Nayyar Rabbani

CAMEL LENS CRYSTALLINS GLYCOSYLATION AND HIGH MOLECULAR WEIGHT AGGREGATE FORMATION IN THE PRESENCE OF FERROUS IONS AND GLUCOSE

The incubation of camel lens cortex homogenate with 100 microM ferrous ions and 5.5 mM glucose under sterile conditions caused rapid protein aggregation, but little or no reaction was seen with either 100 microM ferrous ions or 5.5 mM glucose alone. The formation of glycosylated high molecular weight (HMW) protein aggregates was confirmed by light scattering studies, a decreased level of free -SH groups, incorporation of [14C]-glucose and elution of HMW protein aggregate just after the void volume of a Sephacryl S-1000 column. The bonding involved in the formation of these aggregates was found to be mainly disulfide in nature. Isoelectric focusing (IEF) in the presence and absence of reducing conditions indicated that gamma-crystallins may be involved in the formation of HMW protein aggregates. The modifications observed were found to mimic those seen in cataractous lenses.

PURIFICATION AND CHARACTERIZATION OF ACETYLCHOLINESTERASE FROM DESERT COBRA (WALTERINNESIA AEGYPTIA) VENOM

Acetylcholinesterase (AChE) has been identified and purified from the venom of desert cobra (W aegyptia) to apparent homogeneity using a TSK G 3000 SW gel filtration column and a Mono Q anion-exchange column. AChE was purified to homogeneity as established by sodium dodecylsulfate/polyacrylamide gel electrophoresis. The specific activity of AChE was 357 IU/mg with acetylthiocholine iodide as substrate. The denatured W aegyptia venom AChE displayed a molecular mass of 67000 +/- 3000 Da suggesting it was a single polypeptide. Isoelectric focusing of AChE revealed that the enzyme exists in different isoforms, with isoelectric points ranging between pH 7.4-7.9. The kinetic parameters (Km and Vmax) and IC50 of AChE inhibition by procaine, tetracaine and physostigmine were investigated in the present study.

Kinetic properties of purified carnitine acetyltransferase from the skeletal muscle of Arabian camel (Camelus dromedarius)

The kinetic properties of carnitine acetyltransferase from the skeletal muscle of the Arabian camel (Camelus dromedarius) were studied. The enzyme showed an optimum pH between 7.2 and 8.2. Reciprocal plots of data obtained by varying one substrate concentration while keeping the other constant revealed lines that converged on the abscissa, indicating that the enzyme possible follows a random mechanism of catalysis. The Kms for L-carnitine and acetyl-coenzyme A were 244 and 44 microM respectively, while those for acetyl-DL-carnitine and coenzyme A (Co A) were 307 and 39 microM respectively. The Km for one substrate was found to be independent of the concentration of the second substrate used. Corresponding Vmax values for L-CA, acetyl-Co A, acetyl-DL-carnitine and Co A are 98, 98, 102 and 100 mumol min-1 mg-1 protein respectively. The low Km obtained for acetyl-DL-carnitine suggests an adaptive mechanism in this desert species for enduring prolonged dry spells without food and water.

Effect of trifluoroethanol on α-crystallin: folding, aggregation, amyloid, and cytotoxicity analysis

α‐Crystallin, a member of small heat shock proteins, is the major structural protein within the eye lens and is believed to play an exceptional role in the stability of lens proteins and its transparency. In the current manuscript, we have investigated the effect of an organic solvent, trifluoroethanol (TFE), on the structure and function of α‐crystallin isolated from camel eye lens. Incubation of this protein with TFE changed the secondary and tertiary structures, which resulted in the aggregation of α‐crystallin as evidenced by intrinsic fluorescence, Rayleighs scattering, Thioflavin T assay, and circular dichroism spectroscopic studies. The treatment with different concentrations of TFE led to increased exposure of hydrophobic domains of α‐crystallin, which was observed by 8‐anilino 1‐napthalene sulfonic acid extrinsic fluorescence assay. These results clearly indicate that TFE induced significant changes in the secondary and tertiary structures of α‐crystallin, leading to aggregation and amyloid formation. Furthermore, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide assay established the cytotoxicity of the aggregated α‐crystallin towards HepG2 cell lines through reactive oxygen species production. In conclusion, α‐crystallin protein was found to be susceptible to conformational changes by TFE, suggesting that α‐crystallin, although basically acting like a heat shock protein and functionally displaying chaperone‐like activity, might capitulate to change in lens environment induced by diseased conditions or age‐related changes, resulting in cataract formation. Copyright

