CONFORMATIONAL PROPERTIES OF HUMAN AND RAT APOLIPOPROTEIN-A-IV

Apolipoprotein A-IV was isolated from 4 sources: human and rat lymph and plasma. Conformational properties of the rat and human apoA-IV in solution and denaturation changes induced by guanidine hydrochloride (Gnd.cntdot.HCl) were studied using circular dichroic and fluorescence spectroscopy and analytical sedimentation equilibrium ultracentrifugation. Both rat and human apoA-IV have similar secondary structure with negative maxima in the circular dichroic spectra at 222 nm and 207 nm. No significant difference was found in the .alpha.-helical content of the apoA-IV from rat plasma (33%), rat lymph (37%), human plasma (35%), or human lymph (35%). The denaturation studies with Gnd.cntdot.HCl demonstrated reversibility and the fact that each apoA-IV had a tendency to self-associate in solution and the self-association could be disrupted by low concentration of Gnd.cntdot.HCl (.ltoreq. 0.4 M). Unfolding of the secondary structure of each apoA-IV occurred at higher concentrations of Gnd.cntdot.HCl (midpoint .ltoreq. 1.0 M). The apparent free energy of denaturation of the 4 apoA-IV proteins calculated from changes in the circular dichroic spectra on addition of increasing concentrations of Gnd.cntdot.HCl varied in a range from 3.0 to 4.2 kcal/mol. The fluorescence experiments revealed that apoA-IV from all sources had a maximum fluorescence emission at 342.5 nm, which shifted to the red region on addition of increasing concentrations of Gnd.cntdot.HCl. Fluorescence quenching of apoA-IV by neutral (acrylamide) and negatively charged (iodide ion) quenches demonstrated the increased accessibility of the tryptophanyl residues under the denaturing conditions. There are no significant differences in the solution properties of apoA-IV isolated from rat plasma, rat lymph, human plasma, or human lymph and that apoA-IV from both species is thermodynamically unstable in aqueous solution.