Analysis of metallothioneins by the modified Brdička procedure

Quantitative analysis of metallothioneins (MTs) by the modified Brdička procedure is based on the evaluation of the catalytic hydrogen signal (Cat). The effect of the basic physico–chemical parameters, the temperature and the depolarizer concentration (Co(NH3)6Cl3), on the Cat signal height and the respective calibration straight lines was studied by means of the differential pulse (DP) mode at the hanging mercury drop electrode (HMDE). The temperature was varied from 7 to 18°C, and the depolarizer concentration from 1.2×10−4 to 1.0×10−3 M Co(NH3)6Cl3. The commercially available rabbit liver MT was used as the calibrant. The parameters of the calibration straight lines, the standard errors of the intercept sa and the slope sb were evaluated by means of the least-square method. The hypothesis of a significant difference of the estimated value of the intercept on y-axis compared to the zero value was statistically tested. For those conditions for which this hypothesis was rejected the measuring conditions of the catalytic hydrogen signal are selected and they refer to a buffer 1M (NH4Cl+NH4OH), pH 9.5, (7.0±0.5)°C and the depolarizer concentration (0.8 or 1.0)×10−3 M Co(NH3)6Cl3.