Dilute solution properties of proteoglycan fractions from bovine nasal cartilage

Fresh proteogycans (adult bovine nasal cartilage) isolated from the densest portion of a dissociative density gradient had a weight-average molecular weight of ca. 10⁶ in 4M guanidine hydrochloride (GdnHCI) by light scattering. Fractions of such material obtained by elution with 4M GdnHCI from 2% agarose gel, both normal and cross-linkd, has proteoglycan subunit molecular weights ranging from 0.8 to 2.6 × 10⁶ and root-mean-square radii ranging from 35 to 52 nm in the same solvent. The protein molecular weight per proteoglycan subunit was about 1.2 × 10⁵ and that of keratan sulfate about 1.8 × 10⁵, both independent of total molecular weight. A random-flight “graft copolymer” model having uniform side chains of chondroitin sulfate (40 disaccharides) and keratan sulfate (15 disaccharides) and a random-coil polypeptide back bone was used to estimate the unperturbed radius, whihc was about 19 nm for a mol wt of 1.5 × 10⁶. Experimental light-scattering data for fractions were fitted very well by theoretical curves for the particale scattering factor for both linear and appropriate branched polymers. Examination of coil expansion on the basis of perturbation calculations for branched polymer models suggested that expansion did not account for the experimentally observed radii in terms of unperturbed radii calculated from the model. A possible explanation is that substantial local stiffening of the polypeptide chain due to substitution of side-chain clusters increases the unperturbed radii. The intrinsic viscosity [η] is 4M GdnHCI ranged from 120 to 180 ml/g, and could be interpreted in terms of th eequivalent sphere model; the Flory number has approximately its normal value for flexible linear polymers. The treatment of the sedimentation coefficient by this is less successful, since the Man delkern-Flory parameter β apparently increases with increasing molecular weight; average value are similar to those for flexible polymers, but the variation in β makes this method useful only for rough estimation of molecular weight of proteoglycans. Molecular weights of purified proteoglycans are the same in 0.2M NaCI as in 4M GdnHCI, while crude preparations gave higher molecular weights in 0.2M NaCI, probably because of association due to incomplete removel of “linking” proteins.     

Small-angle x-ray scattering of bovine nasal cartilage proteoglycan in solution

The proteoglycan subunit (PGS) from bovine nasal cartilage was examined in water and in 0.15 N LiCl by small-angle x-ray scattering (SAXS). The molecular weight of 2.5 × 10⁶ and the radius of gyration, R_g = 493 Å, in 0.15 N LiCl, obtained by SAXS, are in good agreement with values reported by others for similar preparations. Values of the radius of gyration of the cross section, mass per unit length, and persistence length of the PGS are also reported. The low value of intrinsic viscosity ([η]) found in 0.15 N LiCl, and a comparison of the experimental distance distribution function to that of the theoretical distance distribution function for sphere, suggest that the PGS in salt solution approaches spherical symmetry. The much higher value of [η] in water suggests a prolate ellipsoid of low axial ratio.     

Motility of bovine spermatozoa studied by laser light scattering

Laser light scattering has been employed to determine the swimming speed distribution and the fraction of motile cells in samples of bovine spermatozoa. As predicted from theory, average trajectory velocities determined by laser light scattering were approximately four times the average translational speed estimated using light microscopy. The proportion of motile spermatozoa decreased with time at the same rate when samples were prepared in either HEPES or phosphate buffers. However, whereas the mean swimming velocity declined slowly in HEPES buffer, it dropped rapidly when phosphate buffer was used. Dilution (in the range 40–0.4×10⁶ spermatozoa·ml^-1) in either of these two buffers reduced the fraction of motile spermatozoa in the sample, but the mean swimming velocity of the remaining active spermatozoa was unchanged. Lowering the temperature from 37° C to 15° C reduced the mean swimming speed by a factor of 2–3 and the fraction of motile cells by a factor of 4–5.     

Interaction between proteoglycan subunit and type II collagen from bovine nasal cartilage, and the preferential binding of proteoglycan subunit to type I collagen.

We studied the interaction of proteoglycan subunit with both types I and II collagen. All three molecular species were isolated from the ox. Type II collagen, prepared from papain-digested bovine nasal cartilage, was characterized by gel electrophoresis, amino acid analysis and CM-cellulose chromatography. By comparison of type I collagen, prepared from papain-digested calf skin, with native calf skin acid-soluble tropocollagen, we concluded that the papain treatment left the collagen molecules intact. Interactions were carried out at 4 degrees C in 0.06 M-sodium acetate, pH 4.8, and the results were studied by two slightly different methods involving CM-cellulose chromatography and polyacrylamide-gel electrophoresis. It was demonstrated that proteoglycan subunit, from bovine nasal cartilage, bound to cartilage collagen. Competitive-interaction experiments showed that, in the presence of equal amounts of calf skin acid-soluble tropocollagen (type I) and bovine nasal cartilage collagen (type II), proteoglycan subunit bound preferentially to the type I collagen. We suggest from these results that, although not measured under physiological conditions, it is unlikely that the binding in vivo between type II collagen and proteoglycan is appreciably stronger than that between type I collagen and proteoglycan.     

