SEPARATION OF METALLOTHIONEIN ISOFORMS BY CAPILLARY ZONE ELECTROPHORESIS

J H BEATTIE, M P RICHARDS, R SELF

    Research output: Contribution to journalArticle

    59 Citations (Scopus)

    Abstract

    The potential of capillary zone electrophoresis (CZE) for the analysis of metallothionein (MT) isoforms was investigated. CZE was performed using two different systems, (1) a laboratory-constructed instrument with an ISCO UV detector and (2) a Waters Quanta 4000 system. Capillaries were of 75 pm I.D. x ca. 1 m in length and loading times were up to 40 s by gravity or 4 s by electrokinetic migration at 30 kV. Samples were dissolved in 10 mM Tris-HCI buffer, pH 9.1, and electrophoresis was performed at 30 kV using a 50 mM Tris-HCI, pH 9.1 running buffer. Detection was by UV absorbance at 185 or 214 nm. Purified and semipurified MT samples were analysed for qualitative assessment of purity, relative isoform abundance and separation characteristics of MT from different species. As progress towards the development of a quantitative assay, the linearity of calibration curves and simple methods of sample preparation for analysis by CZE were investigated. Complete separation of a mixture of the two major MT isoforms was achieved in less than 5 min and the technique was found to be very useful for qualitative analysis of MT. Using a rabbit liver MT standard (500 mug/ml-1), a linear relationship was found between the gravity load time and the integrated peak area. Standard calibration curves were also linear and the detection limit for both CZE instruments under our separation conditions was 1-10 mug MT ml-1. The successful use of two solvent ''traction procedures for tissue samples demonstrated the potential of CZE for routine quantitative analysis of MT.

    Original languageEnglish
    Pages (from-to)127-135
    Number of pages9
    JournalJournal of Chromatography A
    Volume632
    Issue number1-2
    Publication statusPublished - 19 Feb 1993

    Keywords

    • HEMOGLOBIN AFFINITY ASSAY
    • LIVER METALLOTHIONEIN
    • SATURATION METHOD
    • PROTEINS
    • PEPTIDES
    • TISSUES

    Cite this

    BEATTIE, J. H., RICHARDS, M. P., & SELF, R. (1993). SEPARATION OF METALLOTHIONEIN ISOFORMS BY CAPILLARY ZONE ELECTROPHORESIS. Journal of Chromatography A, 632(1-2), 127-135.

    SEPARATION OF METALLOTHIONEIN ISOFORMS BY CAPILLARY ZONE ELECTROPHORESIS. / BEATTIE, J H ; RICHARDS, M P ; SELF, R .

    In: Journal of Chromatography A, Vol. 632, No. 1-2, 19.02.1993, p. 127-135.

    Research output: Contribution to journalArticle

    BEATTIE, JH, RICHARDS, MP & SELF, R 1993, 'SEPARATION OF METALLOTHIONEIN ISOFORMS BY CAPILLARY ZONE ELECTROPHORESIS', Journal of Chromatography A, vol. 632, no. 1-2, pp. 127-135.
    BEATTIE JH, RICHARDS MP, SELF R. SEPARATION OF METALLOTHIONEIN ISOFORMS BY CAPILLARY ZONE ELECTROPHORESIS. Journal of Chromatography A. 1993 Feb 19;632(1-2):127-135.
    BEATTIE, J H ; RICHARDS, M P ; SELF, R . / SEPARATION OF METALLOTHIONEIN ISOFORMS BY CAPILLARY ZONE ELECTROPHORESIS. In: Journal of Chromatography A. 1993 ; Vol. 632, No. 1-2. pp. 127-135.
    @article{47daeb379bc34c05815eb72b609de9f1,
    title = "SEPARATION OF METALLOTHIONEIN ISOFORMS BY CAPILLARY ZONE ELECTROPHORESIS",
    abstract = "The potential of capillary zone electrophoresis (CZE) for the analysis of metallothionein (MT) isoforms was investigated. CZE was performed using two different systems, (1) a laboratory-constructed instrument with an ISCO UV detector and (2) a Waters Quanta 4000 system. Capillaries were of 75 pm I.D. x ca. 1 m in length and loading times were up to 40 s by gravity or 4 s by electrokinetic migration at 30 kV. Samples were dissolved in 10 mM Tris-HCI buffer, pH 9.1, and electrophoresis was performed at 30 kV using a 50 mM Tris-HCI, pH 9.1 running buffer. Detection was by UV absorbance at 185 or 214 nm. Purified and semipurified MT samples were analysed for qualitative assessment of purity, relative isoform abundance and separation characteristics of MT from different species. As progress towards the development of a quantitative assay, the linearity of calibration curves and simple methods of sample preparation for analysis by CZE were investigated. Complete separation of a mixture of the two major MT isoforms was achieved in less than 5 min and the technique was found to be very useful for qualitative analysis of MT. Using a rabbit liver MT standard (500 mug/ml-1), a linear relationship was found between the gravity load time and the integrated peak area. Standard calibration curves were also linear and the detection limit for both CZE instruments under our separation conditions was 1-10 mug MT ml-1. The successful use of two solvent ''traction procedures for tissue samples demonstrated the potential of CZE for routine quantitative analysis of MT.",
    keywords = "HEMOGLOBIN AFFINITY ASSAY, LIVER METALLOTHIONEIN, SATURATION METHOD, PROTEINS, PEPTIDES, TISSUES",
    author = "BEATTIE, {J H} and RICHARDS, {M P} and R SELF",
    year = "1993",
    month = "2",
    day = "19",
    language = "English",
    volume = "632",
    pages = "127--135",
    journal = "Journal of Chromatography A",
    issn = "0021-9673",
    publisher = "Elsevier",
    number = "1-2",

