Metal—protein interaction in carcinus maenas hemocyanin

Abstract

Hemocyanin (Hc) is the respiratory pigment next in importance to hemoglobin for its distribution in two large phyla of Invertebrates: Arthropoda and Mollusca. The characteristic of Hc as oxygen carrier has overshadowed interest in other properties of possible physiological meaning such as the pseudocatalasic, t001 Shell Gill Vas Def. Heart Epider. Co 15.12 1.19 5.16 3.12 2.97 Mn 54.87 12.23 5.11 2.10 4.07 Zn 55.82 21.89 47.38 36.94 83.21 Hepatop. Muscle Hc a Serum a Co 1.48 2.60 1.27 0.38 Mn 1.88 8.52 0.37 Zn 40.51 115.48 22.08 0.88 a μ/ml; for all other values μ/g w.w. t002 n k A( M −1 Co 2+ 3 2.7 × 10 3 Mn 2+ 8 7.8 × 10 3 Zn 2+ 4 1.6 × 10 4 t003 Metal concentration 10 −5 M F/F° 10 −4 M F/F° Co 2+ 0.68 0.51 Mn 2+ 1.18 1.16 Zn 2+ 0.65 0.41 catalasic, the pseudoperoxidasic and pseudophenolasic activities, the possible contribution to the osmotic equilibrium etc. Recently we have obtained indications that Hc could function in Crustacea in transporting metal ions from the external environment to the tissues [1]. In this communication we present preliminary data on the interaction between Hc and some metal ions. In Table I quantitative distribution of Zn 2+, Co 2+ and Mn 2+ in the blood, the shell and tissues of Carcinus maenas is reported. Equilibrium dialysis was carried out and the experimental data were used for calculating, according to the Scatchard procedure [2], the number of sites and the binding constants of Hc with the metal ions. At metal ion concentration higher than 10 −4 M the shape of the Scatchard plot indicates the presence of binding sites with lower affinity. ▪ The binding of Co 2+ and Zn 2+ to Hc leads to a quenching of protein intrinsic fluorescence (λ exc = 280 nm, emission λ max = 330nm). The results are reported in Table III where F° is the value of the fluorescence intensity in the absence of metal. In contrast, the fluorescence increases by about 10% when the protein binds Mn 2+. To assess the nature of these changes, the effect of Co 2+, Zn 2+ and Mn 2+ (chloride salts were used) on the fluorescence properties of tyrosine and tryptophan have been studied in unbuffered water solutions. Zn 2+ and Mn 2+ do not exert any influence on the fluorescence emission of these aromatic amino-acids (Fig.1) As the binding of Zn 2+ and Co 2+ with aminoacids have similar values [3], the significant fluorescence quenching observed upon binding of Co 2+ to Tyr and Trp can be attributed only to a paramagnetism of the cobalt ion. The absence of a ‘paramagnetic ion’ effect in the case of Mn 2+ is probably due to very low values of the binding constant of Mn 2+ with aminoacids as compared with those of the other ions [3]. The almost equal quenching values for Co 2+ and Zn 2+ (Table III) leads to the conclusion that, in our experimental conditions, Hc fluorescence quenching by both ions is due to a conformational modification of the protein, as a consequence of the metal binding. The opposite effect observed in the case of Mn 2+ can be attributed to binding in different sites of the protein which causes a different conformational modification.