Abhijit Chakrabarti

Hemoglobin depletion from red blood cell cytosol reveals new proteins in 2-D gel-based proteomics study

Proteomics studies to identify proteins in the erythrocyte cytosol have been largely affected by the huge abundance of hemoglobin (Hb), which masks the detection of other proteins in the 2‐D gel‐based separation. We have depleted Hb effectively from erythrocyte cytosol using cation exchange chromatography and have detected more than 600 protein spots in the Hb depleted hemolysate using 2‐DE. We have so far identified 59 proteins in the Hb‐depleted cytosol of normal erythrocytes, including 10 proteins not identified before.

EXCITED STATE PROTON TRANSFER FLUORESCENCE OF 3-HYDROXYFLAVONE IN MODEL MEMBRANES

Abstract 3-Hydroxyflavone (3HF), the basic structural moiety of an important group of naturally occurring, biologically active flavonoids, has attracted extensive attention for its intramolecular excited-state proton-transfer (ESPT) and dual fluorescence characteristics. We report here, for the first time, its fluorescence (emission, excitation, polarization anisotropy and lifetime) behaviour in model membranes consisting of small, unilamellar liposomes of synthetic phosphatidylcholine (dimyristoyl phosphatidylcholine (DMPC) and dipalmitoyl phosphatidylcholine (DPPC)). The emission spectrum is conspicuously dominated by the ESPT tautomer fluorescence band. This result, along with other relevant data, indicates that the molecules are embedded in the lipid bilayer matrix facing environments where external H-bonding perturbation effects are minimized. The anisotropy ( r ) versus temperature ( T ) profiles dramatically reveal the thermotropic gel-liquid crystalline phase transition properties of the phospholipids, signifying a novel application of the intrinsic (tautomer) fluorescence of 3HF.

Organization and dynamics of tryptophan residues in erythroid spectrin: Novel structural features of denatured spectrin revealed by the wavelength-selective fluorescence approach

We have investigated the organization and dynamics of the functionally important tryptophan residues of erythroid spectrin in native and denatured conditions utilizing the wavelength‐selective fluorescence approach. We observed a red edge excitation shift (REES) of 4 nm for the tryptophans in the case of spectrin in its native state. This indicates that tryptophans in spectrin are localized in a microenvironment of restricted mobility, and that the regions surrounding the spectrin tryptophans offer considerable restriction to the reorientational motion of the water dipoles around the excited state tryptophans. Interestingly, spectrin exhibits a REES of 3 nm even when denatured in 8 M urea. This represents the first report of a denatured protein displaying REES. Observation of REES in the denatured state implies that some of the structural and dynamic features of this microenvironment around the spectrin tryptophans are retained even when the protein is denatured. Fluorescence quenching data of denatured spectrin support this conclusion. In addition, we have deduced the organization and dynamics of the hydrophobic binding site of the polarity‐sensitive fluorescent probe PRODAN that binds erythroid spectrin with high affinity. When bound to spectrin, PRODAN exhibits a REES of 9 nm. Because PRODAN binds to a hydrophobic site in spectrin, such a result would directly imply that this region of spectrin offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state fluorophore. The results of our study could provide vital insight into the role of tryptophans in the stability and folding of spectrin.

Membrane interaction of erythroid spectrin: Surface-density-dependent high-affinity binding to phosphatidylethanolamine

Density-dependent spectrin binding to dimyristoylphosphatidylcholine/dimyristoylphosphatidylethanolamine (DMPC/DMPE) small uni-lamellar vesicles (SUVs) has been directly evaluated in this work from the increase in the extent of quenching of the tryptophan fluorescence of spectrin at two different temperatures, above and below the main phase transition temperatures (Tm). Results from the binding studies of spectrin to phospholipid SUVs indicated that the binding dissociation constant Kd, increased from 45±7 nM in pure DMPC SUVs to 219±20 nM in DMPC/DMPE (50:50) SUVs, both in the gel and liquid crystalline phase. However, in pure DMPE SUVs the Kd decreased drastically to 0.7±0.2 nM in the gel phase at 18°C and to 2.6±0.7 nM in the fluid phase at 55°C indicating a high affinity binding of spectrin for the bilayer-forming DMPE. The maximum extent of phospholipid-induced quenching and the number of spectrin molecules associated with one SUV particle, evaluated in the present work, led to a model in DMPC/DMPE bilayer membranes indicating the PE-binding site of spectrin to localize at one of the terminal domains of the dimeric spectrin. A direct evidence of the localization of the PE-binding site at one of the terminal ends of the spectrin dimer also came from electron microscopic observation in fluid membranes made of bovine brain PE.

Chaperone Activity and Prodan Binding at the Self-associating Domain of Erythroid Spectrin

