Identification of N-homocysteinylated apolipoprotein Al in normal human serum

Background: In human serum, a portion of homocysteine (Hcy) exists as an N-linked form to the epsilon-amino group of protein lysine residues. N-homocysteinylated proteins differ structurally and functionally from native proteins. The present study strives to develop detection and potential semi-quantification methods for N-homocysteinylated apolipoprotein Al (N-HcyapoAl) in human serum. Methods: Serum treated with or without cysteamine was supplied to isoelectric focusing (IEF) followed by an immunoblot using an anti-apoAl antibody. Cysteamine treatment increased the isoelectric point for N-Hcy-apoAl, but not for unmodified apoAl, due to the presence of -SH group(s) derived from Hcy and the absence of a cysteine residue in the apoAl molecule. N-Hcy-apoAl was semi-quantified from the scanned immunoblot pattern via a computer. Results: After cysteamine treatment, N-Hcy-apoAl in the serum was identified by IEF at the position with a higher pl value compared with intact apoAl. The reproducibility (between assays) of the semi-quantification method was 19.1 % CV (coefficient of variation) for an average ratio 5.9% of N-Hcy-apoAl to the whole apoAl in the serum. Approximately 1.0-7.4% of apoAl was N-homocysteinylated in the serum obtained from 27 healthy subjects. Neither the ratio of N-Hcy-apoAl nor its concentration, calculated by total apoAl concentration, indicated correlation with the so-called total (free and S-linked) Hcy concentration. Conclusions: We directly found that a portion of apoAl in the serum undergoes homocysteinylation in an N-linkage manner, and used this to develop a potential semi-quantification method for N-Hcy-apoAl.