Numbers of receptor sites from Scatchard graphs: facts and fantasies.

Data for ligand and receptor binding presented in the format of a Scatchard graph are compared with the same data shown as bound ligand plotted against the logarithm of free ligand. From this comparison it is apparent that extrapolations in the Scatchard graph to yield total number of receptor sites are generally not correct.

Glycosylation of hemoglobin S by reducing sugars and its effect on gelation.

The binding of various reducing mono- and disaccharides to hemoglobin S has been measured both before and after treatment of the sugar-protein adducts with NaBH4. Incubation of 0.3 M solutions of D-glucose, D-galactose, D-maltose, and lactose, with 2% hemoglobin for 2 h at 37 degrees C, pH 7.2, leads to the incorporation of 1.1, 1.8, 1.8, and 3.3 mol of sugar, respectively, into 1 mol of hemoglobin tetramer (either A or S). Exposure of these aldose-protein adducts to NaBH4 for an additional hour at 10 degrees C increases the binding to 2.0, 3.3, 2.5, and 4.1 mol per mol tetramer, as would be expected if Schiff base linkages were involved in this protein modification reaction. The data suggest a stereochemical requirement for enhanced binding. The dependence of the pre-reduction binding of glucose on the sugar concentration, and on the oxygenation state of hemoglobin has also been examined. Glycosylation of hemoglobin significantly increases the minimum gelling concentration of the deoxy conformation, as measured by sedimentation equilibrium ultracentrifugation. Of the sugar derivatives of hemoglobin S examined by this method, those modified by D-galactose or lactose have minimum gelling concentrations (in the absence of 2,3-diphosphoglycerate) which are comparable to, or greater than, that of fully carbamylated hemoglobinS.

Inhibition of the growth of Plasmodium falciparum in vitro by covalent modification of hemoglobin.

Antimalarial effects might be expected from compounds that modify hemoglobin. Dibromoaspirin and bis(dibromosalicyl) diesters decrease gelation of hemoglobin by specific covalent modification (acetylation and crosslinking) of this protein but do not interfere with oxygen transport. These compounds were toxic to malaria parasites when continuously present in culture, as were drugs with similar pharmacological effects such as indomethacin, ibuprofen, and phenylbutazone. Aspirin and acetaminophen were much less effective. When erythrocytes were pretreated with these compounds prior to parasite exposure, only dibromoaspirin and dibromosalicyl diesters prevented parasite development. The modified hemoglobin was highly resistant to digestion by cathepsin D and parasite proteases, suggesting that covalent modifications of hemoglobin that do not disrupt normal hemoglobin function have antimalarial effects.

O-carbamoylsalicylates: agents for modification of hemoglobins.

To combine the attractive features of cyanate and of O-acetylsalicylate as hemoglobin-modifying agents we have prepared carbamoylsalicylate. This compound is a close analogue of aspirin and also resembles a masked cyanate. O-Carbamoylsalicylate and some related carbamates modify hemoglobin substantially, even at 5 mM concentration.

Raman and infrared spectroscopy of the oxo-bridged iron(III) complex, [Cl3Fe-O-FeCl3]-2 as a spectroscopic model for the oxo bridge in hemerythrin and ribonucleotide reductase.

Vibrational spectroscopic data were collected on the salt [C5H6N]2[Cl3FeOFeCl3] . C5H5N, which has previously been structurally characterized by X-ray crystallography. The modes associated with the oxo bridge were identified by experiments on the 18O-containing species. Spectra for the mu-16O complex contain Raman bands at 870, 458, and 203 cm-1 that shift to 826, 440, and 198 cm-1 in the mu-18O complex. These are respectively assigned to the asymmetric, symmetric, and angle deformations of the bent Fe-O-Fe moiety. A normal mode vibration analysis based on a simple valence force field for the Fe-O-Fe portion of the molecule provides surprisingly good agreement with these experimental frequencies and their assignments. The vibrational data for this simple inorganic complex confirm the assignment of a resonance Raman band around 500 cm-1 in the oxygen-carrying protein hemerythrin and enzyme ribonucleotide reductase as the symmetric stretch of an oxo bridge between two iron(III) centers.

Diaspirins of methylenecitric acid.

A series of bissalicylic esters of methylenecitric acid have been prepared and, as a probe of their potential as antisickling agents, tested for their ability to modify hemoglobin. Substantial acylation of hemoglobin was obtained with these dicarboxylate esters at 1-5 mM concentrations.

Electrostatic effects in acylation of hemoglobin by aspirins.

Carboxylate substituents added to the salicylate ring increase the effectiveness of a variety of aspirins and diaspirins in acylating hemoglobin. Even more effective are a series of monoesters of dicarboxylate derivatives. Bis(5-carbomethoxysalicyl)fumarate and -succinate at 5 mM concentrations modify approximately 100% of the hemoglobin in solution and should alter the aggregation behavior of sickle hemoglobin.

Acylation of hemoglobin by aspirin-like diacyl esters.

Aspirin-like diacyl esters of different steric disposition have been prepared and compared with acetylsalicylate in their abilities to modify hemoglobin.