Metallothionein: structure/antigenicity and detection/quantitation in normal physiological fluids

Abstract

Recent experiments in the application of radioimmunoassay (RIA) in the detection and quantitation of metallothionein (MT) in human sera and urines demonstrate that it is possible to extend the lower limit of practical quantitation from the previous limit of 50-100 pg to 1 pg.RIA of normal sera indicates that the typical range of concentrations of MT is from less than 0.01 ng/mL to about 1 ng/mL, and that concentrations above 2 ng/mL should be considered abnormal. The typical range for normal urines is from less than 1 ng/mL to 10 ng/mL; concentrations above 10 ng/mL should be considered abnormal. A complementary assay, the enzyme-linked immunosorbent assay (ELISA), is under development. The ELISA is a competitive binding assay, detection and quantitation of MT being either by colorimetric or fluorimetric methods. The present useful range for MT quantitation in the ELISA is from about 50-50000 pg (fluorimetric) or 500-5000 pg (colorimetric). Recent experiments using the RIA have identified the principal antigenic determinants of vertebrate MTs as involving the immediate amino terminal residues (-MDPNC-) and the segment including residues 20-25 (-KCKECK- in human MT). Theoretical predictions of secondary structure based on hydrophilicity and sequence analysis indicate that the conformational profile is dominated by tetrapeptide candidates for beta turns (reverse turns) with 2-3 hexapeptide sequences being candidates for helical conformation and 4-5 short sequences (3-5 residues) being candidates for beta chain conformation. The helical candidates are predicted to be unstable and the analysis favors reverse turns for both determinants of vertebrate MT and a sequestered location for the joining region between clusters A and B.