Interactions between mucopolysaccharides and cationic polypeptides in aqueous solution: Chondroitin 4-sulfate and dermatan sulfate

Circular dichroism spectroscopy has been used to study the interactions of both dermatan sulfate and chondroitin 4-sulfate with the cationic polypeptides; poly(L -arginine), poly(L -lysine), and poly(L -ornithine). The results indicate that the mucopolysaccharides have a conformation directing effect on both poly(L -arginine) and poly-(L -lysine) such that these polypeptides adopt the α-helical conformation. The extent of interaction in each polypeptide-polysaccharide system can be judged by the degree of induced helicity and the “melting temperature” at which the interaction is disrupted On comparison of these results with those previously obtained for chondroitin 6-sulfate-polypeptide mixtures, the extent of interaction can be seen to depend on the length of the amino acid side chain and the positions of the anionic groups on the mucopolysaccharide chain. Such considerations place the three mucopolysaccharides in order of increasing interaction: chondroitin 4-sulfate < chondroitin 6-sulfate < dermatan sulfate. These results are correlated with observations that dermatan sulfate is bound more tightly to collagen in connective tissues than are the other two polysaccharides.     

Interactions between mucopolysaccharides and cationic polypeptides in aqueous solution: hyaluronic acid, heparitin sulfate, and keratan sulfate

Circular dichroism spectroscopy has been used to study the interactions of hyaluronic acid, heparitin sulfate, and keratan sulfate with cationic polypeptides. The results indicate that the presence of these mucopolysaccharides has an effect in the conformation of poly(L -lysine) and poly(L -arginine), such that the former adopts the “random” form and the latter takes up the α-helical conformation, rather than the “charged coil” form expected at neutral pH. The relative strengths of the interactions can be judged from the melting temperatures above which they are disrupted. Both the stoichiometry and the strength of the interactions depend on the position, number, and type of anionic groups attached to the polysaccharide backbone. Such considerations place the six common mucopolysaccharides in order of increasing strength of interaction: hyaluronic acid < chondroitin 4-sulfate < heparitin sulfate < chondroitin 6-sulfate < keratan sulfate ? dermatan sulfate. These differences should be paralleled by differences in the interaction of the mucopolysaccharides with collagen and fibrous proteins.     

Comparison of four proteoglycans in terms of their interactions with poly(L-arginine).

Connective tissue proteoglycans undergo interaction with poly(L -arginine) when mixed in dilute neutral aqueous solution. Circular dichroism spectroscopy indicates that the polypeptide adopts the α-helical conformation rather than the extended coil form normally observed at neutral pH. The interactions of a series of proteoglycans with different protein and glycosaminoglycan contents have been compared. The arginine/disaccharide residue ratio at maximum interaction appears to be constant with varying protein content of the proteoglycans that contain chondroitin 4-sulfate. The thermal stability of the proteoglycan interaction is the same as for the component polysaccharide. Thus in terms of the strength of interaction with homopolypeptides, the properties of proteoglycan and the component glycosaminoglycans are the same, and this is likely also to be the case for collagen–proteoglycan systems. The interactions of keratan sulfate-2 have also been investigated. These are similar but have much lower thermal stability than corneal keratan sulfate-1. The results are consistent with the weak interaction of the keratan sulfate-2 component of bovine nasal septum proteoglycan.     

Effects of glycosaminoglycans and proteoglycan on the in vitro assembly and thermal stability of collagen fibrils

The effects of three glycosaminoglycans (chondroitin 6-sulfate, dermatan sulfate, and hyaluronate) and a proteoglycan on the kinetics of fibril formation and on the thermal stability of the in vitro assembled collagen fibrils, under physiological conditions of ionic strength and pH, have been examined. The glycosaminoglycans were found to influence the kinetics of collagen precipitation but not the thermal stability of the in vitro assembled fibrils. The proteoglycan was found to influence the kinetics of collagen precipitation and to reduce the thermal stability of the in vitro assembled fibrils. Comparison of the interaction occurring between chondroitin 6-sulfate and collagen under acidic conditions (0.05M acetic acid) and that occurring under physiological conditions showed that markedly different interaction products were formed under the different conditions.     

