Use of stable isotopically enriched proteins and directly coupled high-performance liquid chromatography inductively coupled plasma mass spectrometry for quantitatively monitoring the transfer of metals between proteins.
Studies have shown that metallothionein (MT) may play an important role in modulating the activity of certain Zn-regulated enzymes under various oxidoreductive conditions by either donating or removing Zn. To better determine the role of MT in interprotein metal transfer, we describe a procedure that uses stable isotopically enriched (67)Zn(7) metallothionein 2 ((67)Zn(7)-MT-2) to quantitatively determine the stoichiometry of transfer of Zn from the protein to a recipient apo-metalloenzyme, apo-carbonic anhydrase (apo-CA) by directly coupled ion exchange high-performance liquid chromatography inductively coupled plasma mass spectrometry. Quantitatively, the transfer of (67)Zn was consistent with the enzymatic activation of the apo-enzyme as judged by its esterase activity and ability to cleave p-nitrophenyl acetate. Maximum enzyme activation occurred at an MT-2:apo-CA molar ratio of 1, implying the release of a single atom of Zn from MT-2. Preincubation of (67)Zn(7)-MT-2 with an excess of oxidized glutathione (GSSG) increased metal donation fourfold, whereas reduced glutathione (GSH) inhibited donation by approximately 50%. By using multiple recipient and donor proteins having different stable isotopic signatures, the technique has the potential for quantitatively studying the kinetic and thermodynamic aspects of Zn transfer between numerous competing ligands in vitro, an important first step toward understanding the regulatory role of this metal in protein functioning and cellular metabolism in vivo.
Department of Biological Sciences and Institute for Integrated Research in Materials, Environments, and Society, California State University, Long Beach, Long Beach, CA 90840, USA. zedmason@csulb.edu
Andrew Z
AZ
Mason
Rhonda
R
Moeller
Kelly A
KA
Thrippleton
Douglas
D
Lloyd
D001059:Apoproteins / Q000032:analysis; D002256:Carbonic Anhydrases / Q000032:analysis; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D019803:Glutathione Disulfide / Q000378:metabolism; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D008668:Metallothionein / Q000737:chemistry* / Q000378:metabolism; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism; D015033:Zinc Isotopes
D001059:Apoproteins; D015033:Zinc Isotopes; D008668:Metallothionein; D002256:Carbonic Anhydrases; D015032:Zinc; D019803:Glutathione Disulfide