Summary
The present article reviews the most significant results obtained over the last five years on the use of HPLC to study mammalian MT polymorphism.
Firstly, it has been shown that reverse phase HPLC with electrochemical (EC, coulometric) detection allowed us to follow the chemical evolution of a trithiolic hexapeptide intrinsic to the MT structure. The EC response is proportional to the number of free thiols, which permits the identification of the reduced and oxidised forms of the peptide which are well separated.
This EC detection was then applied to MT characterisation. Four MTs from rabbit liver and horse kidney were subjected to RP-HPLC (TFA/acetonitile) with on-line UV and EC detection. They were found to exhibit a different polymorphism. Actually, two types of peaks were observed, those equally detected by UV and by EC being attributed to original thiol containing sub-isoforms and those less hydrophobic, detected by UV but hardly at all by EC, being attributed to oxidised forms containing disulphides instead of thiols, hence not detectable in our EC mode. The elutions were then carried out at various temperatures between 25 and 60 °C. This appears to have two main effects: small retention time decrease of all peaks with increasing temperature and large effect on peak “detectability”: when the temperature rises, a drastic alteration of the oxidised peaks is observed. As a result, between 20 and 40°C all peaks are detected and resolved, while at 60 °C only those peaks assumed to be the original sub-isoforms, hence heat-stable, are present.
Previous to that, separation of MT by size exclusion chromatography had shown that they were all partially dimerised (5 to 15% of molar rates of dimers). Although small, these levels of non-monomer MT are not negligible.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
Suzuki KT, Imura N, Kimura M (1993) Metallothionein III. Birkhäuser Verlag, Basel, and references cited therein.
Nordberg M, Kojima Y (1979) Metallothionein and other low molecular weight metal-binding proteins. In: JHR Kägi, M Nordberg (eds): Metallothionein. Birkhäuser Verlag, Basel.
Cherian MG, Chan HM (1993) Biological functions of metallothionein — A review. In: KT Suzuki, N Imura, M Kimura (eds): Metallothionein III. Birkhäuser Verlag, Basel.
Klauser S, Kägi JHR, Wilson KJ (1983) Characterisation of isoprotein patterns in tissue extracts and isolated samples of metallothionein by reverse-phase high-pressure liquid chromatography. Biochem J 209: 71–80.
Richards MP, Steele NC (1987) Isolation and quantification of metallothionein isoforms using reversed-phase high-performance liquid chromatography. J Chromatogr 402: 243–256.
Van Beek H, Baars AJ (1988) Isolation and quantification of cadmium-, zinc- and copper-metallothioneins by high-performance liquid chromatography — atomic absorption spectrometry. J Chromatogr 442: 345–352.
Richards MP (1989) Characterisation of the metal composition of metallothionein isoforms using reversed-phase high-performance liquid chromatography with atomic absorption spectrophotometric detection. J Chromatogr 482: 87–97.
Beattie JH, Richards MP, Self R (1993) Separation of metallothionein isoforms by capillary zone electrophoresis. J Chromatogr 632: 127–135.
Richards MP, Beattie JH (1993) Characterisation of metallothionein isoforms — Comparison of capillary zone electrophoresis with reversed-phase liquid chromatography. J Chromatogr 648: 459–468.
Richards MP (1991) Purification and quantification of metallothioneins by reversed-phase high-performance liquid chromatography. Methods Enzymol 205: 217–238.
Wan M, Kägi JHR, Hunziker PE (1993) Resolution and quantification of four metallothionein isoforms from rabbit liver kidney cells. Protein Express Purif 4: 38–44.
Liu G, Wang W, Shan X (1994) Factor influencing the separation of metallothioneins by capillary zone electrophoresis. J Chromatogr 653: 41–46.
Beattie JH, Richards MP (1994) Separation of metallothionein isoforms by micellar electrokinetic capillary chromatography. J Chromatogr 664: 129–134.
Minami T, Matsubara H, O-higashi M, Otaki N, Kimura M, Kubo K, Okabe N, Okazaki Y (1996) Identification of metallothionein isoforms with capillary zone electrophoresis using a polyacrylamide-coated tube. J Chromatogr 685: 353–359.
Bordin G, Cordeiro Raposo F, Rodriguez AR (1994) Contribution à l’étude du polymorphisme de quelques métallothionéines par chromatographie liquide de haute performance. Can J Chem 72: 1238–1245.
Bordin G, Cordeiro Raposo F, Rodriguez AR (1996) Characterisation of metallothionein isoforms by reverse phase high performance liquid chromatography with on-line UV and electrochemical detection. J Liq Chromatogr Relat Technol 19: 3085–3104.
Bordin G, Cordeiro Raposo F, Rodriguez AR (1998) Effect of temperature variation on metallothionein subisoform separation by reverse phase high performance liquid chromatography. J Liq Chromatogr Relat Technol 21: 2039–2060.
Virtanen V, Bordin G, Rodriguez AR (1996) Separation of metallothionein isoforms with capillary zone electrophoresis using an uncoated capillary column. Effects of pH, temperature, voltage, buffer concentration and buffer composition. J Chromatogr 734: 391–410.
Bordin G, Cordeiro Raposo F, Rodriguez AR (1994) Chromatographic characterisation of a hexapeptide containing three thiol groups, intrinsic to the metallothionein structure. Chromatographia 39: 146–154.
Lee KK, Black JA, Hodges RS (1991) Separation of intrachain disulfide bridged peptides from their reduced forms by reverse phase chromatography. In: CT Mant, RS Hodges (eds): High Performance Liquid Chromatography of peptides and proteins. CRC Press, 389–398.
Suzuki KT, Yamamura M (1980) Isolation and characterisation of metallothionein dimers. Biochem Pharmacol 29: 689–692.
Klein D, Sato S, Summer SH (1994) Quantification of oxidised metallothionein by a cadmium saturation method. Anal Biochem 221: 405–409.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1999 Springer Basel AG
About this chapter
Cite this chapter
Bordin, G., Raposo, F.C., Rodriguez, A. (1999). Mammalian metallothionein sub-isoform separation by RP-HPLC with online UV and electrochemical detection. In: Klaassen, C.D. (eds) Metallothionein IV. Advances in Life Sciences. Birkhäuser, Basel. https://doi.org/10.1007/978-3-0348-8847-9_10
Download citation
DOI: https://doi.org/10.1007/978-3-0348-8847-9_10
Publisher Name: Birkhäuser, Basel
Print ISBN: 978-3-0348-9799-0
Online ISBN: 978-3-0348-8847-9
eBook Packages: Springer Book Archive