High-performance liquid chromatography and photodiode-array detection of the human protein HC (human complex-forming glycoprotein heterogeneous in charge), a chromophore-associated protein

Abstract

Photodiode-array ultraviolet-visible detection has been adapted to our high-performance liquid chromatographic system for the analysis and the characterization of the unknown yellow-brown chromophore associated with the human complex-forming glycoprotein, heterogeneous in charge (Protein HC). By using several post-experiment data processing modes, such as multichromatograms, automatic spectrum analyses or three-dimensional plots, the technique allows a direct verification of purity, quantification, as well as the identification of Protein HC without the necessity for further analytical systems. At least thirteen different shoulders in the absorption spectrum in the visible region between 300 and 480 nm have been identified for urinary Protein HC. However, no chromophore was found to be associated with Protein HC complexed with immunoglobulin A (HC-IgA complex). Comparison of spectra between plasma or urinary protein HC allows one to distinguish spectral differences in its chromophore, at least in the range from 300 to 480 nm. The technique was useful for easy identification of chromophore-containing peptides from the digested Protein HC.