Simultaneous multiple element detection by particle beam/hollow cathode-optical emission spectroscopy as a tool for metallomic studies: determinations of metal binding with apo-transferrin.
Particle beam/hollow cathode-optical emission spectroscopy (PB/HC-OES) is presented as a tool for the determination of metal ion loading in transferrin (Tf). The elemental specificity of optical emission spectroscopy provides a means of assessing metal ion concentrations as well as the relative amounts of metal per unit protein concentration (up to 2 moles of Fe per mole of protein). The PB/HC-OES method allows for the simultaneous detection of metal content (Fe (I) 371.99, Ni (I) 341.41 nm, Zn (I) 213.86 nm, and Ag (I) 338.28 nm in this case), as well as elemental carbon and sulfur (C (I) 156.14 nm and S (I) 180.73 nm) that are reflective of the protein composition and concentration. Quantification for the metal species is based on calibration functions derived from aqueous solutions, with limits of detection for the entire suite being less than 1.0 μM. Determinations in this manner eliminate much of the ambiguity inherent in UV-VIS absorbance determinations of Tf metal binding. Validation of this method is obtained by analyzing loading response of Fe(3+) into Tf using the PB/HC-OES method and comparing the results with those of the standard UV-VIS absorbance method. Maximum Fe(3+) loading of Tf (based on the number of available binding sites) was determined to be 71.2 ± 4.7% by the PB/HC-OES method and 67.5 ± 2.5% for the UV-VIS absorbance method. Element emission ratios between the dopant metals and the carbon and sulfur protein constituents allow for concentration independent determinations of metal binding into Tf. Loading percentages were determined for Ni(2+), Zn(2+), and Ag(+) into Tf with maximum loading values of 19.5 ± 0.4%, 41.0 ± 4.4%, and 141.2 ± 4.3%, respectively. While of no apparent biological significance, Ag(+) presents an interesting case as a surrogate for Pt(2+), whose binding with Tf has shown to be quite different from the other metals. A different mode from the others is indeed observed, and is consistent with conjecture on the Pt(2+) mechanisms. Competitive binding studies not easily performed using absorbance spectroscopy are easily performed by simultaneous, multielement analysis, reflective of the metals and the protein content. In this work, there is clear competition between and Fe(3+) and Zn(2+) for binding in the C-terminus lobe of Tf, while Ni(2+) binds within the N-terminus lobe. Addition of Ag(+) to this mixture does not affect the other metals' distributions, but reflects binding at other protein sites.
Department of Chemistry, Biosystems Research Complex, Clemson University, Clemson, SC 29634-0973, USA.
C Derrick
CD
Quarles
Julia L
JL
Brumaghim
R Kenneth
RK
Marcus
D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism*; D001667:Binding, Competitive; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D019216:Metals, Heavy / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013057:Spectrum Analysis / Q000379:methods*; D014168:Transferrin / Q000737:chemistry / Q000378:metabolism*
D001059:Apoproteins; D019216:Metals, Heavy; D014168:Transferrin; C028284:apotransferrin; D007501:Iron