Vacuum-ultraviolet circular dichroism of chondroitins and their complexes with poly(L-arginine)

The vacuum-ultraviolet circular dichroism (VUCD) of chondroitin and chontroitin-6-sulfate has been measured to 160 nm for films and to 170 nm for D₂O solutions. The pD-dependent dichroic behavior of these glycosaminoglycans in D₂O is similar above 200 nm and is in agreement with previous studies. Near 190 nm, the CD band sign is also dependent on pD. VUCD spectra were recorded for films and solutions of poly(L -arginine). In trifluoroethanol the polypeptide is α-helical, while in D₂O it exists as a random coil. The well-characterized coil–helix transition of poly(L -arginine) during complexation with chondroitin-6-sulfate was observed by VUCD, including the previously inaccessible entire π → π* band. By construction of difference spectra it was also possible to monitor the VUCD of the polysaccharide component during complexation.     

Interaction between chondroitin-6-sulfate and poly-L-arginine in aqueous solution

The interactions between chondroitin-6-sulfate and poly-L -arginine in aqueous salt solution have been investigated by circular dichroism techniques. In the presence of chondroitin-6-sulfate, at neutral pH, poly-L -arginine adopts the α-helical conformation rather than “charged coil” form observed in the absence of mucopolysaccharide. This interaction is at a maximum when the ratio of arginine to disaccharide residues is 2:1. Elevation of the temperature leads to a sharp melting transition at 76.0 ± 1.0°C. This behavior is in marked contrast to that for poly-L -lysine-chondroitin-6-sulfate interactions, which are at a maximum at a 1:1 residue ratio and have a melting transition at 47.0 ± 1.0°C. These results indicate a stronger interaction for poly-L -arginine than for poly-L -lysine. The positive arginine side chains appear to interact with both the negative sulfate and carboxyl residues, while those of the lysines are involved only with the sulfates. Poly-L -ornithine at neutral pH shows no conformation directing interaction with chondroitin-6-sulfate, although a small proportion of α-helix is formed on dilution of the mixture with methanol. The extent of the interaction of cationic polypeptides with chondroitin-6-sulfate increases in the order poly-L -ornithine, poly-L -lysine, poly-L -arginine, i.e., in the order of increasing side-chain length.     

Interactions between chondroitin-6-sulfate and poly-L-lysine in aqueous solution: Circular dichroism studies

The interactions between chondroitin-6-sulfate (chondroitin sulfate C) and poly-L -lysine have been studied as models for investigation of possible complex formation between fibrous proteins and mucopolysaccharides. Results obtained using circular dichroism spectroscopy show that poly-L -lysine adopts the α-helical conformation in dilute aqueous salt solution at pH 7 when mixed with chondroitin-6-sulfate, rather than the “charged-coil” observed in the absence of this mucopolysaccharide. This conformation-directing interaction is at a maximum when the ratio of lysine to disaccharide residues is 1 : 1. Changes in the CD spectrum of a 1 : 1 mixture following increase in the salt concentration, or addition of non-polar solvents, indicate that the interaction is ionic in nature. No such effects are observed for non-sulfated mucopolysaccharides mixed with poly-L -lysine, suggesting that, for chondroitin-6-sulfate, it is the sulfate groups rather than the carboxyls which interact with the amine groups of the polypeptide. Elevation of the temperature leads to disruption of the interactions between the polypeptide and polysaccharide species. A sharp melting transition occurs at 47.0 ± 1.0°C, when the poly-L -lysine reverts to the “charged-coil” conformation. The sharp transition suggests that regular ionic bonds are formed between the polypeptide and polysaccharide. These results suggest that a conformation-directing interaction may occur between sulfated mucopolysaccharides and the polar regions of collagen and other fibrous proteins.     

The binding of chondroitin 6-sulfate to plasma low density lipoprotein

The interaction in vitro of several sulfated glycosaminoglycans with low density lipoproteins (LDL) has been studied. Chondroitin 6-sulfate and heparin were the only ones to produce turbidity when added to LDL in presence of Ca2+. However, when these two glycosaminoglycans were applied to LDL-affinity columns in presence of Ca2+, only chondroitin 6-sulfate was retained. Partially desulfated chondroitin 6-sulfate was not retained on LDL-affinity column, indicating the relevance of sulfate groups in the binding of LDL. Since chondroitin 4-sulfate and heparin, with a sulfate content respectively equal to and greater than that of chondroitin 6-sulfate, are not retained on LDL-affinity columns, the factors relevant to the binding of LDL are probably the conformation of the glycan in solution and the orientation of its sulfate groups.     

