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Biomedical subjects

B Chakrabarti

Publications and source records attributed to B Chakrabarti.

At least 55 records · Page 3Linked to original sources
PubMed1989

Spectral properties of human placental aldose reductase and aldehyde reductase II.

Circular dichroism and fluorescence spectra of aldose reductase (E.C.1.1.1.21) and aldehyde reductase II (E.C.1.1.1.19) purified to homogeneity from human placenta have been studied. The alpha helical content of aldose reductase and aldehyde reductase II was 51% and 56%, respectively, whereas no beta helical structure was found in either case. In the case of aldose reductase, the secondary structure was unaffected at alkaline pH (9.5), whereas a drastic alteration in the structure was observed at 58 degrees C. The secondary structure of aldehyde reductase II, on the other hand, remained unaffected at higher pH and temperature.

Aldehyde Reductase↗
PubMed1988

Heat-induced changes in the conformation of alpha- and beta-crystallins: unique thermal stability of alpha-crystallin.

Of the crystallin proteins of the lens, the principal subunit of the beta-crystallin, beta B2 (beta Bp), has been considered to be the only heat-stable protein because it does not precipitate upon heating. In our recent investigations, however, we have found that the alpha-crystallin from bovine lenses is not only heat stable but also does not denature at temperatures up to 100 degrees C. Using circular dichroism and fluorescence to monitor the conformational changes of alpha- and beta B2-crystallins upon heating, we found that alpha-crystallin maintains a high degree of structure, whereas the beta B2-crystallin shows a reversible sigmoidal order-disorder transition at about 58 degrees C.

Animals↗
PubMed1988

Structure and stability of gamma-crystallins: tryptophan, tyrosine, and cysteine accessibility.

The solute perturbation techniques of fluorescence of tryptophan (Trp) and dye-labeled thiol groups of cysteine as well as phosphorescence of tyrosine (Tyr) were utilized to obtain information on the relative solvent exposure and accessibility of these residues in gamma-crystallins. Both acrylamide and iodide quenchers were used to evaluate the quenching parameters in terms of accessibility and charge characteristics of the proteins. Stern-Volmer plots reveal the presence of more than one class of Trp residues in gamma-III and gamma-IV, and these residues in gamma-II are least accessible compared to the other two. Both steady-state and lifetime quenching studies of the dye-labeled fluorescence indicate that distinct differences also exist among these crystallins in cysteine (Cys) accessibilities. All three proteins, gamma-II, gamma-III, and gamma-IV, show two distinct lifetime components of the dye-labeled Cys residues. Both components of gamma-II undergo dynamic quenching, whereas only the major component of the other two crystallins is affected by the quenchers. Addition of acrylamide causes a decrease in Tyr phosphorescence of gamma-III and gamma-IV, but no change in the emission of gamma-II. The decrease is attributed to the formation of a nonemittive ground-state complex between the acrylamide and Tyr of the proteins; the association constant, Ka, calculated from the emission data, has been considered as a measure of Tyr accessibility. Ka values indicate that Tyr residues in gamma-III are most exposed and accessible compared to those in the other two proteins. Results of quenching by iodide ion reveal significant differences in the surface charge of the proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

High-molecular-weight protein aggregates of calf and cow lens: spectroscopic evaluation.

To gain insight into the molecular features of the high-molecular-weight (HMW) fraction of soluble lens proteins and their changes in aging, we isolated this fraction from the nucleus of calf and cow lenses and measured fluorescence and circular dichroism (CD) properties of the samples. Not only was there an increase in the HMW fraction in the older lens, but there was also an age-related difference in tertiary structure that was clearly manifested in the fluorescence and CD parameters. The far-u.v. CD of low- and high-molecular-weight proteins do not differ significantly in band position and magnitude, but the near-u.v. CD of HMW protein does differ distinctly from that of all other crystallins (alpha, beta and gamma); the entire CD spectrum of this protein is displayed in the negative region. Millipore filtration further revealed that HMW aggregates are essentially a polydisperse population of different conformation (tertiary structure) and that these aggregates are associated by non-convalent interactions. This association is caused mainly by the apolar (hydrophobic) nature of the constituent protein. alpha-Crystallin has more hydrophobic domain along the peptide chain that do other crystallins and thus is likely to be the predominant protein in HMW aggregates.

