Modification of histidine residues leading to the appearance of visible fluorescence

Abstract

The object of this investigation was to study the modification of histidine residues by N-bromosuccinimide leading to the appearance of visible fluorescence.

• 1.

  1. Histidine and tryptophan yield visible fluorescence when treated with N-bromosuccinimide in phosphate buffer at neutral pH. Under the conditions required for the formation of the fluorescent products, tryptophan and tyrosine lose their native ultraviolet fluorescence.

• 2.

  2. Details are presented in peptides yield mmore fluorescence than histidine itself. The amount of fluorescence formed differs from one peptide to another depending on the amino acids adjacent to the histidine residue.

• 3.

  4. The reaction leading to the formation of fluorescent products from histidine residues can experimentally be divided into two distinct steps: (a) Rapid attack of the histidine residue by N-bromosuccinimide to yield a non-fluorescent intermediate. (b) Conversion of the intermediate into a fluorescent compound in a slow reaction cataylsed by phosphate (or pyrophosphate, or hydroxyl ions). Optimal reaction conditions for both steps were determined.

• 4.

  5. The formation of fluorescent products from histidine residues when treated with N-bromosuccinimide may be of use in following the modification of histidine residues in peptides and in proteins. It may also be a means of introducing visible fluorescence into proteins for structural studies.

• 5.

  6. The possible relevance of this work to the problem of oxidative phosphorylation is briefly discussed.