Fig. 1. HPLC elution profile of purified rabbit liver MT-I after mBBr derivatization. Solid line, fluorescence of mBBr-MT-I; dotted line, acetonitrile percentage.

Fig. 2. Effect of acetonitrile content on elution of mBBr-MT. Mobile phase was held for 10 min at 0.3 B, and then raised to various acetonitrile contents by increasing by 1-min the percentage (60–90%) of solvent B. After 10 min under each condition, 0.3 B was re-established in 1-min. Areas of peaks 1 (●) and 2 (○) are shown in arbitrary units under each condition and their relative contributions are indicated in parenthesis.

Fig. 3. HPLC elution profiles of mBBr derivatives of purified rabbit liver MT-I (A), control without MT-I addition (B), and clam extract (C). Solid lines, fluorescence of mBBr-derivatives; dotted lines, acetonitrile percentage.

Fig. 4. Effect of pH on the MT labeling with mBBr.

Fig. 5. Stability of mBBr-MT complex from clam extracts. Data show the mean ± SD of the three independent analyses carried out at each time.

Fig. 6. Electrophoresis of MTs from rabbit liver and clam extract. Proteins were analyzed by SDS-PAGE and the fluorescence of mBBr-labeled (A) and the absorbance of Coomassie-stained proteins (B) was assessed. Lanes 1 and 7: 14 μg of standard proteins of indicated Mr; lane 2: 5 μg of unlabeled rabbit MT-I; lane 3: 5 μg of mBBr-MT-I from rabbit; lane 4: 10 μg of rabbit mBBr-MT-I after RP-HPLC; lane 5: 52 μg of total mBBr-labeled protein from clam extract; lane 6: 12 μg of mBBr-labeled MT from clam extract after RP-HPLC (10 runs).

Fig. 7. MT concentration in clams from sites with different metal levels. The MT content was assayed by RP-HPLC with fluorescent detection (empty bars) and by the colorimetric assay after DTNB reaction (solid bars, data from [28]). The statistical significance of the differences is compared with Almerimar.