Radioimmunoassay of the link proteins associated with bovine nasal cartilage proteoglycan.

Link proteins from bovine nasal cartilage have been purified by preparative polyacrylamide gel electrophoresis in sodium dodecyl sulfate (Baker, J.R., and Caterson, B. (1979) J. Biol. Chem. 254, 2387-2393) and used to raise antisera in rabbits. A sensitive radioimmunoassay procedure utilizing binding of 125I-labeled antigen . antibody complexes to Protein A of Staphylococcus aureus has served to demonstrate the specificity of the antisera for the link proteins. The lack of reactivity with proteoglycan fractions indicates that link proteins and proteoglycan do not share antigenic determinants. This result is in accord with published cyanogen bromide peptide cleavage data (Baker, J.R., and Caterson B. (1977) Biochem. Biophys. Res. Commun. 77, 1-10) which showed proteoglycan and link protein to be structurally dissimilar. The radioimmunoassay procedure has been used to quantitate small amounts of link protein which remain associated with proteoglycan after purification by equilibrium density gradient centrifugation in 4 M guanidine HCl and by gel chromatography in sodium dodecyl sulfate.     

The subunits of chemically treated proteoglycan isolated from bovine nasal cartilage

1. The light fraction of the proteoglycan of bovine nasal cartilage was split by treatment with 0.1m-hydrochloric acid in acetone. The products were separated by gel filtration on 4% agarose and two retarded fractions were detected and isolated. These two fractions were found to have a Stokes radius of 134 and 47 A respectively, as determined by calibration of the column against proteins of known hydrodynamic volumes. 2. The 47 A fraction had a protein content of 4% and a glucosamine/galactosamine ratio 1:23. The 134 A fraction had a protein content of 20% and a glucosamine/galactosamine ratio 1:4.8. 3. The results of the viscometric studies on both fractions suggested that the 134 A fraction alone exhibited the property of undergoing reversible pH-dependent aggregation with a transition point at pH4.9. 4. It was concluded that these fractions could represent subunits of the native cartilage proteoglycan.     

The isolation of collagen-associated proteoglycan from bovine nasal cartilage and its preferential interaction with alpha2 chains of type I collagen.

A collagen complex from bovine nasal cartilage was prepared by extraction of the tissue with 3M-MgCl2 solutions, by using two different procedures. When it was compared with calf skin acid-soluble tropocollagen by polyacrylamide-gel electrophoresis, the 3M-MgCl2-soluble cartilage collagen in the complex appeared to be predominantly type I in nature, consisting of both alpha1 and alpha2 chains. The soluble cartilage collagens were digested with purified bacterial collagenase, and the soluble digests were fractionated on Sepharose 4B. Hydroxyproline-free proteoglycan was isolated in the excluded volume of the column eluate, and this was found to be an aggregate which could be dissociated to link proteins and proteoglycan subunit by equilibrium-density-gradient centrifugation in a CsCl-4M-guanidinium chloride gradient. Interaction with calf skin-soluble tropocollagen was studied by CM-cellulose chromatography. The link-protein system did not interact, but proteoglycan from the bottom of the gradient did interact. In addition, when proteoglycan subunit was allowed to interact with collagen, there was a preferential binding to the alpha2 and beta12 components, and this effect was also observed with the proteoglycan material obtained from the collagenase digests of 3M-MgCl2-soluble cartilage collagen complexes. However, specificity for alpha2 and beta12 chains was not exhibited by chondroitin sulphate glycosaminoglycan, and it is therefore concluded that preference for alpha2 and beta12 chains is a function of the intact proteoglycan structure.     

Self-assembly of melanin studied by laser light scattering

The unknown molecular weight and chemical structure of melanin place the study of these pigments outside the range of the classical biochemical techniques; thus in this paper the problem of characterizing these heterogeneous biopolymers was approached by means of light scattering techniques, static and dynamic. The static technique allowed us to identify the macromolecular properties (MW and R(g)(2)(1/2)) of melanin extracted from sepia inksac and of two synthetic analogues: L-Dopa melanin obtained by autooxidation and by enzymatic oxidation by Tyrosinase. By dynamic light scattering (DLS), the hydrodynamic radius R(h) was measured to monitor the temporal behaviour of the polymerization and aggregation processes and R(h) variation by changing the chemical constraints of the polymerization medium, such as pH and ionic strength. The fractal dimension d of the aggregates of melanin, both natural and synthetic, in the past only recognized during the aggregation of the synthetic one by lowering the pH of the medium, was a useful parameter to further investigate and compare the structure of melanin granules of differing origins, revealing for the natural sample, a structure with clusters that are spherical, not largely hydrated and self-assembled, following a reaction limited aggregation kinetics (d=2.38).     

