Bireswar Chakrabarti

GLYCOSAMINOGLYCANS: STRUCTURE AND INTERACTION

In the last few years, there has been considerable progress in the studies on glycosaminoglycans, a group of acidic polysaccharides present in the intercellular matrix of connective tissue. X-ray diffraction studies have indicated that these polymers can exist in the condensed phase in some helical form. Chiroptical and hydrodynamic measurements have provided significant information regarding the molecular conformation in solution and other physicochemical properties of the polymers. Studies related to the interaction properties of glycosaminoglycans with polypeptides, metal ions, and other molecules are numerous. This review covers mainly the results and their interpretations of both published and as yet unpublished material of the 1970s, but certain previous data are also included. A present-day concept regarding the structure and interaction properties of these molecules on the basis of various physicochemical measurements is presented. The biosynthesis and metabolism of glycosaminoglycans, and the structure of proteoglycans and glycoproteins, are not discussed.

Heat-induced changes in the conformation of α- and β-crystalline: Unique thermal stability of α-crystallin

Of the crystallin proteins of the lens, the principal subunit of the β‐crystallin, βB2 (βBp), 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 α‐crystallin from bovine lenses is not only heat stable but also does not denature at temperatures up to 100°C. Using circular dichroism and fluorescence to monitor the conformational changes of α‐ and βB2‐crystallins upon heating, we found that α‐crystallin maintains a high degree of structure, whereas the βB2‐crystallin shows a reversible sigmoidal order‐disorder transition at about 58°C.

CHANGES IN TERTIARY STRUCTURE OF CALF-LENS α-CRYSTALLIN BY NEAR-UV IRRADIATION: ROLE OF HYDROGEN PEROXIDE

The effect of 300 nm irradiation on the three lens crystallins, α‐, β‐, and γ‐, was studied by using fluorescence and circular dichroism techniques. α‐Crystallin showed a pronounced change in tertiary structure as manifested in fluorescence and circular dichroism measurements. This finding is in agreement with our earlier findings that the tryptophan residues of α‐crystallin are more exposed than those of the other two crystallins. The results of studies using inhibitors specific for the different active species of oxygen suggest that H2O2‐mediated damage is involved in the change of tertiary structure of the proteins. Analyses of circular dichroism spectra indicate that, upon irradiation, the secondary structure of α‐crystallin remains virtually unaltered, and that the change in tertiary structure results primarily from photoinduced damage to the tryptophan residues.

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.

STUDIES ON HUMAN LENS: I. ORIGIN AND DEVELOPMENT OF FLUORESCENT PIGMENTS

Abstract

STRUCTURE AND STABILITY OF γ‐CRYSTALLINS‐IV. AGGREGATION AND STRUCTURAL DESTABILIZATION IN PHOTOSENSITIZED REACTIONS

Abstract— Unlike α‐ and β‐, γ‐crystallins become turbid upon irradiation with 300 nm or white light in the presence of photosensitizers, e.g., methylene blue (MB) or riboflavin (RF). These proteins, however, do not aggregate in the presence of guanidine hydrochloride. Turbidity formation is concentration‐dependent. Except in RF‐sensitized reactions, the onset and rate of turbidity formation are faster in γ‐IV than in the other two crystallins. Labeling the thiol groups of the protein with iodoacetamide does not change the turbidity rate in case of irradiation with 300 nm light, but it does change it in case of RF‐ or MB‐sensitized reaction. The order of rate constants of the decrease in tryptophan emission under aerobic conditions upon 300 nm irradiation and MB‐ and RF‐sensitized reactions is γ‐IV > γ‐III > γ‐II. The rate constants in the absence of air and in the presence of D2O upon MB‐ and RF‐sensitized reactions also indicate that the role of singlet oxygen is significant in the former reaction. We suggest that the photoinduced structural changes occur in two steps. In the first, photooxidation of tryptophan as well as cysteine residues (including the buried cysteines) occurs, leading to small conformational changes of the protein. Conformational changes of the protein, as evident from the near‐UV CD and fluorescence lifetime measurements, subsequently result in aggregation of the protein. As suggested by X‐ray analysis, the perturbation of the cys 78 and 32 by the active species of oxygen appears to be responsible for the destabilization of the protein structure, resulting in rapid aggregation of these crystallins.

