Comparison of the phenol-reagent and bromsulfalein-binding methods for determination of protein and its measurement in mast cells

Abstract

A critical comparison of the phenol-reagent and bromsulfalein-binding methods for the determination of protein revealed no significant difference in sensitivity or reproducibility. Both methods have advantages over others commonly used and are well adapted for use in the microgram range. In the phenol-reagent method, precipitation of protein by trichloroacetic acid prior to analysis was found to be essential for some materials (e.g., normal rat mast cells) and not for others (e.g., neoplastic mouse mast cells, bovine plasma albumin, or human serum albumin). Removal of particular nonprotein amino acids and their derivatives may be required since they contribute to the color measured, but the bromsulfalein-binding method is free of such interference. Use of an empirical factor for standardization based on relation of protein nitrogen to absorbance measured, which compensates for differences in proportion of reactive groups in different proteins and which has been in use for the bromsulfalein method, is also recommended for the phenol-reagent method. Mean protein values of 155 ± 3.2 and 131 ± 2.1 μ g 10 6 cells, respectively, for normal rat and neoplastic mouse peritoneal mast cells were obtained, although the values are subject to significant biological variation. However, over the number of generation transplants of the neoplastic cells studied (Nos. 479–500), no consistent influence on protein content was found. Additional chemical differences between the normal and neoplastic mast cells were shown.