title: Hydrophobic interaction chromatographic separation of proteins in human blood fractions hyphenated to atomic spectrometry as detector of essential elements.
abstract: The binding of metals to proteins in blood fractions was investigated applying hydrophobic interaction chromatography (HIC) for protein separation and graphite furnace atomic absorption spectrometry (GFAAS) as the element specific detector. For the semi-preparative separation of metalloproteins in erythrocytes and blood plasma, a HIC column (Fractogel EMD Phenyl I (S) 150 mm x 10 mm I.D.) was adapted. The separation column was calibrated with the same four standard proteins as used in Pomazal et al. [Analyst 124 (1999) 657]. The sample injection volume and the ammonium sulphate gradient set-up were optimized: 20 or 200 microl, respectively, of blood plasma and of lysed erythrocytes were injected. The separated proteins were collected in 4-ml fractions and analyzed by GFAAS off-line. An optimization of the GFAAS measuring parameters for Cu, Mn, Fe, Zn, Co, Ni, and Cr was performed. For each element, a specific temperature program was optimized with respect to the matrix of the HIC eluate (0.02 M NaH2PO4, 1.8 M (NH4)2SO4). The obtained metal profiles of the eluate were compared with the HIC chromatograms. The limits of detection (LOD) for the elements by GFAAS were: 0.5 ng Cu/ml; 0.2 ng Mn/ml; 1 ng Fe/ml; 0.2 ng Zn/ml; 0.12 ng Co/ml; 0.2 ng Ni/ml; 0.16 ng Cr/ml. The GFAAS method enabled the detection of the proteins of interest via the metals.
authors.affiliation: Institute of Analytical Chemistry, University Vienna, Austria.
authors.forename: Katerina
authors.initials: K
authors.lastname: Pomazal
authors.forename: Christian
authors.initials: C
authors.lastname: Prohaska
authors.forename: Ilse
authors.initials: I
authors.lastname: Steffan
mesh_terms: D001798:Blood Proteins / Q000032:analysis*; D006801:Humans; D008670:Metals / Q000097:blood*; D012680:Sensitivity and Specificity; D013054:Spectrophotometry, Atomic
chemical_list: D001798:Blood Proteins; D008670:Metals