3-POINT CROSS-LINKING - POTENTIAL RED-CELL SUBSTITUTES FROM THE REACTION OF TRIMESOYL TRIS(METHYL PHOSPHATE) WITH HEMOGLOBIN

The symmetrical trifunctional cross-linking reagent trimesoyl tris(methyl phosphate) (3), reacts selectively with amino groups (beta-1Val and beta-82Lys) in the diphosphoglycerate binding site of human hemoglobin A, producing cross-linked tetrameric species in good yield. A major species is triply linked, alpha-alpha-beta-1(82)>B-beta-82, where B symbolizes benzene-1,3,5-tricarbonyl. Both this triply linked species and the doubly linked species, alpha-alpha-1B-beta-82, produced from deoxyhemoglobin have a considerably lower oxygen affinity than does native hemoglobin while maintaining a high degree of cooperativity (n50 = 2.4), making them potentially useful as red cell substitutes, in principle delivering twice as much oxygen as whole blood between P(O2) = 100 and = 40 Torr. The yield of products indicates that triply and doubly linked species form in parallel so that there are independent routes to each. It is proposed that differences in routes are due to stereoisomerism about the amide bonds which form from reaction of the reagent with the protein.