Separation Of Metallothionein Isoforms Research Articles

The influence of experimental conditions and of organic solvents as modifiers on the separation of metallothionein isoforms by capillary zone electrophoresis in an uncoated capillary column

Putative metallothionein (MT) isoforms are readily separated by capillary zone electrophoresis (CZE) with a mixture of tris(hydroxymethyl)aminomethane (Tris) and borate buffers in uncoated polyimide-cladded fused-silica capillary tubing. The influence of buffer concentration and composition, pH, running voltage and temperature on the electroosmotic mobility and on the electrophoretic mobilities of model samples (rabbit liver and horse kidney MT) have been investigated. The use of organic solvents as buffer modifiers and their effect on the separation of putative metallothionein isoforms is also described. It is shown that organic solvent modification significantly enhances the resolution—with organic solvents as modifiers it is possible to obtain a good separation of putative isoforms of rabbit liver MT in one run whereas two separate runs were needed at different pHs or at different temperatures when the separation was performed with Tris-borate buffer without organic-solvents as modifiers were found to be solventspecific although the trends were in the same direction (i.e. decreasing electroosmotic mobility with increasing percentage of organic solvent). Differences in effect on individual putative MT isoforms were observed, indicative of different chemical and physical characteristics of the isoforms. A difference in polymorphism of horse kidney MT and rabbit liver MT is clearly apparent.

Isoform separation of metallothioneins by capillary zone electrophoresis with Tris–tricine buffer in the presence or absence of methanol

A simple capillary electrophoretic method for analysis of metallothionein (MT) isoforms has been developed. Rabbit liver metallothionein (MT) isoforms are readily separated by capillary zone electrophoresis (CZE) with 150 mM Tris(hydroxymethyl)aminomethane (Tris)–150 mM N-Tris(hydroxymethyl)methylglycine (Tricine) buffer (pH 7.50) using an uncoated polyimide cladded fused-silica capillary column thus offering an alternative method for MT separations under original conditions. The effects of buffer concentration, buffer pH and buffer composition on the separation of MT isoforms were investigated. The effect of the addition of organic solvent, MeOH, as buffer modifier on the separation was also studied. Addition of MeOH to the buffer system enhanced the separation and additional information of the heterogeneity within each isoform group was gained. Also the effect of pH on the separation is enhanced when using organic solvent modified buffer. Enhanced information is gained by using photodiode array detection allowing recording of UV spectra of the putative isoform peaks thus offering the possibility of characterisation of the peaks obtained by comparing them to the unique characteristics of metallothionein metal–thiol bonds. The Tris–tricine method presented in this paper allows the separation of peaks that were found to need two separate runs under different conditions for separation to occur when using Tris–borate buffer. Rabbit liver MT, theoretically a mixture of MT-1 and MT-2, exhibited two major peaks, one medium sized peak and several smaller peaks. The experiments indicate that rabbit liver MT exhibited peaks which were not found in either MT-1 or MT-2 isoforms or they were found only as small residues. Both MT-1 and MT-2 samples exhibited one major peak and several smaller peaks. Residues of MT-2 were found in the MT-1 sample and vice versa.

Multiple isoforms of metallothionein are expressed in the porcine liver.

Isogenes are highly homologous to each other and are often difficult to ascertain, as has been the case with metallothionein, a metal-binding protein rich in cysteines. Conventional separation of metallothionein isoforms relied on ion exchange chromatography of the proteins, or screening for the sequences from gene libraries. In this study, a combination of RT-PCR and partial protein sequencing is used in the identification of metallothionein isogenes expressed in porcine liver. By this approach, we have identified expressed coding sequences which constitute 10 new isogenes. Of the four known groups of metallothioneins (MT), phylogenetic analyses place these pig isogenes in the MT-1 group, except two which are identified as being closely related to MT-2, and none in groups 3 and 4. The isogenes are thus namedpMT-1a to -1g, andpMT-2a and -2b. While each of the isogene sequences is unique, two isogenes,pMT-1e1 andpMT-1e2, share an identical amino acid sequence, differing only in specific codons. Two others,pMT-1b andpMT-1g, have a cysteine substituted by arginine, the first such sequence ever detected in MT.pMT-2a andpMT-2b are closely aligned with the MT-2 group of vertebrates, in spite of the absence of a characteristic acidic amino acid at position 10 or 11, common in other mammalian metallothioneins.

Separation of metallothionein isoforms by capillary zone electrophoresis

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 μmI.D. × 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 m M Tris-HCl buffer, pH 9.1, and electrophoresis was performed at 30 kV using a 50 m M Tris- HCl, 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 μg/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 μg MT ml −1. The successful use of two solvent extraction procedures for tissue samples demonstrated the potential of CZE for routine quantitative analysis of MT.

Improved separation of perch liver metallothionein by fast protein liquid chromatography

The separation of metallothionein isoforms by Mono-Q column chromatography by fast protein liquid chromatography is described and compared to separation on a DEAE-Sephadex A-25 ion-exchange column. The separation of two isoforms of rainbow trout ( Salmo gairdneri) metallothionein was possible by DEAE-Sephadex A-25 chromatography, while only one form of perch ( Perca fluviatilis) metallothionein was obtained with this method. However, by applying FPLC on a Mono-Q column it was possible to separate two isoforms of perch metallothionein. When comparing the chromatographic behaviour of rainbow trout and perch metallothioneins it was found that the two isoforms of perch metallothionein were eluted closer together than the two rainbow trout metallothioneins. Both forms of perch and rainbow trout metallothioneins lacked aromatic amino acids and histidine, and had a high cystein content (30 mol%). The molecular weight was estimated to be 6000 for the apothioneins. The results from this study demonstrate the usefulness of FPLC to improve the resolution of metallothionein separations.