New method for the determination of protein N-linked homocysteine

Homocysteine (Hcy) is incorporated into protein via a reaction of the thioester Hcy–thiolactone with ε-amino group of a protein lysine residue. This reaction leads to impairment and alteration of protein’s function and has been implicated in atherothrombotic disease. However, the data regarding N-linked Hcy content in proteins are limited, mostly due to a lack of facile assays. Here I describe a new sensitive assay for the determination of protein N-linked Hcy and demonstrate its utility for individual proteins and biological fluids. N-linked Hcy is liberated from proteins by acid hydrolysis and converted to Hcy–thiolactone, which is then purified and quantified by high-performance liquid chromatography on a cation exchange column. The quantification is by fluorescence after postcolumn derivatization with o-phthaldialdehyde. Using this assay, the levels of N-linked Hcy in individual pure proteins were found to vary from as high as 0.470–0.515 mol/mol protein for human and equine ferritins to as low as 0.00006 mol/mol protein for chicken lysozyme. Hemoglobins from a variety of species contained more N-linked Hcy than did corresponding albumins (0.0127–0.0828 vs. 0.0027–0.0086 mol/mol). Normal human plasma and milk were found to contain submicromolar concentrations of protein N-linked Hcy, whereas cow milk and whey contained micromolar concentrations of protein N-linked Hcy.