Determination of total urinary protein, combining Lowry sensitivity and biuret specificity

Abstract

A highly sensitive method with specificity for the peptide chain backbone was developed for determination of total urinary protein. Interfering substances are removed by gel filtration and cupric ions are stoichiometrically bound to the peptide bonds of protein by the biuret reaction. Ion exchange characteristics of the gel are neutralized, allowing protein copper complex to be separated from excess cupric ions by a second gel filtration step. Copper bound to peptide bonds is colorimetrically determined by use of sodium diethyldithiocarbamate. Nonprotein substances do not interfere unless they simultaneously bind to protein and chelate copper. As little as 1 mg of total protein per deciliter can be determined in heterogeneous biological fluids such as urine.