| Autor: |
Jakubowski H; Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, International Center for Public Health, Newark, NJ 07101, USA. jakubows@umdnj.edu |
| Jazyk: |
angličtina |
| Zdroj: |
Analytical biochemistry [Anal Biochem] 2008 Sep 15; Vol. 380 (2), pp. 257-61. Date of Electronic Publication: 2008 Jun 05. |
| DOI: |
10.1016/j.ab.2008.05.049 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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