Brdicka Reaction Dotaz Zobrazit nápovědu
Metallothionein (MT) as a potential cancer marker is at the center of interest and its properties, functions and behavior under various conditions is intensively studied. In the present study, two major mammalian MT isoforms (MT-1 and MT-2) were separated using capillary electrophoresis (CE) coupled with UV detector in order to describe their basic behavior. Under the optimized conditions, the separation of both isoforms was enabled as well as estimation of detection limits as subunits and units of ng per μL for MT-2 and MT-1, respectively. Further, the effects of thermal treatment and the presence of denaturing agent such as urea on MT-1 and MT-2 isoforms were studied by CE-UV. Thermal treatment caused an increase in the signals of both isoforms. A new parameter called precipitation rate has been defined based on this finding. This parameter can be expressed as a slope of the linear regression of the time dependency curve recalculated on the MT concentration. The thermal precipitation rate for MT-1 and MT-2 was determined as 1.1 and 0.9 ng of MT/min, respectively. The chemical precipitation rate calculated from the linear regression for both isoforms provided the same value of 0.25 ng of MT/min. The results were confirmed by manual spectrometric measurements and by differential pulse voltammetry Brdicka reaction. Based on these results, a model of MT behavior under the conditions studied was suggested.
- MeSH
- biochemické jevy MeSH
- chemická precipitace MeSH
- chemické modely MeSH
- denaturace proteinů MeSH
- elektroforéza kapilární metody MeSH
- králíci MeSH
- lineární modely MeSH
- metalothionein chemie metabolismus MeSH
- močovina chemie MeSH
- molekulární sekvence - údaje MeSH
- nádorové biomarkery analýza chemie MeSH
- protein - isoformy MeSH
- sekvence aminokyselin MeSH
- sekvenční seřazení MeSH
- senzitivita a specificita MeSH
- spektrofotometrie ultrafialová MeSH
- vysoká teplota MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
BACKGROUND: Metallothioneins (MTs) constitute a family of evolutionary conserved low molecular weight proteins with small variations in their amino acid sequences. They play a role in the regulation of trace metals metabolism, in the detoxification of heavy metal ions and in mechanisms controlling growth, differentiation and proliferation of cells. OBJECTIVES: The aim of this study was to evaluate the human and rabbit MTs purity and characterization using advanced analytical approaches. Due to the common use of MT from rabbit liver as a model protein, the properties of the rabbit and human MTs were compared. MATERIAL AND METHODS: Capillary electrophoresis (CE), matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and Brdicka reaction were used for human and rabbit MTs characterization. RESULTS: In chip CE analysis, changes in the range of 5-8 kDa corresponding to the MT monomer, as well as some peaks of 13-14 kDa corresponding to dimers in both species, were observed. Using MALDI-MS, rabbit (MT-2D) and human (MT-1A, MT-1G, MT-1G + Cd and MT-2A) MTs were identified. In the Brdicka reaction analysis, a lower concentration of MTs from both organisms coincided with a decrease in the signal corresponding to MT level (Cat2). However, human MT gave higher Cat2 peak than the same concentration (0.025 mg/mL) of rabbit MT. CONCLUSIONS: The applied methods allowed for the characterization of MTs and gave complementary information about MT isoforms. Altered electrochemical activity of human and rabbit MTs, despite the same number of -sulfhydryl (-SH) groups, was observed, which may be due to different availability of MT cysteinyl groups.
- MeSH
- elektroforéza kapilární metody MeSH
- králíci MeSH
- lidé MeSH
- metalothionein analýza MeSH
- protein - isoformy MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- zvířata MeSH
- Check Tag
- králíci MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- MeSH
- transplantace kůže imunologie MeSH
- transplantační imunologie * MeSH
- Publikační typ
- časopisecké články MeSH
Silver solid amalgam electrode (AgSAE) was used for differential pulse voltammetric (DPV) measurements of cysteine and cysteine-containing peptides, glutathione, gamma-Glu-Cys-Gly and phytochelatin (gamma-Glu-Cys)(3)-Gly (PC3), in the presence of Co(II) ions. It had been established earlier that cysteine-containing peptides and proteins catalyze hydrogen evolution at mercury electrodes in presence of cobalt salts; these processes are known as the Brdicka reaction. DPV signals measured with the AgSAE, the surfaces of which had been modified by mercury meniscus or mercury film, were qualitatively the same as those obtained with the hanging mercury drop electrode (HMDE). With these electrodes the number and the intensity of Brdicka signals of cysteine, glutathione and PC3 differed, making a distinction among them possible. On the other hand, with the polished silver solid amalgam electrode (the surface of which was completely free of liquid mercury) all three compounds produced only one but strikingly intense peak in the region of Brdicka reaction. Using this signal, cysteine, glutathione as well as PC3 could be determined at 10(-8)M level, representing sensitivity up to 2 orders of magnitude better than attained with the mercury-modified AgSAEs or HMDE.
The expression of drebrin, a cytoskeletal protein newly estimated by expression profiling to correlate with the genotype and prognosis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL), was examined by independent methods. After demonstrating its higher expression in TEL/AML1(pos) BCP-ALL by quantitative reverse transcriptase polymerase chain reaction, we developed an anti-drebrin monoclonal antibody (mAb). In a cohort of 86 children with BCP-ALL, we found increased expression of drebrin in TEL/AML1(pos) ALL. In conclusion, relationship of drebrin expression and prognosis or genotype can now be assessed using flow cytometry.
- MeSH
- akutní nemoc MeSH
- fúzní onkogenní proteiny MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- monoklonální protilátky imunologie MeSH
- myši MeSH
- neuropeptidy imunologie metabolismus MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- pre-B-buněčná leukemie genetika imunologie metabolismus MeSH
- prognóza MeSH
- protein PEBP2A2 MeSH
- průtoková cytometrie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
