The estimation of protein by the biuret and Greenberg methods

Abstract

The biuret and Greenberg methods of estimating protein in serum examined within 2 hrs. of collection of blood gave results approx. 2/3 of those given by detns. of protein N by Kjeldahl. This applied both to total protein and to albumin or globulin separated from horse or human sera, oxalated or citrated. As the serum aged the results became higher, the rate of increase being more rapid at higher temps. Spectrophotometric examination of the biuret color gave following maxima: fresh serum protein 545-555 mu, fresh milk protein 555-565 mu, biuret itself 535 mu. As casein solns. aged the biuret maximum shifted to about 570 mu, and subsidiary peaks appeared at about 540 and 600 mu. Density detns. with the spectrophotometer in region of maximum absorption confirmed findings obtained with tintometer and Klett colorimeter that results increase with aging of serum, and extended the findings to milk proteins. Spectrophotometric examination of a set of Lovibond glasses used for protein estimations by Harrison''s method showed a main peak at about 545 mu, and subsidiary peaks at about 460, 605 and 650 mu. The grading of the glasses was not very accurate, and might lead to errors exceeding 16%.