Laser Raman spectroscopic studies of poly(r-cytidylic acid) and poly(r-adenylic acid) complexes with poly(L-lysine) and poly(L-arginine)

Complexes of polyribocytidylic acid and polyriboadenylic acid with poly(L -lysine) and poly(L -arginine) were studied by Raman spectroscopy. The backbones of both polynucleotides are distorted by poly(L -arginine). On the other hand, poly(L -lysine) could distort the backbone of polyriboadenylic acid but not that of polyribocytidylic acid. In general, poly(L -arginine) can increase the order of the base stacking, while poly(L -lysine) causes disordering in the base stacking.     

Interactions of an intact proteoglycan and its fragments with basic homopolypeptides in dilute aqueous solution

The interactions between a proteoglycan and cationic polypeptides have been investigated by the use of circular-dichroism spectroscopy. The interaction produces an induced conformational change for poly(l-arginine) and poly(l-lysine), similar to the effects previously reported for mucopolysaccharide-polypeptide mixtures. For bovine nasal septum proteoglycan, the interactions are similar to those for chondroitin 4-sulphate, which comprises approximately 63% of the total polysaccharide. The results also suggest that the interactions produce a conformational change in the protein core. Similar studies for the Smith-degradation product show that the protein core can adopt a substantial alpha-helical content and is capable of interactions with poly-(l-arginine). The interactions for chondroitin sulphate ;doublets' are significantly different from those for the separated chains, indicating that the arrangement of the polysaccharide side chains in pairs (and larger groups) along the protein backbone contributes to the interaction properties of the intact proteoglycan.     

Spin-label studies of the pH-induced random coil to α-helix conformational transition in poly(L-lysine)

Poly(L -lysine) of various molecular weights between 2700 and 475,000 was spin-labeled. From the electron spin resonance spectra, the degree of freedom of the nitroxide was determined by calculation of the rotational correlation time as the poly(L -lysine) underwent the pH-induced random coil to α-helix conformational transition. In general, the rotational correlation time of the nitroxide increased as the pH was increased, indicating a more restricted environment for the spin label when poly(L -lysine) is deprotonated. For the high-molecular-weight poly(L -lysine) this corresponds to the formation of the α-helix and indicates that the side chain–side chain interaction and decreased segmental motion of the backbone (slightly) restricts the motion of the spin label. For the 2700-molecular-weight poly(L -lysine), previously shown not to assume a helical conformation at high pH, the increase in the rotational correlation time of the spin label indicates that the side chain–side chain interaction takes place after deprotonation but without helix formation. This may indicate that helix formation per se is not needed to produce the observed effect even with the high-molecular-weight polymers. The rotational correlation time of the spin label at a particular pH did not depend on the molecular weight of the poly(L -lysine) over the 200-fold range of molecular weights. This indicates that the rotational correlation time reflects the rotational mobility of the spin label in a localized environment and not the rotational diffusion of the entire macromolecule.     

Mucopolysaccharide-polypeptide interactions: Effect of the position of the sulfate group

The differences in the interaction in solution of poly(l-lysine) with chondroitin 6-sulfate (chondroitin sulfate C) and with chondroitin 4-sulfate (chondroitin sulfate A) have been studied by circular dichroism spectroscopy. Both mucopolysaccharides force the poly(l-lysine) to adopt the α-helix in solution rather than the charged coil form expected at neutral pH. The observed spectra indicates that the polypeptide is at least 80% helical when the 6-sulfate form is present, but only about 20% α-helical in the presence of chondroitin 4-sulfate. Thus chondroitin66-sulfate has a stronger conformation directing effect on poly(l-lysine) than does the 4-sulfate, which is probably due to the different positions of the sulfate group on the polysaccharide c chain.     

The solution conformation of poly(L-lysine). A Raman and infrared spectroscopic study.

