Structural changes in metallothionein isoforms revealed by capillary electrophoresis and Brdicka reaction.
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.
Department of Chemistry and Biochemistry, Faculty of Agronomy, Mendel University in Brno, Brno, Czech Republic.
Marketa
M
Ryvolova
David
D
Hynek
Helena
H
Skutkova
Vojtech
V
Adam
Ivo
I
Provaznik
Rene
R
Kizek
D000595:Amino Acid Sequence; D000818:Animals; D001669:Biochemical Phenomena; D014408:Biomarkers, Tumor / Q000032:analysis / Q000737:chemistry; D011232:Chemical Precipitation; D019075:Electrophoresis, Capillary / Q000379:methods*; D006358:Hot Temperature; D016014:Linear Models; D008668:Metallothionein / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D020033:Protein Isoforms; D011817:Rabbits; D012680:Sensitivity and Specificity; D016415:Sequence Alignment; D013056:Spectrophotometry, Ultraviolet; D014508:Urea / Q000737:chemistry
D014408:Biomarkers, Tumor; D020033:Protein Isoforms; D014508:Urea; D008668:Metallothionein