Recombinant human bone morphogenetic protein-2 (rhBMP-2) promotes bone growth but must be retained at the delivery site for optimal efficacy in vivo. rhBMP-2 release from a collagen-based matrix has shown favorable pharmacokinetics. The present study assessed binding affinity and binding saturation of rhBMP-2 to a collagen matrix as a function of solution and rhBMP-2 isoform variables. Results indicate that rhBMP-2 binds to the collagen matrix with affinities on the order of 10(3) to 10(4) M-1. Maximum binding, v, was primarily a function of pH for heterogeneous rhBMP-2 and the extended (T-266/T-266) isoform. However, binding saturation of the < Q(283)/ < Q(283) isoform was unaffected by pH. Overall, binding saturation was higher than the calculated saturation of a rhBMP-2 monolayer, suggesting both hydrophobic and ionic interactions in a multilayer formation. The contributions of pH and ionic strength to the linkage free energy of interaction was on the order of 1.3 kcal mol(-1) and similar to 0.3 kcal mol(-1), respectively. This thermodynamic approach can serve to optimize interactions between therapeutic proteins and delivery systems.