Spectrin, the major constituent protein of the erythrocyte membrane skeleton, exhibits chaperone activity by preventing the irreversible aggregation of insulin at 25 °C and that of alcohol dehydrogenase at 50 °C. The dimeric spectrin and the two subunits, α-spectrin and β-spectrin prevent such aggregation appreciably better, 70% in presence of dimeric spectrin at an insulin:spectrin ratio of 1:1, than that in presence of the tetramer of 25%. Our results also show that spectrin binds to denatured enzymes α-glucosidase and alkaline phosphatase during refolding and the reactivation yields are increased in the presence of the spectrin derivatives when compared with those refolded in their absence. The unique hydrophobic binding site on spectrin for the fluorescence probe, 6-propionyl-2-(dimethylamino)naphthalene (Prodan) has been established to localize at the self-associating domain with the binding stoichiometry of one Prodan/both dimeric and tetrameric spectrin. The other fluorescence probe, 1-anilinonaphthalene-8-sulfonic acid, does not show such specificity for spectrin, and the binding stoichiometry is between 3 and 5 1-anilinonaphthalene-8-sulfonic acid/dimeric and tetrameric spectrin, respectively. Regions in α- and β-spectrins have been found to have sequence homology with known chaperone proteins. More than 50% similarities in α-spectrin near the N terminus with human Hsp90 and in β-spectrin near the C terminus with human Hsp90 and Escherichia coli DnaJ have been found, indicating a potential chaperone-like sequence to be present near the self-associating domain that is formed by portions of α-spectrin near the N terminus and the β-spectrin near the C terminus. There are other patches of sequences also in both the spectrin polypeptides, at the other termini as well as in the middle of the rod domain having significant homology with well known chaperone proteins.

Eryptosis in hereditary spherocytosis and thalassemia: role of glycoconjugates.

The present work is aimed to study the mechanism of faster erythrocyte clearance in hereditary spherocytosis (HS), a heterogeneous disorders characterized by alterations in the proteins of the red cell membrane skeleton along with different kinds of thalassemia. The maximum exposure of phosphatidylserine (PS) is found in HS compared to those in both α- and β-thalassemia. Interestingly, in HS more PS exposed cells were found in younger erythrocytes compared to normal and the thalassemics where aged cells showed higher loss of PS asymmetry. Loss of sialic acid and GlcNAc bearing glycoconjugates, presumably the glycophorins, was also found upon aging. The loss of PS asymmetry together with the cell surface glycoproteins mediated by membrane vesiculation, seemed to play key role in early clearance of erythrocytes from circulation following a mechanism similar to HbEβ-thalassemia.

Polarity Estimate of the Hydrophobic Binding Sites in Erythroid Spectrin: A Study by Pyrene Fluorescence

The apparent dielectric constant, ε, of the hydrophobic pyrene binding sites in erythroid spectrin and human serum albumin (HSA) were estimated using the linear relationship [Turro, N.J., Kuo, P.L., Somasundaran, P. and Wong, K. (1986). J. Phys. Chem. 90, 288–291] between the ratio of the first (373 nm) and the third (384 nm) vibronic peak intensities (l1/l3) and the dielectric constant of the bulk medium. Binding of the hydrophobic fluorescent probe, pyrene, to erythroid spectrin and HSA was determined from concentration dependent change in the ratio l1/l3 from the emission spectra. Pyrene binds to spectrin (Kapp = 6.2 × 106 M−1) with a higher affinity than that of HSA (Kapp = 3.7 × 104 M−1) and the binding in both cases are saturable. The ε for spectrin and HSA was estimated to be 7 ± 2.1 and 5.4 ± 1.6 respectively. A case study with spectrin, covalently labeled with pyrene maleimide, have been presented for aging of pyrene-labelled spectrin showing the potential of the use of vibrational peak ratios (l1/l3) in the study of polarity of microenvironments in the neighborhood of cysteine residues of a protein. Large changes in the pyrene spectral components indicated conformational changes in the cysteine microenvironment of the protein upon storage at 4°C.

Differential regulation of redox proteins and chaperones in HbEβ‐thalassemia erythrocyte proteome

Purpose: In (hemoglobin, Hb) HbEβ‐thalassemia, HbE (β‐26 Glu→Lys) interacts with β‐thalassemia to produce clinical manifestation of varying severity. This is the first proteomic effort to study changes in protein levels of erythrocytes isolated from HbEβ‐thalassemic patients compared to normal.

Loss of phospholipid membrane asymmetry and sialylated glycoconjugates from erythrocyte surface in haemoglobin E β-thalassaemia

This study aimed to investigate any correlation between the extent of phosphatidylserine (PS) asymmetry and sialylated glycoconjugate levels with the faster clearance of circulating erythrocytes in haemoglobin E (HbE) β‐thalassaemia. Erythrocytes from peripheral blood samples of different HbEβ‐thalassaemia patients showed loss of PS asymmetry measured by annexin V binding using flow cytometry. Maximum PS exposure was found when HbE was 50–60% and HbF was <20% indicating a possible correlation with severity of the disease. Separation of erythrocytes into aged and younger cells showed higher loss of PS asymmetry in the younger erythrocytes of HbEβ‐thalassaemia patients when compared with normal blood, where PS asymmetry was lost only in the older cells. Sialylated glycoconjugate measurement using the lectins wheatgerm agglutinin and pokeweed mitogen showed loss of sialic acid and N‐acetyl‐d‐glucosamine‐bearing glycoproteins in the order normal

The tertiary amine local anesthetic dibucaine binds to the membrane skeletal protein spectrin

The quinoline‐based tertiary amine dibucaine has been shown to bind the membrane skeletal protein spectrin with a dissociation constant of 3.5×10−5 M at 25°C. Such binding is detected by monitoring the quenching of the tryptophan fluorescence intensity with increasing concentrations of dibucaine only and not with the benzene‐based local anesthetics procaine, tetracaine and lidocaine. Binding of dibucaine also indicated changes in the tertiary structure of spectrin indicated by a circular dichroism spectrum in the near‐UV region due to absorption of the aromatic side chains. The thermodynamic parameters associated with the binding indicated the interaction of dibucaine and spectrin to be enthalpy‐driven and insensitive to an increase in the ionic strength of the buffer.