Determination of Protein in Urine by the Biuret Method

A method for the determination of protein in urine by means of the biuret reaction was described by Hiller (1) and by Hiller, McIntosh, and Van Slyke (2), in which pure biuret prepared by Kahlbaum was used as a standard for visual calorimetry. Since Hiller (1) the biuret method has been used for the determination of urinary proteins by Price (3) and by Lehmann (4), and has been widely used in many forms and modifications for the determination of plasma proteins. A review of the literature on the biuret method will not be given here, but may be found in the papers of KiintzeI and Droscher (5) and of Robinson and Hogden (6). The latter workers studied the conditions necessary for the production of a stable color which bears a quantitative relationship to the protein concentration and also published transmission curves for the biuret color. The present paper describes a photometric method for the determination of urinary protein in which the entire procedure is carried out in a single test-tube, which serves as a cuvette for the photoelectric spectrophotometer. The method can be used even when the urine contains Evans blue excreted after blood volume determinations. Since pure preparations of biuret are not now obtainable on the market, solutions of urinary proteins, of concentration determined by accurate Kjeldahl analysis, are used to prepare standard optical density curves, and the use of a chrome-alum solution for checking the curves is detailed.