Advanced procedures for separation and analysis of low molecular weight inhibitor (NCX_IF) of the cardiac sodium-calcium exchanger

A low molecular weight inhibitor (NCX_IF) of the cardiac Na/Ca exchanger, isolated from the calf ventricle tissue, is capable of regulating the muscle strip's contractility and relaxation without involving the β-activation pathway. The structural analysis of NCX_IF requires highly purified preparations that fulfill the demanding requirements for mass spectra and NMR analyses. No such preparation is yet available. To this end, new HPLC procedures were developed by a combination of the reverse phase, normal phase, and HILIC (hydrophilic liquid chromatography) techniques. The specific activity of NCX_IF is 10⁵ times higher in the purified preparations (as compared to the crude extract) showing a 2-5% yield of total inhibitory activity and 20-100 μg content of final material. The purification yield reveals that 1 kg ventricle muscle contains 0.1-0.2 mg NCX_IF, meaning that the tissue concentrations of NCX_IF may reach 10 ^-7-10^-6 M. The diode-array scanning of purified preparations of NCX_IF shows a homogeneous 3D peak with a maximal absorption at 202 nm. These spectral properties may represent a five-membered ring (e.g., proline, histidine) and/or simple chemical groups (like amine, carbonyl, ester, etc.), but not an aromatic ring or complex conjugates (alkyne, alkene, aldehyde, etc.). NCX_IF does not respond to phenol/sulfur reagent, suggesting that it lacks reducing (aldo) sugar. NCX_IF shows a faint response to fluorescamine, meaning that it may contain an amino group (or its derivative). It is believed that a combination of presently developed procedures with LC/MS and LC/MS/MS may provide a useful tool for structural analysis of NCX_IF.