Kanenobu Kubo

Study of metallothionein using capillary zone electrophoresis

Metallothioneins (MTs) have many different functions in tissues, but the roles of individual isoforms are still not entirely clear. Capillary zone electrophoresis (CZE) is a powerful method for the separation of substances because of its small sample requirement, rapid analysis, high sensitivity and high resolution. The separation and identification of mammalian MT-1, MT-2, and MT-3 and class III MTs by CZE has been reported. Uncoated and polyacrylamide-coated capillary tubes were recently used for the separation of MTs, and a UV detector is usually employed for observations of peaks of MTs. Small changes to the structure and metal components of MTs are reflected in the migration times of the peaks. N-acetylated and non-acetylated MTs can be separated and identified by CZE-mass spectrometry (MS). In addition, metal complexes with MTs can be characterized by CZE-proton-induced X-ray emission (PIXE) detector and CZE-inductively coupled plasma (ICP)-MS. For the quantification of an MT isoform, the peak area of UV absorption is used, but the technique has problems. One is lack of a purified isoform standard. The other is the need for a suitable internal standard substance. CZE-ICP-isotope dilution (ID)-MS is also reported to be able to quantify MT isoforms. CZE combined with other techniques is very effective for separation and quantitative and qualitative analyses of MT isoforms in biological materials.

Sodium dodecyl sulfate-protein polypeptide complexes in 8 M urea with special reference to sodium dodecyl sulfate-polyacrylamide gel electrophoresis

The effects of 8 M urea on the complexes formed between sodium dodecyl sulfate and protein polypeptide were found to be as follows: (1) The maximum amount of SDS bound is reduced by almost half, and the minimum equilibrium concentration of SDS necessary to reach saturation was nearly doubled; (2) The apparent content of alpha-helical structure deduced from CD measurement is reduced to only 50--70% of that in the presence of sodium dodecyl sulfate alone; (3) The effective size of the sodium dodecyl sulfate-protein polypeptide complex deduced from viscosity measurements is increased, but is still smaller than the effective size of the protein in 8 M urea alone.

Determination of metallothionein-1/metallothionein-2 ratios in the mouse liver and pancreas by capillary zone electrophoresis using a polyacrylamide-coated capillary at neutral pH.

Metallothionein (MT) isoforms, MT-1 and MT-2, in biological specimens are clearly separated by capillary zone electrophoresis (CZE) using a polyacrylamide-coated capillary. The effectiveness of CZE analysis in the study of MT isoforms in biological specimens is discussed. We did two experiments to determine the MT-1/MT-2 ratio in biological specimens. The ratio of MT-1/MT-2 can be determined by CZE under a neutral pH without any detergents. One of these studies is time-dependent changes of the MT-1/MT-2 ratio in the cytosol of the pancreas and liver in mice after Zn or Cd injection. In the pancreas, both isoforms were detected in the control mice and the ratio of MT-1/MT-2 was below 1.0. When Zn was injected, the maximum peak areas of both isoforms were obtained at 24 h, and the ratios increased over a value of 1.0 at 3 h and peaked at 10 h. However, in the Cd-injected mice, the peak areas of both isoforms increased up to 72 h, and the ratios were below 1.0 up to 72 h. On the contrary, neither isoform was detected in the livers of control mice. The ratios of Zn-injected mice liver were near the value 1.0 between 6 and 72 h, although the areas of both isoforms showed peaks at 48 h. The ratios of Cd-injected mice livers were detected to be over 1.0 from 10 h, but there were no significant difference between 10 and 72 h, and the areas of both isoforms showed peaks at 24 h. The other experiment investigated the ratio in each fraction of cell fractionation. Cell fractionation was done in the livers of Zn-treated mice. Twenty-four hours after the injection, the ratio of MT-1/MT-2 was 0.80+/-0.12 and 1.19+/-0.21 (mean+/-SD) in nuclear and cytosol fractions, respectively. Neither isoform was detected in mitochondrial or microsomal fraction. From the present results, CZE analysis is a suitable method for observation of the ratio of MT-1/MT-2 in biological specimens, and dynamic changes in both isoforms can be detected.

Electrophoretic behavior of micellar and monomeric sodium dedecyl sulfate in polyacrylamide gel electrophoresis with reference to those of SDS-protein complexes

Abstract Electrophoretic behavior of sodium dodecyl sulfate (SDS) in polyacrylamide gels has been examined at various gel concentrations. Micellar SDS is subject to significant molecular sieving from the gels while monomeric SDS is virtually free from the effect. The two forms are in a rapid equilibrium with each other. The gel concentration, therefore, has a signifieant effect on the electrophoresis of SDS added in SDS-polyacrylamide gel electrophoresis. The simplified procedure of Stoklosa and Latz (1974, Biochem. Biophys. Res. Commun. 58 , 74–79), in which SDS is added only in sample solutions, has been criticized based on the results obtained.

Simple visualization of protein bands in SDS-polyacrylamide gel electrophoresis by the insoluble complex formation between SDS and a cationic surfactant.

Abstract Protein bands in SDS-polyacrylamide gel electrophoresis can be visualized as white bands by the mere immersion of a gel, after electrophoresis, into a dilute aqueous solution of cationic surfactant with which SDS forms an insoluble complex. Because the time-consuming and laborious procedure of staining is eliminated, this procedure is particularly suited for quick detection of protein bands after electrophoresis

Rapid identification of metallothionein isoforms in liver cytosol fraction by capillary zone electrophoresis using EDTA.