Proteoglycans of bovine articular cartilage. Studies of the direct interaction of link protein with hyaluronate in the absence of proteoglycan monomer

When link protein binds to hyaluronate in the absence of proteoglycan monomer a high molecular weight complex is formed. Two assay procedures have been developed to examine the formation of the complex and the rate and stoichiometry of binding of link protein to hyaluronate in the complex. In the first, the complex is isolated by differential centrifugation, and the stoichiometry of binding of link protein to hyaluronate in the sedimented complex is determined. In the second assay, which involves turbidimetry, the rate of complex formation (delta A420/min) is determined, and the amount of complex formed is determined in terms of the maximum turbidity (A420,max) attained. The effects of temperature, pH, initial total solute concentration, and the ratio by weight of link protein to hyaluronate on the amount of complex formed and on the rate of complex formation were examined. There is a linear correlation between the amount of complex formed as determined by turbidity and by differential centrifugation. Using these assays, we examined the specificity of the binding of link protein to hyaluronate and the capacity of hyaluronate oligosaccharides to competitively inhibit the binding of link protein to hyaluronate. Hyaluronate decasaccharide is the oligosaccharide of minimum size that strongly inhibits the binding of link protein to hyaluronate. Proteoglycan monomers dissociate from hyaluronate as the pH is decreased from pH 7 to pH 5. Turbidimetric studies show that the rate of binding of link protein to hyaluronate increases with decreasing pH. The binding affinity of proteoglycan monomers for hyaluronate is decreased at pH 5, whereas the binding affinity of link protein for hyaluronate is not. This difference in the effect of pH on the stability of binding of link protein to hyaluronate, compared with proteoglycan monomer, explains in part the capacity of link protein to stabilize the binding of proteoglycan monomer to hyaluronate at pH 5.     

The isolation and characterization of the link proteins from proteoglycan aggregates of bovine nasal cartilage.

A preparation containing the link proteins may be obtained from bovine nasal cartilage by extraction with 4 M guanidine hydrochloride and by equilibrium density gradient centrifugations of the extract as commonly employed in the isolation of proteoglycan monomers. In the present paper, protein-rich proteoglycans have been removed from such a preparation to give purified link proteins by chromatography on Sepharose CL-6B in 1% sodium dodecyl sulfate. The individual link proteins, which in order of increasing electrophoretic mobility are termed link proteins 1, 2, and 3, have been separated and isolated in a subsequent preparative gel electrophoresis step. The link proteins present in largest amount, link proteins 1 and 2, have essentially the same amino acid compositions, and following partial digestion with the V8 protease from Staphylococcus aureus and analytical electrophoresis in sodium dodecyl sulfate, their peptide patterns closely resemble each other. Therefore,it is probable that link proteins 1 and 2 are structurally similar. Link protein 1 contains more carbohydrate than link protein 2 (9.5% and 3.0%, respectively) and it is suggested that the major difference between them is in carbohydrate content.     

Cytoplasmic streaming in Chara corallina studied by laser light scattering.

An apparatus is described by means of which the power versus frequency spectrum of the photomultiplier current can be obtained for laser light scattered by streaming cytoplasm in the algal cell Chara corallina. A Doppler peak is noted in the spectrum which is abolished when cytoplasmic streaming is arrested by electrical stimulation. For 5 cells of Chara, this simple laser-Doppler velocimeter gave streaming velocities (46-7 mum s-1, S.D. +/- 4-8 at 20 degrees C) similar to those obtained for the same cells using the light microscope (44-3 mum s-1, S.D. +/- 5-3 at 20 degrees C). A narrow distribution of streaming velocities is indicated. The technique described provides a rapid, quantitative assay of the in vivo rheological properties of cytoplasm.     