High-affinity binding of NADPH to camel lens ζ-crystallin

Fluorescence spectrum of camel lens ζ-crystallin, a major protein in the lens of camelids and histicomorph rodents, showed maximum emission at 315 nm. This emission maximum is blue shifted compared to most proteins, including α-crystallin, and appeared to be due to tryptophan in highly hydrophobic environment. Interaction of NADPH with ζ-crystallin quenched the protein fluorescence and enhanced the fluorescence of bound NADPH. Analysis of fluorescence quenching suggested high-affinity interaction between NADPH and ζ-crystallin with an apparent Km<0.45 μM. This value is at least an order of magnitude lower than that suggested by activity measurements. Analysis of NADPH fluorescence showed a biphasic curve representing fluorescence of free- and bound-NADPH. The intersection between free- and bound-NADPH closely paralleled the enzyme concentration, suggesting one mole of NADPH was bound per subunit of the enzyme. Phenanthrenequinone (PQ), the substrate of ζ-crystallin, also was able to quench the fluorescence of ζ-crystallin, albeit weaker than NADPH. Quantitative analysis suggested that ζ-crystallin had low affinity for PQ in the absence of NADPH, and PQ binding induced significant conformational changes in ζ-crystallin.

Glycation Induced Generation of Amyloid Fibril Structures by Glucose Metabolites.

The non-enzymatic reaction (glycation) of reducing sugars with proteins has received increased interest in dietary and therapeutic research lately. In the present work, the impact of glycation on structural alterations of camel serum albumin (CSA) by different glucose metabolites was studied. Glycation of CSA was evaluated by specific fluorescence of advanced glycation end-products (AGEs) and determination of available amino groups. Further, conformational changes in CSA during glycation were also studied using 8-analino 1-nephthlene sulfonic acid (ANS) binding assay, circular dichroism (CD) and thermal analysis. Intrinsic fluorescence measurement of CSA showed a 22 nm red shift after methylglyoxal treatment, suggesting glycation induced denaturation of CSA. Rayleigh scattering analysis showed glycation induced turbidity and aggregation in CSA. Furthermore, ANS binding to native and glycated-CSA reflected perturbation in the environment of hydrophobic residues. However, CD spectra did not reveal any significant modifications in the secondary structure of the glycated-CSA. Thioflavin T (ThT) fluorescence of CSA increased after glycation, illustrated cross β-structure and amyloid formation. Transmission electron microscopy (TEM) analysis further reaffirms the formation of aggregate and amyloid. In summary, glucose metabolites induced conformational changes in CSA and produced aggregate and amyloid structures.

Hydrophobicity of the NADPH binding domain of camel lens ζ-crystallin

Abstract Interaction of camel lens ζ-crystallin with the hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) enhanced the ANS fluorescence and quenched the protein fluorescence. Both of these events were concentration-dependent and showed typical saturation curves suggesting specific ANS-ζ-crystallin binding. Quantitative analysis indicated that 1 mole ζ-crystallin bound at most 1 mole ANS. NADPH but not 9,10-phenanthrenequinone (PQ) was able to displace ζ-crystallin-bound ANS. These results suggested the presence of a hydrophobic domain in ζ-crystallin, possibly at the NADPH binding site. α-Crystallin as well as NADPH protected ζ-crystallin against thermal inactivation suggesting the importance of this site for enzyme stability. The NADPH:quinone oxidoreductase activity of ζ-crystallin was inhibited by ANS with NADPH as electron donor and PQ as electron acceptor. Lineweaver-Burk plots indicated mixed-type inhibition with respect to NADPH, with a K i of 2.3 μM. Secondary plots of inhibition with respect to NADPH indicated a dissociation constant ( K ′ I ) of 12 μM for the ζ-crystallin-NADPH-ANS complex. The K i being smaller than K ′ I suggested that competitive inhibition at the NADPH binding site was predominant over non-competitive inhibition. Like ANS-ζ-crystallin binding, inhibition was dependent on ANS concentration but independent of incubation time.

