The Binding of T-1824 and Structurally Related Diazo Dyes by the Plasma Proteins

The purpose of the present study was to find out if the differences in disappearance rates of T-1824, trypan blue, niagara sky blue and niagara sky blue 6B depend upon the binding of these dyes by the plasma proteins (Gregersen and Rawson, 1943). The affinity of the various dyes for the plasma proteins was investigated: 1, with the electrophoresis method of Tiselius (1937) ; 2, by the ultracentrifuge; 3, by the effects of the plasma proteins upon the spectral absorption of the dyes, and 4, by a cellophane-staining test. ELECTROPHORESIS EXPERIMENTS. The protein solutions were prepared for electrophoresis as follows: 4 cc. of serum or plasma were diluted with 12 cc. of a 0.2 M phosphate buffer, varying in pH from 7.40 to 7.60, and containing 0.15 M sodium chloride. The mixture was dialyzed at 5OC. in a cellophane bag for two or more days against two liters of the buffer. The buffer solution was changed at least once during the dialysis period. The dye was added from stock solutions before dialysis2 Figure la shows the electrophoretic pattern of normal titrated human plasma. The pattern in figure lb is produced by the same plasma containing T-1824 at a concentration of 0.004 per cent. The light absorption of the dye causes a welldefined shaded area. The fact that the shading begins with the ascending albumin boundary and ends with the descending albumin boundary demonstrates that the dye migrates entirely with the albumin. The same results were obtained with trypan blue, niagara sky blue, niagara sky blue 6B and also with brilliant vital red. In dog serum, as in human plasma, all of the dyes also migrated entirely with the albumin fraction. At the close of each dye-plasma experiment the protein boundaries were pushed by clockwork and plunger in order to separate the alpha, beta and gamma globulins on the descending side from the rest of the solution. Chemical and spectrophotometric examination of the globulin solution showed the presence of protein and the absence of dye. The electrophoretic pattern of human plasma containing 0.098 per cent T-1824 (after dialysis) showed the dye boundary to begin with the ascending albumin boundary and to end with the descending beta globulin boundary. Hence if sufficient dye is present it will be bound by the alpha and beta globulins as well as by the serum albumin. The descending gamma globulin was free of dye. At the end of four hours of electrophoresis, the fractions listed in table 1 were