Identification of metallothionein (MT) isoforms on capillary zone electrophoresis (CZE) analysis was studied using a linear polyacrylamide-coated capillary at pH 7.4 and EDTA. The CZE system was able to separate standard (purified and commercially available) MT specimens into their isoforms within 10 min. The peaks of MT-1 and MT-2 isoforms disappeared on addition of EDTA to the specimen, and the disappearance was shown to be time-dependent and dose-dependent, although the reason why the peaks decreased is still unclear. A heat-treated cytosol fraction prepared from Zn-injected mouse liver showed many major and minor peaks on CZE analysis. Two major peaks were identified to be MT-1 and MT-2, respectively, by co-injection with the purified MT isoforms. When EDTA was added to the cytosol fraction, the two major peaks, MT-1 and MT-2, and three other minor peaks disappeared time-dependently. Therefore, each MT isoform in the cytosol fraction can be identified by the addition of EDTA, also the peaks are identified by the corresponding migration times of purified MTs. Unknown substances like MT sub-isoforms may also be detected, although this question warrants clarification. From these results, it was concluded that the addition of EDTA is useful for identification of MT isoforms in cytosol fractions on CZE analysis.

Capillary zone electrophoresis of albumin‐depleted human serum using a linear polyacrylamide‐coated capillary: Separation of serum α‐and β‐globulins into individual components

The separation of human serum globulins into individual components was investigated by capillary zone electrophoresis (CZE) using a linear polyacrylamide‐coated capillary at pH 7.4. Prior to CZE analysis of globulin components present in serum, it was found that it was necessary to remove albumin. Preparation of albumin‐depleted human serum with a HiTrap Blue column allowed the detection of α‐ and β‐globulin components as a series of peaks. Almost all the peaks, both narrow and broad, observed in CZE analysis could be assigned to six globulin components (α1‐acid‐glycoprotein, α1‐antitrypsin, haptoglobin, α2‐macroglobulin, Gc‐globulin, and transferrin) by using the technique of antibody‐based indirect detection. The CZE results, obtained from serum preparations from three healthy adults and six patients, showed that the CZE system might be capable of detecting qualitative differences among individuals with regard to individual globulin components.

Identification of metallothionein isoforms on capillary zone electrophoresis by adding anti-metallothionein antibody.

The aim of this study was to identify metallothionein (MT) isoforms in mouse liver by using capillary zone electrophoresis (CZE). Purified MT-1 and MT-2 isoforms were completely separated by CZE using a polyacrylamide-coated tube at physiologic pH. There were two peaks in the cytosol fraction prepared from zinc-injected mouse liver, in which the migration times corresponded with those of purified MT-1 and MT-2 isoforms. When anti-MT monoclonal antibody was added with the purified MT-1 or MT-2 solution, the peaks decreased. Furthermore, the two peaks in the cytosol prepared from Zn-injected mouse liver decreased in a time-dependent manner from the electropherogram after the addition of the antibody. Therefore, those peaks were identified as MT-1 and MT-2 isoforms, respectively. In conclusion, the addition of anti-MT monoclonal antibody to the cytosol fraction of tissues is an effective method for identification of MT isoforms after separation using CZE.

Solubilization of oil-soluble dyes by sodium dodecyl sulfate-protein polypeptide complexes with reference to SDS-polyacrylamide gel electrophoresis

Sodium dodecyl sulfate binds to the linear polypeptide derived from a protein to form micelle-like clusters (Takagi, T., Tsujii, K. and Shirahama, K. (1975) J. Biochem. 78, 939--947) which are expected to solubilize lipophilic materials. Such clusters were found to afford the complexes strong solubilizing power against oil-soluble dyes, comparable or slightly superior to SDS micelles, by conventional solubilizing technique (equilibration with solid dye) and by gel chromatography technique first applied to measure solubilization by such complexes. Solubilization behavior of the bound SDS was insensitive to the kind of protein polypeptide, in contrast to the variety found in the pioneering similar study on the complexes between SDS and initially native proteins (Steinhardt, J., Stocker, N., Carroll, D. and Birdi, K.S. (1977) Biochemistry 16, 718--725). The knowledge of the solubilizing power of SDS-protein polypeptide complexes seems to be valuable in design of experiments and interpretation of results obtained in application of SDS-polyacrylamide gel electrophoresis to samples containing lipophilic materials.

Separation of metallothionein isoforms of mouse liver cytosol by capillary zone electrophoresis

Metallothionein (MT) isoforms of mouse liver cytosol were separated by capillary zone electrophoresis (CZE) using a polyacrylamide-coated tube at neutral pH, samples prepared from non-treated, heat-treated, and ethanol-precipitated specimens were compared. The liver was homogenized in three kinds of media, 0.25 M sucrose containing 100 mM Tris-HCl buffer at pH 7.4 (BS), BS containing 1% ascorbic acid (BS-C), and BS containing 5 mM beta-mercaptoethanol (BS-M). Mouse liver was used 24 h after subcutaneous injection of 50 mg Zn kg(-1). In the non-treated specimen of the cytosol fraction, the MT-2 isoform was separated in all three media, while the MT-1 isoform was difficult to identify. In the ethanol-precipitated specimen, MT isoforms were separated well using either BS or BS-C. However, when BS-M was used, a small MT-2 peak was obtained the MT-1 peak could not be identified. MT-1 isoform in the heat-treated specimen was difficult to identify. In contrast, MT-2 isoform was separated well in all three kinds of media. In the non-treated specimen of the control liver cytosol, the MT-2 isoform was detected using all three media, the MT-1 peak was undetected. Based on these results, MT isoforms can be detected in the crude cytosol fraction of liver using CZE combined with a polyacrylamide-coated tube at neutral pH.