Talanta 69 (2006) 556–564 Novel UV assay for protein determination and the characterization of copper–protein complexes by mass spectrometry G. Drochioiu a,∗ , N.E. Damoc b , M. Przybylski b,∗∗ a Faculty of Chemistry, “Al. I. Cuza” University of Iasi, 11 Carol I, Iasi 700506, Romania b Laboratory of Analytical Chemistry, Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany Received 26 January 2005; received in revised form 4 October 2005; accepted 25 October 2005 Available online 29 November 2005 Abstract 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 240 nm. Copper absorbance slowly increases from 240 to 190 nm where a blank with the reagents is recommended. A maximum absorbance for the copper–protein complex was found at 226 nm 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, 1–1.5 ml of 1.8% KOH solution with 0–20 g ml -1 protein is treated with 25 mg of copper phosphate powder. The mixture is powerfully stirred, centrifuged, and the absorbance of the supernatant is measured at 226 nm in 1 cm quartz cuvettes against a blank of the reagents. The color system obeys Beer’s law in the range 0.1–20 g ml -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. © 2005 Elsevier B.V. All rights reserved. Keywords: Protein determination; Biuret reaction; UV detection; Copper complex; Mass spectrometry 1. Introduction Quantification of protein has been one of the most universal denominators and several methods have been devised to mea- sure protein in microgram quantities [1–3]. Measurements of protein concentrations have been obtained by several different techniques such as micro-Kjeldahl method [4], near infrared spectroscopy [5], electrochemical procedures [6], Rayleigh light scattering [7], particle beam hollow cathode optical emission spectroscopy [8], turbidimetry [9], radio iodination [10], or spectroscopic techniques such as NMR [11]. Elemental analy- ∗ Corresponding author. Tel.: +40 232 201278; fax: +40 232 201313. ∗∗ Corresponding author. Tel.: +49 7531 88 2249. E-mail addresses: gabidr@uaic.ro (G. Drochioiu), michael.przybylski@uni-konstanz.de (M. Przybylski). sis by X-ray fluorescence spectrometry [12], atomic absorption spectrophotometry [13], atomic fluorescence spectrometry [14], and optical emission spectroscopy [15] have also been used for detection of proteins by performing a digestion to dissociate them and then monitoring the metal elements that were bound to the protein molecules. Nevertheless, the widely used meth- ods have their disadvantages [7,16]. Proteins in dilute solutions could also be determined by near infrared fluorescence recovery [17], pulse-polarography [18,19], and capillary gas chromatog- raphy [16], with electrospray ionization magnetic mass spec- trometry [20] or by capillary zone electrophoresis at 214 nm [21]. Some of these analytical techniques are quite new and do not so far belong to the usual apparatus in biochemical labo- ratories. Protein concentrations of 0.10–15.3 g ml -1 could be determined by their enhancement effect on the Rayleigh light scattering of carboxyarsenazo [7]. This method is simple, rapid, 0039-9140/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2005.10.026