MODIFICATION OF HUMAN HEMOGLOBIN WITH METHYL ACYL PHOSPHATES DERIVED FROM DICARBOXYLIC-ACIDS - SYSTEMATIC RELATIONSHIPS BETWEEN CROSS-LINKED STRUCTURE AND OXYGEN-BINDING PROPERTIES

Human hemoglobin was reacted with five dicarboxylic acid bis(methyl phosphate) reagents under different ligand conditions. The bis(methyl phosphate) reagents tested were derived from fumaric, isophthalic, terephthalic, trans-stilbene-3,3'-dicarboxylic, and trans-stilbene-4,4'-dicarboxylic acids. These acyl phosphate mixed anhydrides are anionic electrophiles and will react with N-terminal amino and lysyl epsilon-amino groups to form amides. The major and many of the minor reaction products that result have been isolated and structurally characterized by globin chain and peptide analysis. Products which are not cross-linked linked, intrachain linked, and interchain singly and doubly cross-linked occur in proportions which depend upon the reaction conditions and reagent. Modifications of the beta chains were limited to the amino groups of beta1Val, beta82Lys, and, to a minor extent, beta144Lys. In the case of the smaller reagents, the amino groups of alpha1Val, alpha99Lys, and, to a minor extent, alpha139Lys were modified. The oxygen binding affinities of most of the major modified hemoglobins have been measured and are characterized by P50 values from about 1/2 to over 5 times that of unmodified human hemoglobin. Most show strong cooperativity with Hill coefficients (n) of 2.0 or greater. Several of the products that are cross-linked between the beta1Val of one chain and the beta82Lys of the other chain have oxygen affinities in a physiologically useful range for oxygen transport and delivery. An inverse linear correlation has been found between the log of P50 and bridging distances for the hemoglobins cross-linked between beta1Val of one chain and the beta82Lys of the other chain. There is a positive correlation (with a smaller slope) for the bridge length of the beta82Lys cross-linked hemoglobins with the log of their P50's.