Expression, purification and characterization of a biologically active and thermally stable human lysyl oxidase

Lysyl oxidase (LOX), a promising therapeutic target for the progression of cancer and fibrosis, has not been well characterized yet. A major difficulty faced in LOX characterization is its lack of solubility in common buffers. In this study, mature LOX (mLOX) was cloned, purified and its purity was ascertained by mass spectroscopy. Through screening various buffers, 0.2 M glycine-NaOH buffer with 10% glycerol pH 8.0 was identified to maintain mLOX in its soluble state. About 67% of the refolded mLOX was found to be in copper bound state after His-tag removal. Catalytic properties Km and kcat were found to be 3.72 × 10−4 M and 7.29 ×103s−1. In addition, collagen cross-linking in ARPE-19 cells was augmented on exposure to mLOX, endorsing its biological activity. Circular Dichroism revealed that mLOX comprises 8.43% of α-helix and 22% of β-strand and it was thermally stable up to 90°C. Disulfide linkage imparts the structural stability in LOX which was experimentally ascertained with intrinsic and extrinsic fluorescence studies.

Homocysteine & its metabolite homocysteine-thiolactone & deficiency of copper in patients with age related macular degeneration - A pilot study.

Background & objectives: Age related macular degeneration (ARMD) is a leading cause of blindness, particularly in persons above 60 yr of age. Homocysteine is implicated in many ocular diseases including ARMD. This study was undertaken to assess the status and relationship between plasma homocysteine, homocysteine - thiolactone, homocysteinylated protein and copper levels in patients with ARMD. Methods: A total of 16 patients with ARMD and 16 age-matched controls were recruited for the study. Plasma glutathione, homocysteine, homocysteine - thiolactone and extent of homocysteine conjugation with proteins, copper and thiobarbituric acid reactive substances were measured. Results: Homocysteine levels were elevated with increase in homocysteine-thiolactone, thiobarbituric acid reactive substances and a decrease of glutathione. The levels of homocysteinylated protein were elevated in ARMD. The elevated homocysteine, homocysteine-thiolactone correlated with the decrease in copper level. Interpretation & conclusions: Elevated homocysteine and its metabolite homocysteine-thiolactone and decreased levels of copper may play an important role in the pathogenesis of ARMD.

Determination of carbonyl group content in plasma proteins as a useful marker to assess impairment in antioxidant defense in patients with Eales' disease.

PURPOSE: Formation of protein carbonyl groups is considered an early biomarker for the oxidant/antioxidant barrier impairment in various inflammatory diseases. We evaluated the intensity of free radical reactions in patients with Eales' disease, an idiopathic inflammatory condition of the retina. METHODS: Twenty patients with Eales' disease in active vasculitis stage, 15 patients with Eales' disease in healed vasculitis stage and 20 healthy control subjects were recruited for the study. Plasma protein carbonyl groups,plasma glutathione (GSH) superoxide dismutase (SOD) activity and thiobarbituric acid reactive substances (TBARS) were determined in erythrocytes. RESULTS: Plasma protein carbonyl content was elevated by a factor of 3.5 and 1.8 respectively in active and healed vasculitis stages. The increase of carbonyl group content in active and healed stage of patients with Eales' disease correlated with diminished SOD activity and GSH content. There was also increased accumulation of TBARS in active and healed vasculitis stages of Eales' disease, and this correlated with diminished SOD activity. CONCLUSION: Our results showed that protein carbonyl group content increases with severity of Eales' disease. The increase in carbonyl content correlated with diminished antioxidant status. This confirms an earlier report that free radical mediated tissue damage occurs in Eales' disease. The determination of protein carbonyl content may be used as a simple biomarker to monitor the efficacy of antioxidant supplementation in controlling retinal vasculitis in patients with Eales' disease.

Trace elements iron, copper and zinc in vitreous of patients with various vitreoretinal diseases.

PURPOSE: To measure the concentrations of iron, copper and zinc in human vitreous and to interpret their levels with various vitreoretinal diseases like proliferative diabetic retinopathy, retinal detachment, intraocular foreign body, Eales' disease and macular hole. METHODS: Undiluted vitreous fluid collected during pars plana vitrectomy was used to measure trace elements using an atomic absorption spectrophotometer. RESULTS: The level of vitreous iron increased threefold in Eales' disease (1.85 +/- 0.36 pg/ml), 2.5-fold in proliferative diabetic retinopathy (1.534 +/- 0.17 pg/ml) and 2.3-fold in eyes with intraocular foreign body (1.341 +/- 0.25 pg/ml) when compared with macular hole (0.588 +/- 0.16 pg/ml). This was statistically significant (P < 0.05). Zinc was found to be low in Eales' disease (0.57 +/- 0.22 pg/ ml) when compared with other groups, though the difference was not statistically significant. CONCLUSION: The increased level of iron with decreased zinc content in Eales' disease confirms the earlier reported oxidative stress mechanism for the disease. In proliferative diabetic retinopathy and intraocular foreign body the level of iron increases. This is undesirable as iron can augment glycoxidation, which can lead to increased susceptibility to oxidative damage, in turn causing vitreous liquefaction, posterior vitreous detachment and ultimately retinal detachment and vision loss.