The physiological functions of hyaluronan (HA) in the extracellular matrix of vertebrate tissues involve a range of specific protein interactions. In this study, the interaction of HA with the Link module from TSG-6 (Link_TSG6) and G1 domain of aggrecan (G1), were investigated by a biophysical analysis of translational diffusion in dilute solution using confocal fluorescence recovery after photobleaching (confocal FRAP). Both Link_TSG6 and G1 were shown to bind to polymeric HA and these interactions could be competed with HA8 and HA10 oligosaccharides, respectively. Equilibrium experiments showed that the binding affinity of Link_TSG6 toHAwas maximal at pH 6.0, and reduced dramatically above and below this pH. In contrast, G1 had maximum binding at pH 7.0-8.0 and moderate to strong binding affinity over a much broader pH range (5.5-8.0). The KD determined for Link_TSG6 binding to HA showed a 100-fold increase in binding affinity between pH 7.4 and 6.0, whereas G1 showed a 75-fold decrease in binding affinity over the same pH range. The sharp difference observed in their pH binding suggests that pH controls the physiological function of TSG-6, with a low affinity for HA at neutral pH, but with increased affinity as the pH falls below pH 7. TSG-6 and aggrecan interact with HA through structurally homologous domains and the difference in pH-dependent binding can be understood in terms of differences in the presence and topographical distribution of key regulatory amino acids in Link_TSG6 and in the related tandem Link domains in aggrecan G1. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.