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 epsilon-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.
Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, International Center for Public Health, Newark, NJ 07101, USA. jakubows@umdnj.edu
Hieronim
H
Jakubowski
D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D005293:Ferritins / Q000032:analysis; D006710:Homocysteine / Q000031:analogs & derivatives / Q000032:analysis* / Q000097:blood / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D008892:Milk / Q000737:chemistry; D011506:Proteins / Q000032:analysis*; D013050:Spectrometry, Fluorescence / Q000379:methods
D011506:Proteins; D006710:Homocysteine; D005293:Ferritins; C007957:homocysteine thiolactone