THE BINDING OF T-1824 AND STRUCTURALLY RELATED DIAZO DYES BY THE PLASMA PROTEINS

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Abstract
The electrophorcsis expts. show that T-1824, niagara sky blue 6B, trypan blue. and niagara sky blue in serum or plasma arc wholly bound by the albumin fraction when they are present in low cones., i.e., about 0.004% or less. This no longer holds, however when the dye conc. exceeds certain limits representing the binding capacity of the albumin. The evidence indicates that each mole of albu-min can bind a max. of 8 to 14 moles of T-1824. Ultra-centrifugation of serum albumin solns. containing 5 moles of T-1824 to 1 mole of albumin also demonstrates that the dye is bound by the protein. The dye comes down with the albumin forming a single boundary leaving the supernatant buffer solution colorless. Addition of plasma to aqueous soln.s. of the dyes shifts the point of maximum absorption toward the red end of the spectrum and changes the contour of the spectral absorption curve (Gregersen and Gibson, 1937). These effects are caused mainly by the albumin fraction. There is, however, some difference in the effect of albumin on the absorption of the 4 dyes. With the 2 dyes, niagara sky blue and trypan blue, the optical density falls as the albumin conc. is in-creased to the point where its molecular conc. is 1/2 that of the dye. Further increase in the albumin conc. is without much effect. With T-1824 and niagara sky blue 6B the maximal effect of the addition of albumin is reached at much lower concs., approx. 1/10 moles of albumin per mole of dye, and the optical density then increases as the albumin conc. is raised still further. This ratio for the maximum number of T-1824 molecules bound by a molecule of albumin is in agreement with the value obtained from the electrophoretic studies. The cellophane staining tests reveal differences in the affinities of the four dyes for albumin. There is a direct relationship between the rates at which these 4 dyes leave the circulation (Gregersen and Rawson, 1943) and their tendency to stain cellophane. Thus although all 4 dyes are preferentially bound in the plasma by albumin an explanation for their different disappearance rates is found in their different affinities for albumin.

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