    }

    TY - JOUR

    T1 - SEPARATION OF METALLOTHIONEIN ISOFORMS BY CAPILLARY ZONE ELECTROPHORESIS

    AU - BEATTIE, J H

    AU - RICHARDS, M P

    AU - SELF, R

    PY - 1993/2/19

    Y1 - 1993/2/19

    N2 - The potential of capillary zone electrophoresis (CZE) for the analysis of metallothionein (MT) isoforms was investigated. CZE was performed using two different systems, (1) a laboratory-constructed instrument with an ISCO UV detector and (2) a Waters Quanta 4000 system. Capillaries were of 75 pm I.D. x ca. 1 m in length and loading times were up to 40 s by gravity or 4 s by electrokinetic migration at 30 kV. Samples were dissolved in 10 mM Tris-HCI buffer, pH 9.1, and electrophoresis was performed at 30 kV using a 50 mM Tris-HCI, pH 9.1 running buffer. Detection was by UV absorbance at 185 or 214 nm. Purified and semipurified MT samples were analysed for qualitative assessment of purity, relative isoform abundance and separation characteristics of MT from different species. As progress towards the development of a quantitative assay, the linearity of calibration curves and simple methods of sample preparation for analysis by CZE were investigated. Complete separation of a mixture of the two major MT isoforms was achieved in less than 5 min and the technique was found to be very useful for qualitative analysis of MT. Using a rabbit liver MT standard (500 mug/ml-1), a linear relationship was found between the gravity load time and the integrated peak area. Standard calibration curves were also linear and the detection limit for both CZE instruments under our separation conditions was 1-10 mug MT ml-1. The successful use of two solvent ''traction procedures for tissue samples demonstrated the potential of CZE for routine quantitative analysis of MT.

    AB - The potential of capillary zone electrophoresis (CZE) for the analysis of metallothionein (MT) isoforms was investigated. CZE was performed using two different systems, (1) a laboratory-constructed instrument with an ISCO UV detector and (2) a Waters Quanta 4000 system. Capillaries were of 75 pm I.D. x ca. 1 m in length and loading times were up to 40 s by gravity or 4 s by electrokinetic migration at 30 kV. Samples were dissolved in 10 mM Tris-HCI buffer, pH 9.1, and electrophoresis was performed at 30 kV using a 50 mM Tris-HCI, pH 9.1 running buffer. Detection was by UV absorbance at 185 or 214 nm. Purified and semipurified MT samples were analysed for qualitative assessment of purity, relative isoform abundance and separation characteristics of MT from different species. As progress towards the development of a quantitative assay, the linearity of calibration curves and simple methods of sample preparation for analysis by CZE were investigated. Complete separation of a mixture of the two major MT isoforms was achieved in less than 5 min and the technique was found to be very useful for qualitative analysis of MT. Using a rabbit liver MT standard (500 mug/ml-1), a linear relationship was found between the gravity load time and the integrated peak area. Standard calibration curves were also linear and the detection limit for both CZE instruments under our separation conditions was 1-10 mug MT ml-1. The successful use of two solvent ''traction procedures for tissue samples demonstrated the potential of CZE for routine quantitative analysis of MT.

    KW - HEMOGLOBIN AFFINITY ASSAY

    KW - LIVER METALLOTHIONEIN

    KW - SATURATION METHOD

    KW - PROTEINS

    KW - PEPTIDES

    KW - TISSUES

    M3 - Article

    VL - 632

    SP - 127

    EP - 135

    JO - Journal of Chromatography A

    JF - Journal of Chromatography A

    SN - 0021-9673

    IS - 1-2

    ER -