding to IgG at 4°C, is predominantly due to ANS binding to the Fab regions. At 4°C, heat-aggregated lgG has 2.9 i; 0.1 binding sites for ANS and the dissociation constant for the interaction was 190 +_ 20 uM. It would appear that heat-treated IgG has undergone conformational changes creating new ANS binding sites. However, it is not possible to localize these conformational changes to either the PC or Fab regions from the above information. There was eight fold enhancement in the fluorescence of ANS bound to heat-aggregated lgG compared to untreated IgG and a three-four fold enhancement in fluorescence of ANS when bound to heat-treated Fc compared to normal Fc. It would appear that at least some of the heat—induced conformational changes occur in the Fc region of IgG. Chemically crosslinked IgG aggregates were also titrated with ANS and there was a two fold enhancement in fluorescence of ANS bound to trimer-tetramer IgG compared to monomer IgG. Two fold increases in fluorescence were seen for ANS bound to chemically crosslinked Fab and Fe aggregates compared to their respective monomers. Cleavage of the chemical crosslinker, dithiobis(succinimidyl)propionate by 30 mM dithiothreitol reduced the difference in fluorescence of ANS bound to trimer IgG and monomer IgG by 65%. It was concluded that cleavage of the chemical crosslinker causes the aggregated IgG to revert to a similar conformation as monomer IgG, at least in so far as its ANS binding properties. The extent of fluorescence energy transfer from tryptophan residues to bound ANS also increased with increasing size of IgG, Fab and Fe aggregates. These results, collectively, suggest that aggregation of IgG induces conformational changes in IgG leading to the exposure of hydrophobic patches on the surface of both the Fe and Fab regions. The increases in fluorescence of ANS bound to IgG and Fc aggregates were correlated with increasing binding of Clq to the aggregates, and also the complement activating capacity of the IgG aggregates. ANS had no effect on the binding of protein A to aggregated IgG at concentrations sufficient for inhibition of Clq binding.From analysis of the dissociation constants for ANS binding to Clq and the Fc region of IgG and the concentrations of ANS required for inhibition of the Clq : IgG interaction, it would appear that the mode of inhibition is via ANS binding to the Clq binding site on IgG. Thus, it would appear that the ANS binding sites on the Fc region are at or close to the Clq binding site and therefore it is possible that some of the aggregation induced conformational changes in IgG occur at the Clq binding site. Both the increased fluorescence of ANS bound to IgG or Fe and Clq binding to aggregated, compared to monomeric IgG or F0, could be reversed by treating the IgG or PC aggreagtes with 1 M urea. This treatment had little effect on : the tryptophan fluorescence of either IgG or Clq ; the fluorescence of ANS bound to Clq ; nor the binding of Clq to the cation exchange resin, Bio-Rex 70. On the basis of these results it was concluded that that urea-mediated disruption of the conformational changes in IgG, induced by aggregation, leads to a loss in the Clq binding activity of IgG. This implies that the conformational changes in IgG may be necessary for Clq binding and complement activation.