Interactions of a Fluorescently Labeled Peptide with Kringle Domains in Proteins

The tripeptide Lys-Cys-Lys has been synthesized and covalently labeled at the cysteine sulfhydryl with 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonic acid to produce a fluorescent labeled peptide (FLP). When excited at 340 nm, the FLP fluorescence strongly with maximal intensity at 405 nm. Addition of proteins containing the kringle lysine-binding domain, such as human lipoprotein (a) and plasminogen kringle 4, significantly attenuate the fluorescence intensity of the FLP. Other proteins, such as bovine serum albumin, did not affect the quantum yield of FLP fluorescence. When human lipoprotein (a) is bound to a lysine-Sepharose affinity column, FLP was found to effectively elute the protein, indicating that the peptide can compete with lysine for the kringle-binding site on lipoprotein (a). The data suggest that FLP binds specifically to kringles through the lysine residues on the peptide, and that binding significantly affects the fluorescence from the labeled peptide. These properties of FLP make it a potentially useful tool for studying the relative affinity of different kringles for lysine binding, which is thought to be an important mechanism for kringle-target protein interactions.