Separation of three mouse metallothionein isoforms by free-solution capillary electrophoresis

M P Richards, G K Andrews, D R Winge, J H Beattie

    Research output: Contribution to journalArticle

    18 Citations (Scopus)

    Abstract

    We have used free-solution capillary electrophoresis (FSCE) to separate three distinct mouse metallothionein (MT) isoforms, MT-1, MT-2 and MT-3. FSCE was conducted in an uncoated fused-silica capillary (57 cmXSO mu m I.D., 50 cm to detector) using 50 mM sodium phosphate buffer adjusted to pH 7.0 or 2.0. At neutral pH, each of the three isoform peaks were well resolved from a mixture with the order of migration (MT-1>MT-2>MT-3) related to the net negative charge on the protein. At acidic pH, the migration order was reversed with MT-3 migrating fastest, suggesting MT-3 had a higher net positive charge than MT-2 or MT-1. UV absorbance spectra (190-300 nm) confirmed the presence of Zn in MT-1 and MT-2. MT-3, which was saturated with Cd to stabilize the protein, gave a spectrum characteristic of the Cd-S charge transfer (shoulder at ca. 250 nm). At pH 2.0, the absorbance spectra for all three mouse MTs were characteristic of the metal-free form of the protein (apothionein). Thus, FSCE conducted at neutral pH separates MT isoforms with their metals intact, whereas at pH 2.0, both the Zn and the Cd dissociate from the protein during the run.

    Original languageEnglish
    Pages (from-to)327-331
    Number of pages5
    JournalJournal of Chromatography B: Biomedical Sciences and Applications
    Volume675
    Issue number2
    Publication statusPublished - 26 Jan 1996

    Keywords

    • metallothioneins
    • proteins
    • ZONE ELECTROPHORESIS
    • BRAIN

    Cite this

    Separation of three mouse metallothionein isoforms by free-solution capillary electrophoresis. / Richards, M P ; Andrews, G K ; Winge, D R ; Beattie, J H .

    In: Journal of Chromatography B: Biomedical Sciences and Applications, Vol. 675, No. 2, 26.01.1996, p. 327-331.

    Research output: Contribution to journalArticle

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    N2 - We have used free-solution capillary electrophoresis (FSCE) to separate three distinct mouse metallothionein (MT) isoforms, MT-1, MT-2 and MT-3. FSCE was conducted in an uncoated fused-silica capillary (57 cmXSO mu m I.D., 50 cm to detector) using 50 mM sodium phosphate buffer adjusted to pH 7.0 or 2.0. At neutral pH, each of the three isoform peaks were well resolved from a mixture with the order of migration (MT-1>MT-2>MT-3) related to the net negative charge on the protein. At acidic pH, the migration order was reversed with MT-3 migrating fastest, suggesting MT-3 had a higher net positive charge than MT-2 or MT-1. UV absorbance spectra (190-300 nm) confirmed the presence of Zn in MT-1 and MT-2. MT-3, which was saturated with Cd to stabilize the protein, gave a spectrum characteristic of the Cd-S charge transfer (shoulder at ca. 250 nm). At pH 2.0, the absorbance spectra for all three mouse MTs were characteristic of the metal-free form of the protein (apothionein). Thus, FSCE conducted at neutral pH separates MT isoforms with their metals intact, whereas at pH 2.0, both the Zn and the Cd dissociate from the protein during the run.

    AB - We have used free-solution capillary electrophoresis (FSCE) to separate three distinct mouse metallothionein (MT) isoforms, MT-1, MT-2 and MT-3. FSCE was conducted in an uncoated fused-silica capillary (57 cmXSO mu m I.D., 50 cm to detector) using 50 mM sodium phosphate buffer adjusted to pH 7.0 or 2.0. At neutral pH, each of the three isoform peaks were well resolved from a mixture with the order of migration (MT-1>MT-2>MT-3) related to the net negative charge on the protein. At acidic pH, the migration order was reversed with MT-3 migrating fastest, suggesting MT-3 had a higher net positive charge than MT-2 or MT-1. UV absorbance spectra (190-300 nm) confirmed the presence of Zn in MT-1 and MT-2. MT-3, which was saturated with Cd to stabilize the protein, gave a spectrum characteristic of the Cd-S charge transfer (shoulder at ca. 250 nm). At pH 2.0, the absorbance spectra for all three mouse MTs were characteristic of the metal-free form of the protein (apothionein). Thus, FSCE conducted at neutral pH separates MT isoforms with their metals intact, whereas at pH 2.0, both the Zn and the Cd dissociate from the protein during the run.

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