The Raman and infrared spectra of poly(L -lysine) and poly(DL -lysine) in solution are reported and the effects of various salts are investigated. The results demonstrate that α-helix formation in solution is induced by specific salts and the spectral data support the hypothesis of regions of local order for poly(L -lysine) in aqueous solutions of low ionic strength.     

Conformation of poly(L-arginine). II. Complexes with polyanions

Conformaitons of poly(L -arginine)/polyanion complexes were studies by CD measurements. The polyanions were the homoplolypeptides poly(L -glutamic acid) and poly(L -aspartic acid); the synthetic polyelectrolytes and polyethylenesulfonate; and the polynucleotides were native DNA, denatured DNA, and poly(U). It was found that poly(L -arginine) forms the α-helical conformation by interacting with the acidic homopolypeptides and the synthetic anionic polyelectrolytes. In each complex, poly(L -glutamic acid) is in the α-helical conformation, whereas poly(L -aspartic acid) is mostly in the random structure. The poly(L -glutamic acid) complex changed into the β-sheet structure at the transition temperature about 65°C in 0.01M cacodylate buffer (pH 7). Even in the presence of 5M urea, this complex remained in the α-helical conformation at room temperature. The existence of the stable complex of α-helical poly(L -arginine) and α-helical poly(L -glutamic acid) was successfully supported by the model building study of the complex. The α-helix of poly(L -arginine) induced by binding with polyacrylate was the most stable of the poly(L -arginine)-polyanion complexes examined as evidenced by thermal and urea effects. The lower helical content of the polyethylenesulfonate-complexed poly(L -aginine) was explained in terms of the higher charge density of the polyanion. On the other hand, native DNA, denatured DNA, and poly(U) were not effective in stabilizing the helical structure of poly(L -arginine). This may be due to the rigidity of polyanions and to the steric hindrance of bases. Furthermore, the distinitive structual behavior of poly(L -arginine) and poly(L -lysine) regarding polyanion interaction has been noticed throughout the study.     

Conformational studies on Cu(II) polycomplexes of pyridoxal Schiff bases of polypeptides

Structures of Cu(II) complexes of pyridoxal Schiff bases with poly(L -lysine), poly(L -ornithine), and poly(L -α,γ-diaminobutyric acid) were investigated by absorption spectra, CD, and conformational analysis. Although the polypeptides retain their typical right-handed α-helical conformation, opposite Cotton effects were found for the poly(L -lysine) and poly(L -ornithine) polycomplexes in the whole range of wavelengths from 600 to 250 nm. As in the analogous derivatives of salicyladehyde, this effect seems to be due to a stereospecific binding of the square planar Cu(II)-bis-pyridoxylideneimine group to the α-helical matrix. Circular dichroism spectrum of poly(L -α,γ-diaminobutyric acid) polycomplex is similar to that found for poly(L -lysine) derivative, but indicates large tetrahedral distortion of the square-planar coordination of copper ion.     

Physicochemical characteristics of the glycosaminoglycan-lysosomal enzyme interaction in vitro. A model of control of leucocytic lysosomal activity.

1. The activities of 30 different lysosomal enzymes were determined in vitro in the presence of the sulphated glycosaminoglycans, heparin and chondroitin sulphate, all the enzymes being measured on a density-gradient-purified lysosomal fraction. 2. Each enzyme was studied as a function of the pH of the incubation medium. In general the presence of sulphated glycosaminoglycans induced a strong pH-dependent inhibition of lysosomal enzymes at pH values lower than 5.0, with full activity at higher pH values. However, in the particular case of lysozyme and phospholipase A2 the heparin-induced inhibition was maintained in the pH range 4.0-7.0. 3. For certain enzymes, such as acid beta-glycerophosphatase, alpha-galactosidase, acid lipase, lysozyme and phospholipase A2, the pH-dependent behaviour obtained in the presence of heparin was quite different to that obtained with chondroitin sulphate, suggesting the existence of physicochemical characteristic factors playing a role in the intermolecular interaction for each of the sulphated glycosaminoglycans studied. 4. Except in the particular case of peroxidase activity, in all other lysosomal enzymes measured the glycosaminoglycan-enzyme complex formation was a temperature-and time-independent phenomenon. 5. The effects of the ionic strength and pH on this intermolecular interaction reinforce the concept of an electrostatic reversible interaction between anionic groups of the glycosaminoglycans and cationic groups on the enzyme molecule. 6. As leucocytic primary lysosomes have a very acid intragranular pH and large amounts of chondroitin sulphate, we propose that this glycosaminoglycan might act as molecular regulator of leucocytic activity, by inhibiting lysosomal enzymes when the intragranular pH is below the pI of lysosomal enzymes. This fact, plus the intravacuolar pH changes described during the phagocytic process, might explain the unresponsiveness of lysosomal enzymes against each other existing in primary lysosomes as well as its full activation at pH values occurring in secondary lysosomes during the phagocytic process.     