Structure and stability of γ-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.

Optical characteristics of carboxyl group in relation to the circular dichroic properties and dissociation constants of glycosaminoglycans

Circular dichroism studies of glycosaminoglycans including chemically transformed heparins at various pH values reveal that carboxyl chromophore plays an important role in the dichroic behavior of the polymers. With decreasing pH, iduronic acid-containing glycosaminoglycans show increased negative ellipticity near 220 nm whereas the polymers containing glucuronic acid display enhanced negative dichroism near 230 nm and decreased negative dichroism around 210 nm. The pH-dependent optical properties have been utilized to determine the pKa values of uronic acid moieties. The acid strengths of the iduronic acid-containing glycosaminoglycans are inherently smaller than those of corresponding glucuronic acid-containing polymers. Glycosaminoglycans in which the amino sugars are linked with iduronic acid display a very weak n leads to pi* amide transition, or none. The rotational strength at 210 nm of these polymers is largely due to iduronic acid moieties. The CD variations above 200 nm with change in pH do not indicate any major conformational transition of the molecules but the difference between dermatan sulfate and heparin can be attributed to difference either in iduronic acid conformation or in intersaccharide linkages.

Role of singlet oxygen in the degradation of hyaluronic acid.

To investigate the effect of singlet oxygen on the molecular properties of hyaluronic acid, the polymer was irradiated in the presence of a dye sensitizer for singlet oxygen. Viscosity and circular dichroism techniques were used to monitor these changes. The relative viscosity of the polymer solution decreased steadily with increasing duration of irradiation, indicating an apparent decrease in molecular weight of hyaluronic acid. Circular dichroism measurements of the irradiated sample, however, did not show any appreciable change in the secondary structure, but do suggest that the generated singlet oxygen changes the tertiary structure and that this change is followed by a minor depolymerization.

SENSITIZER‐INDUCED CONFORMATIONAL CHANGES IN LENS CRYSTALLIN—I. PHOTODYNAMIC ACTION OF METHYLENE BLUE AND N‐FORMYLKYNURENINE ON BOVINE α‐CRYSTALLIN

Abstract— Fluorescence and circular dichroic properties of bovine a‐crystallin have been monitored to detect changes in the structural integrity of the protein following photoreactions in the presence of sensitizer, either methylene blue or N‐formylkynurenine. Methylene blue‐sensitized photooxidation causes a change in the tertiary structure as manifested in the near‐UV CD; this is observed within 0.5 h of irradiation during which time tryptophan emission decreases rapidly. Using inhibitors specific for active species of oxygen, it has been shown that singlet oxygen predominantly causes this change but the sensitizer molecules also have some role in this process. Upon 6 h of irradiation in the presence of methylene blue under both aerobic and anaerobic conditions, the thiol groups that were in a non‐polar region of the protein are exposed to polar environments. In conformity with these fluorescence results. near‐UV CD (tertiary structure) suffers a drastic alteration whereas the far‐UV CD (secondary structure) remains virtually unchanged. The studies with inhibitors indicate that sensitizer molecule itself is primarily responsible for this process. This major change in the conformation has been explained by suggesting that a large portion of the protein unfolds in the photosensitized reaction, thereby altering microenviron‐ments, orientation, and intermolecular interactions of different amino acids. N‐formylkynurenine also shows some changes in the near‐UV CD, presumably, caused by H2O2 generated in the photosensitized reaction. But the major alteration in the microenvironments of thiol groups and in the near‐UV CD, as observed in the case of methylene blue, does not occur even when the protein is irradiated for 6 h in the presence of N‐formylkynurenine and air.