Inhibition of camel lens ζ-crystallin by aspirin and aspirin-like analgesics

Abstract Camel lens ζ-crystallin was reversibly inhibited to various degrees by aspirin (acetyl salicylic acid) and the aspirin-like analgesics: paracetamol (acetaminophen) and ibuprofen (2-(4-isobutyl phenyl)-propionic acid). Among these, aspirin was the most potent inhibitor, causing nearly complete inhibition in a dose-dependent, but time-independent manner. Analysis of inhibition kinetics revealed that aspirin was uncompetitive inhibitor ( K i 0.64 mM) with respect to NADPH and non-competitive inhibitor ( K i 1.6 mM) with respect to the substrate, 9,10-phenanthrenequinone (PQ). Multiple-inhibition analysis showed that aspirin and pyridoxal 5′ phosphate (PAL-P), a lysine specific reagent, simultaneously bound to a critical lysine residue located towards the NADPH binding region. Consistent with this, NADPH was able to substantially protect ζ-crystallin against aspirin, whereas PQ did not provide any protection. The results suggested that an essential lysine residue was the locus of aspirin binding. The inhibition of ζ-crystallin by aspirin and aspirin-like analgesics was reversible thus eliminating acetylation as a mechanism for inhibition. Reversible binding of aspirin to this lysine may cause steric hindrance resulting in uncompetitive inhibition with respect to NADPH.

Characterization of colchicine binding with normal and glycated albumin: In vitro and molecular docking analysis

The transport of more than 90% of the drugs viz. anticoagulants, analgesics, and general anesthetics in the blood takes place by albumin. Hence, albumin is the prime protein needs to be investigated to find out the nature of drug binding. Serum albumin molecules are prone to glycation at elevated blood glucose levels as observed in diabetics. In this piece of work, glycation of bovine serum albumin (BSA) was carried out with glyceraldehyde and characterized by molecular docking and fluorometry techniques. Glycation of BSA showed 25% loss of free amino groups and decreased protein fluorescence (60%) with blue shift of 6 nm. The present study was also designed to evaluate the binding of colchicine (an anti-inflammatory drug) to native and glycated BSA and its ability to displace 8-analino-1-nephthalene sulfonic acid (ANS), from the BSA–ANS complex. Binding of ANS to BSA showed strong binding (Ka = 4.4 μM) with native conformation in comparison to glycated state (Ka = 8.4 μM). On the other hand, colchicine was able to quench the fluorescence of native BSA better than glycated BSA and also showed weaker affinity (Ka = 23 μM) for glycated albumin compared with native state (Ka = 16 μM). Molecular docking study showed that both glyceraldehyde and colchicine bind to common residues located near Sudlow’s site I that explain the lower binding of colchicine in the glycated BSA. Based on our results, we believe that reduced drugs-binding affinity to glycated albumin may lead to drugs accumulation and precipitation in diabetic patients.

Expression, Purification and Properties of Redox-Sensitive Eye Lens Zeta-Crystallin of Arabian Camel

The high protein concentration, unique composition and complex geometry of the lens makes it transparent. α-, β-, and γ-crystallins are present in all the lenses. In addition, taxon-specific crystallins are present in lenses in bulk quantity. Zeta (ζ)-crystallin is an NADPH-dependent quinone oxidoreductase, which constitutes nearly 10 % of the total eye lens protein in the evolutionary divergent animals (Camel, guinea pig and Japanese frog eye lenses) living in different ecological conditions. ζ -Crystallin is also present in human and other animal lenses but at catalytic amount. The physiological role of γ-crystallin in the eye lens is not well understood, however, truncated ζ-crystallin causes congenital cataract in guinea pig. In earlier study, redox regulated reversible activity of ζ-crystallin was reported. In this study, recombinant camel ζ-crystallin was overexpressed in E.coli and purified to homogeneity. Effect of different concentrations of reducing agent, dithiothretol (DTT) on the quinone oxidoreductase activity of recombinant ζ-crystallin was studied by enzymatic assay. To evaluate the effect of the reducing agent on the ζ-crystallin conformation, we have used far-UV and near-UV CD, intrinsic fluorescence, ANS binding assay and size exclusion chromatography. Our results showed that nearly 50% of the of ζ-crystallin activity was lost at 50 µM DTT. However, no detectable changes in secondary structure were observed. No changes in the tertiary structure and surface hydrophobicity of ζ-crystallin were detected; however, marginal changes were seen at saturating concentration of DTT (1 mM).