DETERMINATION OF CELL-SURFACE CHARGE BY PHOTOMETRIC TITRATION

A colloid titration method has been frequently used to determine the n umber of charged residues at the cell surface. Here we present a new v ersion of this technique, based on photometric measurements of a metac hromatic shift in the maximum absorption of toluidine blue as it binds to the cell surface. The major improvements are: (1) simplified metho dology and (2) increased precision of equivalence point determination. The data are analyzed using Gran's theory, which allows measurements to be taken at regular intervals instead of being concentrated around the equivalence titration point. We used this method to characterize t he cell surface charge of three populations of rat mast cells: (1) per itoneal mast cells (PMC), (2) bone marrow-derived mast cells (BMMC) an d (3) a rat cultured mast cell line (RCMC). Our results indicate that PMC have (4.23+/-0.59) X 10(8), while BMMC (8.58+/-0.26) X 10(7) negat ively charged residues per cell. The results for RCMC were similar to those for BMMC. Taking into account the size differences between PMC a nd BMMC, the average charge density of PMC was also significantly high er than that of BMMC. The differences in cell surface charge were anal yzed in the light of different sensitivities of mast cells to polycati onic secretagogues. (C) 1997 Elsevier Science B.V.