Histidine residues underlie Congo red binding to A beta analogs

The binding mechanism of Congo red (CR) to Alzheimer's disease (AD) amyloid fibrils (A beta) in terms of binding affinity and number of sites was quan titated from absorption spectroscopy (at 200-700 nm) by measuring the conce ntration of CR bound (CR-B) to AD A beta assemblies as a function of CR con centration and pH in 80% ethanol. The rationale for the use of this high co ncentration of ethanol derives from its use in histological screens for amy loid in tissue sections. Moreover, free CR can be separated from bound CR b y filtration in ethanolic but not aqueous medium. The A beta analogs studie d here included: (1) peptides having different lengths: A beta 1-40, A beta 11-28, A beta 13-28, A beta 19-28, A beta 11-25; (2) wildtype, control seq uences of A beta 1- 40 and sequences having different natural amino acid su bstitutions: primate Pr1-40, rodent Ro1-40, hereditary cerebral haemorrhage with amyloidosis, Dutch type (HCHWA-D) Du1-40, primate reverse sequence Pr 40-1; and (3) A beta 11-25 sequences having different substitutions: H13D, H14D, and D23K. Negative-staining showed that A beta 1-40 fibrils in buffer were indistinguishable om those in buffered ethanolic medium. For all amyl oid analogs except A beta 19-28, which has no histidine residues and showed no CR binding over the entire pH range 4.0-9.5, CR-B decreased as a functi on of increasing pH. The decrease was steepest at about pH 5 and became zer o above pH 7. For analogs having the same number of histidines, CR-B fell o n the same binding curve, indicating that histidine residues are the likely binding sites for CR in this medium. The pH titration of the binding was p arameterized by the stoichiometry of dye to the sites, the number of histid ines per molecule, the binding dissociation constant K-d, and the apparent proton dissociation constant pK of the histidine; and the calculated pH-tit ration curves were found to fit the observed ones. For the peptides having 1-3 histidines the average pK was 5.0-5.5, which was similar to the expecte d pK of histidine in low dielectric medium (80% ethanol), and the K-d's wer e 2.8-5.9 mu M. That histidine residues underlie CR binding in A beta amylo id is consistent with previous findings that A beta peptides sediment as fi brillar assemblies at pH similar to 3-7 and bind Congo red over the same pH range in aqueous medium. Further the conformation near the binding motif H is13-His14-Gln15-Lys16 in A beta assemblies is not greatly altered in 80% e thanol.