TY - JOUR
T1 - Characterization of the adduct formed from the reaction between homocysteine thiolactone and low-density lipoprotein
T2 - Antioxidant implications
AU - Ferguson, Eric
AU - Hogg, Neil
AU - Antholine, William E.
AU - Joseph, Joy
AU - Singh, Ravinder Jit
AU - Parthasarathy, Sampath
AU - Kalyanaraman, B.
N1 - Funding Information:
The authors thank Dr. Jimmy Feix for his critical reading of the manuscript and continued interest in this work. This work was supported by NIH Grants HL47250 and RR01008.
PY - 1999/4
Y1 - 1999/4
N2 - Homocysteine thiolactone is a cyclic thioester that is implicated in the development of atherosclerosis. This molecule will readily acylate primary amines, forming a homocystamide adduct, which contains a primary amine and a thiol. Here, we have characterized and evaluated the antioxidant potential of the homocystamide-low-density lipoprotein (LDL) adduct, a product of the reaction between homocysteine thiolactone and LDL. Treatment of LDL with homocysteine thiolactone resulted in a time-dependent increase in LDL-bound thiols that reached approximately 250 nmol thiol/mg LDL protein. The thiol groups of the homocystamide-LDL adduct were labeled with the thiol-reactive nitroxide, methanethiosulfonate spin label. Using paramagnetic relaxing agents and the electron spin resonance spin labeling technique, we determined that the homocystamide adducts were predominately exposed to the aqueous phase. The homocystamide-LDL adduct was resistant to myoglobin- and Cu2+- mediated oxidation (with respect to native LDL), as measured by the formation of conjugated dienes and thiobarbituric acid reactive substances, and the depletion of vitamin E. This antioxidant effect was due to increased thiol content, as the effect was abolished with N-ethylmaleamide pre-treatment. We conclude that the reaction between homocysteine thiolactone and LDL generates an LDL molecule that is more resistant to oxidative modification than native LDL. The potential relationship between the homocystamide-LDL adduct and the development of atherosclerosis is discussed.
AB - Homocysteine thiolactone is a cyclic thioester that is implicated in the development of atherosclerosis. This molecule will readily acylate primary amines, forming a homocystamide adduct, which contains a primary amine and a thiol. Here, we have characterized and evaluated the antioxidant potential of the homocystamide-low-density lipoprotein (LDL) adduct, a product of the reaction between homocysteine thiolactone and LDL. Treatment of LDL with homocysteine thiolactone resulted in a time-dependent increase in LDL-bound thiols that reached approximately 250 nmol thiol/mg LDL protein. The thiol groups of the homocystamide-LDL adduct were labeled with the thiol-reactive nitroxide, methanethiosulfonate spin label. Using paramagnetic relaxing agents and the electron spin resonance spin labeling technique, we determined that the homocystamide adducts were predominately exposed to the aqueous phase. The homocystamide-LDL adduct was resistant to myoglobin- and Cu2+- mediated oxidation (with respect to native LDL), as measured by the formation of conjugated dienes and thiobarbituric acid reactive substances, and the depletion of vitamin E. This antioxidant effect was due to increased thiol content, as the effect was abolished with N-ethylmaleamide pre-treatment. We conclude that the reaction between homocysteine thiolactone and LDL generates an LDL molecule that is more resistant to oxidative modification than native LDL. The potential relationship between the homocystamide-LDL adduct and the development of atherosclerosis is discussed.
KW - Antioxidants
KW - Electron spin resonance
KW - Homocystamide adduct
KW - Homocysteine
KW - Homocysteine thiolactone
KW - Lipid peroxidation
KW - Low-density lipoprotein
KW - Protein thiols
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U2 - 10.1016/S0891-5849(98)00287-1
DO - 10.1016/S0891-5849(98)00287-1
M3 - Article
C2 - 10232841
AN - SCOPUS:0032953962
SN - 0891-5849
VL - 26
SP - 968
EP - 977
JO - Free Radical Biology and Medicine
JF - Free Radical Biology and Medicine
IS - 7-8
ER -