Abstract

Aim—To obtain further data on the structure and conformation of calprotectin, a prominent leucocyte protein found in many species.

Methods—The binding of Ca²⁺ to calprotectin was studied by means of equilibrium dialysis using ⁴⁵Ca as tracer. The thermal stability and denaturation kinetics of calprotectin were studied by means of differential scanning calorimetry. Con-comitant alterations in optical activity resulting from different conditions were measured. A computer program calculated the parameters to fit different models of protein structure. Ultraviolet spectroscopy gave absorbtion spectra. Sedimentation velocity studies and molecular weight determinations by the low speed (sedimentation) equilibrium technique were performed.

Results—A maximum of six calcium ions were bound per calprotectin molecule at 0·7 mM calcium chloride. The apparent dissociation constants were calculated. Ca²⁺ ions increased the denaturation temperature by 26°K. The enthalpy of denaturation was also increased by Ca²⁺. Addition of Ca²⁺ to the buffers caused a gradual change in the near UV circular dichroism spectrum, while only minor changes were seen at wavelengths of 210-240 nm. A gradual increase in the sedimentation coefficient was observed on addition of calcium chloride. The extinction coefficient at 279nm was determined: E₂₇₉= 2·53·10⁴ M⁻¹ cm⁻¹.

Conclusions—Calprotectin can bind six calcium ions. Upon binding, the protein shows distinct conformational changes and increased thermal stability. The former may be of importance for its function, while the biological significance of the latter is unknown.