Spectroscopic characterization by photodiode array detection of human urinary and amniotic protein HC subpopulations fractionated by anion-exchange and size-exclusion high-performance liquid chromatography

Abstract

A procedure for spectroscopic characterization and partial fractionation of human protein HC populations by high-performance liquid chromatography-photodiode array ultraviolet-visible detection is reported. Human protein HC from urine or amniotic fluid fractionated by anion-exchange HPLC in a protein Pak DEAE 5PW appeared to be heterogeneous as judged by the asymmetric elution pattern, consisting of a continuous irregular broad peak with several shoulders distributed along the whole chromatogram. Selected fractions containing shoulders were rechromatographed and finally six symmetrical homogeneous peaks with different retention times were obtained from each protein HC preparation. The direct automatic absorption spectra analyses at each peak maximum, indicated that all of the homogeneous peaks seemed to be protein HC, all of them associated to the same chromophore although with different stoichiometry ratios. Isoelectric focusing showed that each peak was composed of a limited number of subpopulations of protein HC with different isoelectric points. Size microheterogeneity has been also demonstrated in both urinary and amniotic protein HC preparations by a combination of size-exclusion HPLC on a TSK 3000 SW6 column and photodiode array detection. Partial fractionation of human albumin on an analytical anion-exchange Mono-Q PC 1.6/5 column, has allowed the identification of heterogeneous chromophore-containing populations displaying significant absorption in the visible region in resemblance to that of protein HC.