Aging↗
PubMed1988

Monitoring in situ circular dichroism of the intact vitreous: a new approach.

For the first time, an attempt has been made to study the vitreous humor in situ by circular dichroism (CD). The vitreous, an avascular and acellular gel-like tissue, is optically transparent and homogeneous, and, thus, light scattering is minimal. The macromolecular components of this tissue, hyaluronate (HA), collagen and noncollagenous protein (NC-P), appear to exist in the matrix in a nonoriented fashion. As a result, no linear dichroism was observed. A typical CD of the vitreous shows a minimum at 206 nm with a shoulder at 220 nm and one small positive peak at approximately 252 nm. Gaussian analysis resolves this spectrum into four component bands. CD analysis of individual components reveals that NC-P makes the major contribution to the dichroic strength of the vitreous; contributions of HA and collagen, on the other hand, are small. The positive peak arises largely from ascorbic acid in the vitreous. CD measurement of the intact vitreous appears to be a useful technique for assessing the structure and changes of the constituent molecules in the normal and diseased vitreous.

Animals↗
PubMed1987

Structure and stability of gamma-crystallins. Denaturation and proteolysis behavior.

The denaturation behavior of bovine lens gamma-crystallin fractions II, III, and IV and their susceptibility to proteolysis in vitro was compared to determine whether differences in their stability could play a role in cataract formation. Tertiary and secondary structure changes induced by increasing concentrations of urea, guanidine hydrochloride, and sodium dodecyl sulfate and by increasingly alkaline pH were followed by near-UV and far-UV circular dichroism, Trp fluorescence emission, and exposure of sulfhydryl groups. Major differences were found in the denaturation and proteolysis behavior of the three gamma-crystallin fractions. In general, the unfolding of gamma-II and gamma-III crystallins is rather gradual, suggesting the presence of intermediate unfolding states; in contrast, the order-disorder transition of gamma-IV crystallin is abrupt. The gamma-IV crystallin fraction is the most stable in urea and guanidine hydrochloride, but is most susceptible to nonspecific proteolysis and alkaline pH denaturation. Differences in denaturation and proteolysis behavior are attributed to the inherent differences in the tertiary structures of these crystallins.

Animals↗
PubMed1987

Structure and stability of gamma-crystallins. II. Differences in microenvironments and spatial arrangements of cysteine residues.

The gamma-crystallin fractions II, III and IV from calf eye lens were treated with the thiol-specific fluorescent probe 2-(4'-maleimidylanilino)naphthalene-6-sulfonate (MIANS), in order to determine the reactivity of the seven (gamma-II) or six (gamma-III, gamma-IV) cysteine residues. Two classes of reactive cysteines were distinguished by variations in fluorescence intensity with increasing molar excess of the probe, and approximately three cysteines were nonreactive in each gamma-crystallin. From the position of the emission maximum, it is apparent that MIANS-labeled cysteines of gamma-IV are in the least hydrophobic environment. Fluorescence energy transfer was observed from tryptophan to MIANS-labeled cysteines in both gamma-II and gamma-III crystallins, with efficiencies of 86% and 89%, respectively, but not in gamma-IV crystallin. We suggest that the spatial arrangements and microenvironments of cysteine residues of gamma-crystallins are sufficiently different from each other to account for the variations in fluorescence characteristics of the MIANS-labeled proteins and the lack of energy transfer in gamma-IV crystallins.

Anilino Naphthalenesulfonates↗
PubMed1987

Effects of visible-light irradiation on vitreous structure in the presence of a photosensitizer.

