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Hemoglobin quenching of the fluorescence intensity of 12-(9-anthroyl)stearic acid (AS) embedded in the red blood cell membrane occurs through an energy transfer mechanism and can be used to measure the binding of hemoglobin to the membrane. The binding of hemoglobin to red cell membranes was found to be reversible and electrostatic in nature. Using a theory of energy transfer based on Förster formulation, the quantitative data for the binding were derived. The number of binding sites was found to be 1.4 +/- 0.2 X 10(6) molecules per cell and the binding constant was 0.85 X 10(8) M-1.

The bimane fluorescent labels, monobromobimane, dibromobimane, and monobromotrimethylammoniobimane, are derivatives of syn-9,10-dioxabimane:1,5-diazabicyclo[3.3.0]octa-3,6-diene-2,8-dione. They efficiently label hemoglobin (reactive thiol groups), membrane proteins, and glutathione of normal human red cells under physiological conditions. Monobromobimane and dibromobimane are effective on intact cells while red cell membranes may be impermeable to the positively charged monobromotrimethylammoniobimane, the latter being effective only on lysed cells. These bimane labels provide a class of labeling agents that may have wide applicability in biological materials. syn-9,10-Dioxabimanes, a new class of compounds, are highly fluorescent (1). Exceptions to this generalization are the essentially nonfluorescent bromoderivatives, illustrated by a monobromobimane (mBBr) (I), a dibromobimane (bBBr) (II), and a monobromotrimethylammoniobimane (qBBr) (III). We have now found that these three bromobimanes are highly efficient labeling agents for cells, proteins, and small molecules like glutathione, giving rise to highly fluorescent derivatives. The fluorescent derivatives are very stable in air and under irradiation. The labeling procedures are simple and can be carried out rapidly under physiological conditions. We shall describe in the present article the use of these reagents on normal human red cells. MATERIALS AND METHODSBromobimanes. The synthesis of the bromobimanes is described elsewhere (1). mBBr and bBBr were dissolved in acetonitrile and the 50 mM stock solutions were stored in the dark; the solutions are stable for at least 2 months. The salt, qBBr, was dissolved in aqueous buffer (pH 7.4) shortly before use; the 1-4 mM solutions have a half-life at 220C of approximately 4 hr.Red Cells. Human blood, anticoagulated with heparin, was obtained from normal individuals. The buffy coat was removed after centrifugation; the red cells were washed twice with 135 mM NaCI/10 mM phosphate buffer, pH 7.4, and resuspended in the same buffer to a packed cell volume of 5-10%. Ghosts were obtained from the cells by the procedure of Steck and Kant (2). Hemoglobin solutions were prepared by the centrifugation of lysed red cells at 22,000 X g for 30 min, followed by dialysis of the membrane-free supernatants against 10 mM phosphate buffer, pH 7.4.Reactions of Bromobimanes with Intact Cells, Lysates, and Hemoglobin Solutions. The reagents were added in a ratio of 5-20 ,umol of reagent per ml of packed red cells (1-4 moles of reagent per mole of hemoglobin). Usually 10-20 Aul of the stock solution was used per ml of cell suspension of 5-10% packed cell volume. The reagent solution was placed in a dry test tube and the-sample (cell suspension, hemoglobin solution) was added with rapid mixing. A stock solution (50 mM) of the mBBr reagent could be diluted with buffer to 1-2 mM before it was mixed with a sample. The bBBr compound, which is less soluble in water, was not diluted. qBBr was dissolved in buffer immediately before use...

The binding of hemoglobin to the red cell membrane was characterized over a wide range of free hemoglobin concentrations by measurement of membrane bound and supernatant hemoglobin. Scatchard analysis of the binding data revealed two classes of sites: high affinity sites with a binding constant of 1 X 10(8) M-1 and 1.2 X 10(6) sites per cell, and a second, low affinity class of sites with a binding constant of 6 X 10(6)M-1 and 6 X 10(6) sites per cell. The low affinity sites are shown to be nonspecific and appear to be a result of the ghost preparation. The high affinity sites are shown to be specific to the inner surface of the red cell membrane. The competition of hemoglobin and glyceraldehyde-3-phosphate dehydrogenase suggests band III proteins as a potential binding site for hemoglobin.