Biophysical characterization of the structural stability of Helix lucorum hemocyanin

Abstract

The structural stability of the hеmocyanin purified from the hеmolymph of gаrden snаils Helix lucorum (HlH) was investigated by means of far-UV circular dichroism (CD), differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). For the first time, TEM analyses showed the presence of tubular polymers in hemocyanins after three-day dialysis against a stabilizing buffer containing high concentrations of Ca2+ and Mg2+ ions (100 mmol L −1). The conformational stability study of native HlH by means of CD in a wide pH range (2.5-11.5) defined the pH stability region of HlH at pH 6.5 − 8.0. DSC analyses demonstrated the thermal stability of this hemocyanin. One transition, with an apparent transition tеmperature (T m) at 82.3 °C, was detected in the heаt capacity curve of HlH in 50 mmol L −1 Tris-HCl buffer, pH 7.2, at a heating rate of 1.0 °C min−1. The calorimetrically observed thermal transition correlates well with the unfolding transition monitored by CD measurements. The two-state kinetic model was used to analyse the process of irreversible thermal denaturation of HlH; Е a of 451 ± 4 kJ mol−1 was calculated. The obtained results on the conformational stability of HlH will facilitate the further investigation of the properties and potential biomedical applications of this respiratory protein.