Novel UV assay for protein determination and the characterization of copper-protein complexes by mass spectrometry.

A very simple, highly selective and sensitive assay of proteins based on the biuret absorption in the ultraviolet region has been developed. The well-known biuret assay is based on the reaction of proteins with copper ions under strong alkaline conditions to form a copper-protein complex. Yet, copper ions may seriously interfere with the determination if the measurement is made in the UV range. In the present approach, proteins mobilize copper ions from insoluble salts at different pH values, and the copper-protein complexes are investigated by UV spectrophotometry and mass spectrometry. Upon using copper phosphate, free copper ions do not interfere with the determination from 540 to 240nm. Copper absorbance slowly increases from 240 to 190nm where a blank with the reagents is recommended. A maximum absorbance for the copper-protein complex was found at 226nm and high pH value. The stoichiometries of the copper-protein complexes measured directly with a mass spectrometer are pH dependent: half of the peptides without any histidine residue chelate just a single Cu(2+) ion at pH 7.4 but each such peptide mobilizes from 1 to 6 Cu(2+) ions at pH 10.3. To determine proteins, 1-1.5ml of 1.8% KOH solution with 0-20mugml(-1) protein is treated with 25mg of copper phosphate powder. The mixture is powerfully stirred, centrifuged, and the absorbance of the supernatant is measured at 226nm in 1cm quartz cuvettes against a blank of the reagents. The color system obeys Beer's law in the range 0.1-20mugml(-1) protein at this wavelength. The molar absorptivity value proved to be a characteristic of each protein being analyzed. Therefore, individual proteins should be used to plot calibration curves. This assay proved to be over 100 times more sensitive than the classical biuret procedure. The method is highly selective and the determination is little affected by the presence of other substances. All other important analytical parameters were studied and practical applicability of the method has been verified by the analysis of some biological materials.