The cytospectrophotometrical determination of proteins by the tetrazonium coupling reaction. II. Calibration of the staining method.

The protein content of identical samples from 5 different cell types (lymphocytes of the thymus and of the bursa of FABRICIUS of the chicken, EHRLICH ascites tumor cells, YOSHIDA ascites tumor cells, and rat liver cells) have been determined both macroscopically, by the method of LOWRY (1951), and microspectrometrically, by the tetrazonium coupling method of NOHAMMER (1978). In all the different cell types, a strong correlation is shown between the protein values determined by the 2 methods. By comparing the values thus measured, a conversion factor has been obtained such that an extinction of 0.4235, determined microspectrometrically, corresponds to a protein mass of 1 pgm. To test the accuracy of the microspectrometric method of protein determination at the lowest extreme point, the protein content of rat liver mitochondria has been measured and a mean value of 0.239 pgm protein per mitochondrion obtained. This corresponds well with the value of 0.233 pgm per mitochondrion as determined macroscopically by GEAR (1972).