Differential fluorescence labeling of cysteinyl clusters uncovers high tissue levels of thionein

Abstract

The isolation of thionein (T) from tissues has not been reported heretofore. T contains 20 cysteinyl residues that react with 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide to form fluorescent adducts. In metallothionein (MT) the cysteinyl residues, which are bound to zinc, do not react. However, they do react in the presence of a chelating agent such as EDTA. The resultant difference in chemical reactivity provides a means to measure T in the absence of EDTA, (MT + T) in its presence, and, of course, MT by difference. The 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide derivative of T can be isolated from tissue homogenates by HPLC and quantified fluorimetrically with a detection limit in the femtomolar range and a linear response over 3 orders of magnitude. Analysis of liver, kidney, and brain of rats reveals almost as much T as MT. Moreover, in contrast to earlier views, MT in tissue extracts appears to be less stable than T. The existence of T in tissues under normal physiological conditions has important implications for its function both in zinc metabolism and the redox balance of the cell.

Figure 1

Fluorescence of the ABD-F derivatives of MT and T under different reaction conditions. Final concentrations: 0.6 μM T or zinc MT, 0.2 M potassium borate-Cl⁻, pH 7.5, 2.7 mM ABD-F. Reaction mixtures were incubated for 5 min at 50°C, and then analyzed by HPLC as described in the legend of Fig. 2. Samples: 1, T + TCEP; 2, T + TCEP + EDTA; 3, MT + TCEP; 4, MT + TCEP + Zn; 5, MT + TCEP + EDTA; 6, MT + EDTA; 7, T + EDTA. Concentrations of TCEP, EDTA, and Zn(NO₃)₂: 18 mM, 6 mM, and 3 μM, respectively. Data are given as percentages relative to sample 5 (n = 3).

Figure 2

Separation of ABD-T by reversed-phase HPLC. Samples were treated with acetonitrile and then derivatized with ABD-F in the presence of TCEP and EDTA as described in Materials and Methods. Chromatography was performed with a Hypersil C4 column. Mobile phase: A, 5 mM Tris⋅HCl, pH 7.5; B, 50% 2-propanol in A. Flow rate: 1 ml/min. The column was washed with 100% A for 2 min, followed by a linear gradient from 0 to 75% B for 15 min and another wash with 75% B. Excitation and emission wavelengths were 384 nm and 510 nm, respectively. The fluorescence detector gain was set at ×100 scale expansion. Injection volume was 50 μl. The dotted line is the chromatogram of the ABD-F derivative of purified MT (23 pmol). The solid line is the chromatogram of the derivatized rat liver sample (2 mg total protein).

Figure 3

Relationship between fluorescence peak area and amount of ABD-T injected into the HPLC system. Different concentrations of zinc MT-2 were derivatized and then subjected to HPLC analysis as described in the legend of Fig. 2. Errors are too small to be displayed in the plot (n = 2).

Figure 4

SDS/PAGE of ABD-T fractions from rat liver and ABD-T. The sample was prepared as described in Materials and Methods, subjected to HPLC as described in the legend of Fig. 2. The ABD-T peak fraction was pooled, and then analyzed on a 10–20% Tris-Tricine SDS gel with fluorescence detected on a UV transilluminator. Lane 1, prestained protein standard. Lane 2, ABD-T fraction from rat liver. Lane 3, ABD-F modified, purified rabbit zinc MT-2.

Figure 5

Stability of MT and T in rat liver extract. Rat liver extract was stored on ice for different lengths of time. The amounts of total MT (MT + T), MT, and T in the liver extract were measured as described in Materials and Methods. Analyses were performed in duplicate.