Two types of urate binding sites on hemocyanin from the crayfish Astacus leptodactylus: an ITC study

The oxygen binding behaviour of hemocyanins from Crustacea is regulated by small organic compounds such as urate and L-lactate. We investigated the bi nding characteristics of urate and the related compound caffeine to the 2 x 6-meric hemocyanin of A. leptodactylus under fully oxygenated conditions e mploying isothermal titration calorimetry (ITC). An analysis of urate and c affeine binding based on a model of n identical binding sites resulted in a pproximately four binding sites for caffeine and eight for urate. This resu lt suggests that the binding process for these effecters is more complex th an this most simple model. Therefore, we introduced a number of alternative models. Displacement experiments helped to select the appropriate model. B ased on these experiments, at least two different types of binding sites fo r urate and caffeine exist on the 2 x 6-meric hemocyanin of A. leptodactylu s. The two binding sites differ strongly in their specificity towards the t wo analogues. It can be hypothesized that two different subunit types (beta and gamma) are responsible for the two types of binding sites. (C) 2001 Pu blished by Elsevier Science B.V.