SPECTROSCOPIC CHARACTERIZATION BY PHOTODIODE-ARRAY DETECTION OF HUMANURINARY AND AMNIOTIC PROTEIN HC SUBPOPULATIONS FRACTIONATED BY ANION-EXCHANGE AND SIZE-EXCLUSION HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A procedure for spectroscopic characterization and partial fractionati on of human protein HC populations by high-performance liquid chromato graphy-photodiode array ultraviolet-visible detection is reported. Hum an protein HC from urine or amniotic fluid fractionated by anion-excha nge HPLC in a protein Pak DEAE 5PW appeared to be heterogeneous as jud ged by the asymmetric elution pattern, consisting of a continuous irre gular broad peak with several shoulders distributed along the whole ch romatogram. Selected fractions containing shoulders were rechromatogra phed and finally six symmetrical homogeneous peaks with different rete ntion 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 the m associated to the same chromophore although with different stoichiom etry ratios. Isoelectric focusing showed that each peak was composed o f a limited number of subpopulations of protein HC with different isoe lectric 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 dete ction. Partial fractionation of human albumin on an analytical anion-e xchange Mono-Q PC 1.6/5 column, has allowed the identification of hete rogeneous chromophore-containing populations displaying significant ab sorption in the visible region in resemblance to that of protein HC.