Conformation of hyaluronate in neutral and alkaline solutions.

Increasing the pH of a neutral salt solution of sodium hyaluronate to 12.5 produces a rapid drop in viscosity which is reversible upon restoring the pH to neutrality. Light scattering data showing a decrease in radius of gyration with no change in molecular weight and negative results with chondroitin and other acidic glycosaminoglycans suggest that the conformational change is specific for hyaluronate molecules.     

An X-ray diffraction study of poly-L-homoarginine hydrochloride.

An X-ray study of the synthetic polypeptide poly(L -homoarginine hydrochloride) has been made to investigate whether, like the chemically related polypeptides poly(L -lysine hydrochloride), poly(L -arginine hydrochloride), and poly(L -ornithine hydrobromide), it can undergo conformational transitions merely from variations in its degree of hydration. X-ray photographs of powder and oriented specimens containing one to 15 molecules of water per L -homoarginine hydrochloride residue showed that this polymer forms only a β-pleated-sheet structure. The pleated sheets, formed by antiparallel polypeptide chains hydrogen-bonded to each other, are piled up along the b axis in an alternating sequence (“sandwich structure”). This structure did not appreciably change with variations of the degree of hydration, and the observed reflections at 56% relative humidity (1.8 molecules of water per residue) could be indexed satisfactorily in terms of a monoclinic unit cell, of space group P2₁, with a = 9.34 Å, b = 40.07 Å, c = 6.94 Å, and γ = 106°. These dimensions are shown by models to be compatible with the proposed structure, and the calculated density of 1.27 g/cm³ agrees well with the experimental value of 1.29 g/cm³. Removal of the last molecule of water results in a very diffuse pattern, while specimens containing 20 molecules of water per residue show only reflections due to water.     

Conformation of hyaluronate in neutral and alkaline solutions

Increasing the pH of a neutral salt solution of sodium hyaluronate to 12.5 produces a rapid drop in viscosity which is reversible upon restoring the pH to neutrality. Light scattering data showing a decrease in radius of gyration with no change in molecular weight and negative results with chondroitin and other acidic glycosaminoglycans suggest that the conformational change is specific for hyaluronate molecules.     

Chromatography of glycosaminoglycans on hydrophobic gel. Correlation between chromatographic behavior of glycosaminoglycans on phenyl-sepharose CL-4B and their solubility in the presence of high concentrations of ammonium sulfate

The effect of bound sulfate groups and uronic acid residues of glycosaminoglycans on their behavior in chromatography on hydrophobic gel was examined by the use of several pairs of depolymerized chondroitin, chondroitin 4- or 6-sulfate, and dermatan sulfate having comparable degree of polymerization. Chromatography on Phenyl-Sepharose CL-4B in 4.0-2.0 ammonium sulfate containing 10m hydrochloric acid showed that: (a) The retention of depolymerized chondroitin 4- or 6-sulfate on the gel varies with the temperature, whereas the depolymerized samples of chondroitin and dermatan sulfate does not show a temperature dependence (this is not the case for hyaluronic acid or dextrans). (b) Among depolymerized samples of chondroitin and chondroitin 4- and 6-sulfate that have a similar degree of polymerization, chondroitin 4- and 6-sulfate showed the highest retention. (c) The retention on the gel of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate decreased in this order. The solubility in ammonium sulfate solution of the polysaccharides agreed well with the chromatographic behavior, suggesting that the fractionation by the hydrophobic gel largely depends on the ability to precipitate on the gel rather than on the hydrophobic interaction between gel and polysaccharide.