Sensitized photo-induced changes of vitreous structure were investigated using both in vivo and in vitro model systems. In the former, rabbit eyes were injected with the photosensitizer riboflavin, and in the latter, calf vitreous samples were treated with riboflavin or Methylene Blue prior to irradiation with white light. The active species of oxygen, i.e. singlet oxygen, superoxide anion, hydroxyl radical and hydrogen peroxide, generated by the photodynamic action of the sensitizer, caused significant liquefaction of the calf vitreous in vitro. There was little liquefaction of the rabbit vitreous in vivo, suggesting the presence of a protective mechanism in vivo. hyaluronidase induced significantly greater liquefaction in vitro than either Methylene Blue or riboflavin. This study suggests that loss of gel vitreous structure can result from extensive depolymerization of hyaluronidase by hyaluronidase and less drastic conformation and molecular weight changes in the photosensitized reactions. Although light-induced liquefaction was less marked than enzyme-induced liquefaction, the mechanism of the former is more pertinent to age-related vitreous synchysis.

Animals↗
PubMed1987

Polar-apolar characteristics and fibrillogenesis of glycosylated collagen.

To find the effect of carbohydrate on collagen fibrillogenesis, type I skin collagen was glycosylated by glycosyltransferase, and type II cartilage collagen was deglycosylated by glycosidase. The secondary structures remained unchanged, but the tertiary structures were altered, as shown by increased TNS fluorescence of the bound probe in the glycosylated form. Since TNS binds preferentially to the hydrophobic region of a protein molecule, glycosylation caused an apparent increase in the available hydrophobic sites for the dye. Glycosylation also resulted in a longer lag time and a slower growth rate of fibrillogenesis, although the amount of fibrils formed was unchanged. Deglycosylation resulted in a shorter lag time and an increased rate of fibrillogenesis. Neither glycosylation nor deglycosylation changed the stability of the molecule, as was evident from the melting temperature.

Animals↗
PubMed1987

Conformational differences between hyaluronates of gel and liquid human vitreous: fractionation and circular dichroism studies.

Post-mortem human vitreous samples (liquid and gel) of comparable intrinsic viscosity values (n approximately equal to 3000 cc/g) were chromatographed on DEAE-Sephacel columns at 4 degrees C using a linear salt gradient ranging from 0----0.4 M NaCl. All samples examined produced numerous discrete hyaluronic acid (HA) fractions. The HA fractions from liquid vitreous were eluted at lower salt concentrations than those from gel vitreous. Several HA fractions from vitreous analyzed by circular dichroism (CD) displayed CD minima at 210 nm, with an ellipticity value of 14 to 16 X 10(3) deg X cm2d mole-1. However, all HA fractions from liquid vitreous showed lower ellipticity values and a weak positive signal above 240 nm. This signal was absent in HA fractions from gel vitreous. Results suggest that subtle but definite conformational differences involving carboxylic groups exist between gel and liquid vitreous hyaluronate.

Chemical Fractionation↗
PubMed1985

Structure and stability of gamma-crystallins. I. Spectroscopic evaluation of secondary and tertiary structure in solution.

The three major bovine gamma-crystallin fractions (gamma-II, gamma-III and gamma-IV) are known to have closely related (80-90%) amino acid sequences and three-dimensional folding of the polypeptide backbone. Their chiroptical and emission properties, as measured by circular dichroism (CD) and fluorescence, are now shown to differ distinctly. The far-ultraviolet CD spectra indicate that all three gamma-crystallins have predominantly beta-sheet conformation (45-60%) with only subtle differences in secondary structure. The fluorescence emission maxima of gamma-II, gamma-III and gamma-IV, due to the four tryptophan residues, appear at 324, 329 and 334 nm, respectively, suggesting that tryptophan residues are buried in environments of decreasing hydrophobicity. Corresponding differences in quantum yield may be due to fluorescence quenching by neighboring sulfur-containing residues. Titratable tyrosines are maximal for gamma-III, as manifested from difference absorption spectra at alkaline pH. The near-ultraviolet CD spectra differ in position, magnitude and sign of tryptophan and tyrosine transitions. In addition, a characteristic CD maximum at 235 nm, presumably due to tyrosine-tyrosine exciton interactions, differs in magnitude for each gamma-crystallin. This study shows that the environment and interactions of the aromatic residues of the individual gamma-crystallin fractions are quite different. These variations in tertiary structure may be significant, in terms of stability of gamma-crystallins towards aggregation and denaturation, for understanding lens transparency and cataract formation in general.

Animals↗