title: A high-performance liquid chromatography method for determining transition metal content in proteins.
abstract: Transition metals are common components of cellular proteins and the detailed study of metalloproteins necessitates the identification and quantification of bound metal ions. Screening for metals is also an informative step in the initial characterization of the numerous unknown and unclassified proteins now coming through the proteomic pipeline. We have developed a high-performance liquid chromatography method for the quantitative determination of the most prevalent biological transition metals: manganese, iron, cobalt, nickel, copper, and zinc. The method is accurate and simple and can be adapted for automated high-throughput studies. The metal analysis involves acid hydrolysis to release the metal ions into solution, followed by ion separation on a mixed-bead ion-exchange column and absorbance detection after postcolumn derivatization with the metallochromic indicator 4-(2-pyridylazo)resorcinol. The potential interferences by common components of protein solutions were investigated. The metal content of a variety of metalloproteins was analyzed and the data were compared to data obtained from inductively coupled plasma-atomic emission spectroscopy. The sensitivity of the assay allows for the detection of 0.1-0.8 nmol, depending on the metal. The amount of protein required is governed by the size of the protein and the fraction of protein with metal bound. For routine analysis 50 microg was used but for many proteins 10 microg would be sufficient. The advantages, disadvantages, and possible applications of this method are discussed.
authors.affiliation: Department of Chemistry, University of Toronto, Lash Miller Chemical Laboratories, 80 St. George St., Toronto, Ont., Canada M5S 3H6.
authors.forename: Anelia
authors.initials: A
authors.lastname: Atanassova
authors.forename: Robert
authors.initials: R
authors.lastname: Lam
authors.forename: Deborah B
authors.initials: DB
authors.lastname: Zamble
mesh_terms: D000818:Animals; D002256:Carbonic Anhydrases / Q000032:analysis; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004926:Escherichia coli / Q000201:enzymology; D006801:Humans; D008667:Metalloproteins / Q000032:analysis*; D008670:Metals / Q000032:analysis*; D012118:Resorcinols; D013482:Superoxide Dismutase / Q000032:analysis; D014168:Transferrin / Q000032:analysis; D028561:Transition Elements / Q000032:analysis*
chemical_list: D008667:Metalloproteins; D008670:Metals; D012118:Resorcinols; D014168:Transferrin; D028561:Transition Elements; D013482:Superoxide Dismutase; D002256:Carbonic Anhydrases; C028398:4-(2-pyridylazo)resorcinol