Studies on the interaction of newly secreted proteoglycan subunits with hyaluronate in human articular cartilage

Newly secreted proteoglycans from adult human cartilage do not interact well with hyaluronate, but attain this ability with time in the extracellular matrix. The conversion process occurs in all types of cartilagenous matrix, as newborn cartilage cultures, chondrosarcoma cultures and adult chondrocyte cultures each secreted proteoglycan subunits which exhibited the delayed aggregation phenomenon. However, the rate of conversion is probably dependent upon the structure of the surrounding matrix and the cell type. In vitro, link protein appears to enhance an initial change in the hyaluronate-binding region of the newly secreted proteoglycan subunits to allows stronger interaction with hyaluronate. In a second step, which is pH- and temperature-dependent, the change becomes irreversible. Thus, in addition to its role in stabilizing the interaction of mature proteoglycan subunits with hyaluronate, link protein may also aid in promoting the conversion of the newly synthesized proteoglycan subunit to a form that is capable of strong interaction with hyaluronate.     

Self-association of long-acting insulin analogues studied by size exclusion chromatography coupled to multi-angle light scattering

Two structurally very different insulin analogues analysed here, belong to a class of analogues of which two have been reported to have a protracted action through self-assembly to high molar mass in subcutis. The process of self-association of insulin analogues Lys(B29) (N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin and Lys(B29) (N(ε)-lithocholyl) des(B30) human insulin was investigated using size exclusion chromatography (SEC) in connection with multi-angle light-scattering. Self-assembly to high molar mass was obtained by exchanging the formulation containing phenolic preservatives with an isotonic eluent during SEC. It was shown that increasing amounts of zinc in the formulations of the two analogues increased the size of the self assemblies formed during gel filtration. The addition of 0.2 mM phenol to the elution buffer slowed down the self-association process of zinc containing formulations and shed light on the initial association process. The results indicated that a dihexamer is a possible building block during self-association of Lys(B29) (N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin. Surprisingly, in the absence of zinc the two analogues behaved very differently. Lys(B29) (N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin was in equilibrium between oligomers smaller than a hexamer, whereas Lys(B29) (N(ε)-lithocholyl) des(B30) human insulin self-associated and formed even larger complexes than in the presence of zinc.     

Interactions of cartilage proteoglycans with hyaluronate. Inhibition of the interaction by modified oligomers of hyaluronate.

Oligomers of hyaluronic acid were prepared by digestion of hyaluronic acid from rooster combs with testicular hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35), leech head hyaluronidase (hyaluronate 3-glycanohydrolase, EC 3.2.1.36), and with fungal hyaluronidase (hyaluronate lyase from Streptomyces hyalurolyticus). The oligomers were fractionated by gel permeation, using Sephadex G-50. Oligomers isolated after incubation of the hyaluronic acid with the testicular hyaluronidase were further modified. To prepare oligomers with N-acetylglucosamine at both ends, terminal nonreducing glucuronic acid residues were removed with beta-glucuronidase. Reducing terminal N-acetylglucosamine residues were removed by reaction under mildly alkaline conditions. The reducing terminal N-acetylglucosamine residues were also reduced with sodium borohydride to form N-acetylglucosaminitol. The potentials of the various oligosaccharides to bind to the proteoglycan from bovine nasal septum cartilage were estimated by determining their effectiveness as inhibitors of the proteoglycan-hyaluronate interaction. The present study shows that, to bind maximally to the proteoglycan, the hyaluronate oligosaccharide must be at least 10 sugar residues in length and be terminated at the nonreducing and reducing ends with a glucuronate residue and an N-acetylglucosamine residue, respectively. Sugar residues extended beyond this basic decasaccharide, do not interact with the hyaluronate binding site on the proteoglycan.     

Studies on the polydispersity and heterogeneity of cartilage proteoglycans. Identification of 3 proteoglycan structures in bovine nasal cartilage.

1. Three chondroitin sulphate components were isolated from adult bovine nasal cartilage after treatment with alkaline NaB3H. Average molecular weights of 13000, 18 600 and 28 000 were obtained for chondroitin sulphate species representing 10, 52 and 38% (w/w) of the total chondroitin sulphate respectively. Each chondroitin sulphate pool has a narrow molecular-weight distribution. 2. A proteoglycan subunit preparation, isolated from one nasal cartilage by extraction and density-gradient fractionation in dissociative solvents, partitioned on a CSCl density gradient according to size and composition. Variation of proteoglycan molecular weight across the gradient was directly related to the average chondrotin sulphate chain length, which in turn reflected the relative proportion of the three chondroitin sulphate pools in each proteoglycan fraction. Consideration of proteoglycan molecular parameters, compositions and behaviour on sedimentation leads to a proposal that nasal cartilage contains 3 distinct proteoglycan pools, each of which has a constant number of chondroitin sulphate side chains of different average molecular weight. 3. Molecular-weight distribution parameters for these proteoglycan preparations indicate that all serine residues on the protein core capable of initiating chondroitin sulphate biosynthesis are occupied and that proteoglycan polydispersity results directly from the polydispersity of the attached chondroitin sulphate component.