The interaction of Fura-2 with Ca^2＋ is studied using steady fluorescence technique. The effect of pH on the spectra behavior of Fura-2 in the presence of Ca^2＋ is investigated, the excitation maxima （340 nm） and the isobestic point （360 nm） for the fluorescence spectra of Fura-2 depend on pH. At different temperatures the apparent dissociation constants （ Kd ） of Fura-2-Ca^2＋ complex are examined, Kd is found to decrease with increasing temperatures （20 ℃, 37 ℃, 50 ℃ ） and △His calculated to be 21.16 kJ/mol by using the Van＇ t Hoff equation at pH 7.4 for all the temperatures tested. The determination of intracellular Ca^2＋ concentration （ [Ca^2＋ ] i ） in lymphocyte is developed by using Fura-2 as a fluorescence probe in the presence of Cefotaxime at 37 ℃ and pH 7.4.
The effect of penicillin on the human peripheral lymphocytes was studied by steady fluorescent technique and ratiometric fluorescence dye, Fura-2, The change of the free calcium concentration in cytosol was examined under different conditions. A characterization of Fura-2-Ca interaction in an isotonic saline solution showed that Ca^2＋ formed a 1：1 Fura-2-Ca complex with the apparent dissociation constant 1.81×10^-7 mol/L. The mechanism, by which penicillin induced the decrease of [Ca^2＋]i , was discussed in detail. The low dose of penicillin might modify the lymphocytes＇ immunology response by interfering the increase in the intracellular free calcium concentration.
Hai Yan WANGDan Dan WANGChun Gui ZHAOYe Hong ZHOUShao Min SHUANGChuan DONG
