Binding of lipoic acid induces conformational change and appearance of a new binding site in methylglyoxal modified serum albumin.

The binding of lipoic acid (LA), to methylglyoxal (MG) modified BSA was studied using isothermal titration calorimetry in combination with enzyme kinetics and molecular modelling. The binding of LA to BSA was sequential with two sites, one with higher binding constant and another comparatively lower. In contrast the modified protein showed three sequential binding sites with a reduction in affinity at the high affinity binding site by a factor of 10. CD results show appreciable changes in conformation of the modified protein as a result of binding to LA. The inhibition of esterase like activity of BSA by LA revealed that it binds to site II in domain III of BSA. The pH dependence of esterase activity of native BSA indicated a catalytic group with a pK(a) = 7.9 +/- 0.1, assigned to Tyr411 with the conjugate base stabilised by interaction with Arg410. Upon modification by MG, this pK(a) increased to 8.13. A complex obtained by docking of LA to BSA and BSA in which Arg410 is modified to hydroimidazolone showed that the long hydrocarbon chain of lipoic acid sits in a cavity different from the one observed for unmodified BSA. The molecular electrostatic potential showed that the modification of Arg410 reduced the positive electrostatic potential around the protein-binding site. Thus it can be concluded that the modification of BSA by MG resulted in altered ligand binding characteristics due to changes in the internal geometry and electrostatic potential at the binding site.

Malondialdehyde, a lipid-derived aldehyde alters the reactivity of Cys34 and the esterase activity of serum albumin.

Malondialdehyde (MDA), one of the key end products of lipid oxidation is elevated in a variety of diseases. It is now well established that MDA can modify proteins in vivo. This paper describes the effects of modification of albumin by MDA and peroxidized linolenic acid on the reactivity of Cys34, a crucial residue conferring antioxidant properties. BSA (10 mg/ml) was incubated with MDA (1 mM) for 72 h in phosphate buffer (100 mM, pH 7.4). BSA was also incubated for three days with lipid samples, which have already undergone peroxidation for 2, 5, 7, and 9 days respectively. The reactivity of Cys34 after modification was monitored using cystamine and 5,5'-dithiobis(2-nitro benzoic acid) (DTNB). The Kobs for the reaction was found to be different between native and MDA modified protein clearly indicating that modification affects the reactivity of Cys34. The individual rate constant (K1) for reaction with DTNB varied significantly between albumin and modified albumin suggesting that loss in reactivity was due to changes at Cys34. However, (K2), the rate constant for reaction of protein with cystamine, determined from a plot of Kobs versus cystamine concentration did not change. This study further shows that modification results in significant loss of the esterase like activity of albumin. Since albumin plays a crucial role in the antioxidant defence due to its abundance (approximately 0.6 mM) in serum, these findings have implications in disease states where increased levels of MDA and oxidative stress drastically may affect the antioxidant capacity of serum.

DNA damage by free radical production by aminoguanidine.

Aminoguanidine (AG), a prototype therapeutic dicarbonyl scavenger, is the most potent drug available today to inhibit the formation of advanced glycation endproducts (AGEs) and to reverse glycation-mediated damage in normal aging. This paper examines the ability of AG to cause damage to supercoiled plasmid DNA in the presence of the transition metal, Fe(+3). Damage to DNA was dependent on the concentrations of both the transition metal and AG. We could detect hydroxyl radical as well as hydrogen peroxide during the incubation of AG with Fe(+3). Thus this finding further cautions against the indiscriminate use of AG in clinical prophylaxis in diabetes and questions its use as a therapeutic agent.

Glucose, glycation and aging.

Glycation, a deleterious form of post-translational modification of macromolecules has been linked to diseases such as diabetes, cataract, Alzheimer's, dialysis related amyloidosis (DRA), atherosclerosis and Parkinson's as well as physiological aging. This review attempts to summarize the data on glycation in relation to its chemistry, role in macromolecular damage and disease, dietary sources and its intervention. Macromolecular damage and biochemical changes that occur in aging and age-related disorders point to the process of glycation as the common event in all of them. This is supported by the fact that several age-related diseases show symptoms manifested by hyperglycemia. Free radical mediated oxidative stress is also known to arise from hyperglycemia. There is evidence to indicate that controlling hyperglycemia by antidiabetic biguanides prolongs life span in experimental animals. Caloric restriction, which appears to prolong life span by bringing about mild hypoglycemia and increased insulin sensitivity further strengthens the idea that glucose via glycation is the primary damaging molecule.