Reagents needed:

From Sigma:

p-hydroxymercuriphenyl sulfonate

4-(2-pyridylazo)resorcinol (PAR)

Dialyse protein extensively against TNG buffer, 10mM Tris pH 8.0, 200mM NaCl, 5% glycerol

Dilute proteins to somewhere between 3-8µM

To 1ml of diluted protein, add 10µl of 20mM p-hydroxymercuriphenyl sulfonate solution and 10µl of 10mM PAR solution.

Read A₅₀₀ using spectrophotometer and use extinction coefficient of 6.6 X 10⁴ mol^-1 cm^-1 for the Par· Zn²⁺ complex to calculate concentration of Zn²⁺