Metallothionein Measurement Experiment

Summary

Metallothionein is a short cysteine-rich peptide produced by microorganisms and plants as a metal-binding protein with a high affinity for a wide range of heavy metals. It is a protein with low molecular mass and very high content of cysteine residues and metals. The metals to which it binds are mainly cadmium, copper and zinc, and it is widely found in a wide range of organisms from microbes to humans, and its structure is highly conserved.

Operation method

Cadmium-hemoglobin method

Principle

MT is a low molecular weight protein that contains high amounts of cysteine and has a high affinity for metals.

Materials and Instruments

Bovine hemoglobin
Cadmium chloride Tris-HCl Sucrose
Spectrophotometer Centrifuge

Move

I. Experimental steps

1. Tissue was added to 0.25 mol/l sucrose solution at 1:4 (w/v) to prepare a homogenate, and centrifuged at 18 000 g for 20 min (4°C).

2. 0.5 ml of supernatant was taken and 1.9 ml of 0.03 mol/l Tris-HCl buffer (pH 8.0) was added.

3. Add 1 ml CdCl2 (10 ppm) and mix well, incubate for 5 min at room temperature.

4. Add 0.2 ml of 2% bovine hemoglobin, boil water bath for 1 min, and centrifuge at 1,000 g for 5 min at room temperature.

5. Repeatedly add bovine hemoglobin, heat and centrifuge 3 times.

6. Determine the Cd content of the supernatant by graphite method in an atomic absorption spectrophotometer.

7. Tissue MT content was calculated as 6 g of atomic Cd bound moleculable MT.

8. Protein content of the supernatant was determined by the Bradford method.

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