Owl monkey vitreous: A novel model for hyaluronic acid structural studies

Circular dichroism (CD) studies have been made on hyaluronic acid (HA) obtained from dialyzed owl monkey vitreous. The protein content of these samples is low enough not to interfere with the CD measurements of hyaluronic acid. The ellipticity values of vitreous HA are higher than those of HA from other tissues, indicating a higher degree of preferred order. Since the purification procedures involve only dialysis, the owl monkey vitreous can be a model tissue for structural studies of HA close to its native state.     

Circular dichroism of cephalopod rhodopsin and its intermediates in the bleaching and photoregeneration process.

In the bleaching process of cephalopod rhodopsin, a new intermediate was found in the conversion process from lumirhodopsin to metarhodopsin. This intermediate of octopus has an absorption peak at about 475 nm and has been named as M475. The circular dichroism value of M475 is too small to be evaluated. On the other hand, lumirhodopsin shows a negative CD at 470 nm, a positive CD at 350 nm and a large positive CD band with three peaks at 280, 287 and 295 nm. Such a large CD band in the ultraviolet region is not observed in rhodopsin, M475 and metarhodopsin. This CD seems to be mainly due to tryptophan and tyrosine residues restricted in free rotation in the protein moiety of lumirhodopsin. The intermediate in the photoregeneration process of cephalopod rhodopsin, P380, has a positive CD band at the main peak, 380 nm, and also a large positive CD band in the ultraviolet region like lumirhodopsin.     

Conformational transition of hyaluronic acid in aqueous-organic solvent monitored by vacuum ultraviolet circular dichroism

The chiroptical transition of hyaluronic acid (HA) in aqueous-organic solvent has been investigated by circular dichroism (CD) spectroscopy into the vacuum ultraviolet region. The CD of HA changes dramatically, monitoring a cooperative transition as the dielectric constant of an aqueous solution is reduced by adding organic solvents. This transition results in a high-intensity CD band at 188 nm, indicating an ordered structure in the mixed solvent. Heating HA in the mixed solvent also causes a cooperative transition, reducing the CD to that found for the polymer in aqueous solution. In contrast, heating HA in aqueous solution results in small, noncooperative changes in the CD spectrum. This indicates an unordered structure in aqueous solution. The CD as the dielectric constant is reduced exhibits isodichroic points, showing that there are only two environments for chromophores contributing to the CD. This is confirmed by singular value decomposition of CD spectra recorded as a function of solvent composition, which shows the spectra to contain only two principal components. The data describing the thermally induced transition of HA in mixed solvent are not consistent with infinite cooperativity. The van't Hoff relation yields thermodynamic parameters for the conformational transition in terms of the cooperative unit of -60 kcal mol-1 for delta H degrees and -180 eu mol-1 for delta S degrees.     

Circular dichroism of squid rhodopsin and its intermediates

Circular dichroism (CD) and absorption spectra of squid (Todarodes pacificus) rhodopsin, isorhodopsin and the intermediates were measured at low temperatures. Squid rhodopsin has positive CD bands at wavelengths corresponding the - and β-absorption bands at liquid nitrogen temperature (CD maxima: 485 nm at -band and 348 nm at β-band) as well as at room temperature (CD maxima: 474 nm at -band and 347 nm at β-band). The rotational strength of the -band has a molecular ellipticity about twice that of cattle rhodopsin. The CD spectrum of bathorhodopsin displays a negative peak at 532 nm, the rotational strength of which has an absolute value slightly larger than that of rhodopsin. The reversal in sign at -band of the CD spectrum may indicate that the isomerization of retinal chromophore from twisted 11-cis form to twisted 11-trans form has occurred in the process of conversion from rhodopsin to bathorhodopsin. Lumirhodopsin has a small negative CD band at 490 nm, the maximum of which lies at 25 nm shorter wavelengths than the absorption maximum (515 nm), and a large positive CD band near 290 nm, which is not observed in rhodopsin and the other intermediates. This band may be derived from a conformational change of the opsin. In the process of changing from lumirhodopsin to LM-rhodopsin, the CD bands at visible and near ultraviolet regions disappear. Both alkaline and acid metarhodopsins have no CD bands at visible and near ultraviolet regions.     

Glycosaminoglycan conformations and changes on periodate oxidation

The progressive periodate oxidation of glycosaminoglycans (GAG), including hyaluronate (HA), chondroitins (CH) (chondroitin, chondroitin 4- and 6-sulfate), dermatan sulfate (DS), and keratan sulfate (KS), were monitored by CD and high performance liquid chromatography (HPLC) using a size-exclusion column. The rate of oxidation also was measured and calculated using first- and second-order kinetics, and the data appear to fit better with first-order kinetics. In both HA and CH, the n - pi amide band at 208 nm decreases in intensity upon oxidation, but in HA it becomes positive after 16 h of periodate treatment. In CH, the band disappears, and the pi - pi amide band below 200 nm becomes optically active. Concomitantly, a second negative band near 290 nm appears for these two oxidized GAG. Oxidation causes a slight change in the CD of DS. It ordinarily displays a very weak n - pi band at 210 nm, but instead shows an intense pi - pi amide band near 190 nm. CD of KS remains unaffected by periodate. Kinetic studies, however, show a higher oxidation rate for DS than HA and CH. With the exception of KS, all other oxidized polymers shown an apparent decrease in molecular weight (higher peak retention time) in HPLC analysis. Both CD and HPLC results have been attributed to a major conformational change of HA and CH, and a minor one for DS. The ease and extent of periodate oxidation as well as the changes in molecular properties following periodate treatment are critically dependent on the configuration of the individual GAG rather than the oxidation rate. There is a distinct difference in the conformational change between HA and CH, as manifested by their dichroic behavior, that was attributed to the equatorial disposition of C-4 hydroxyl group in HA and axial disposition CH.     

Circular dichroism of rhodopsins and porphyropsins

Circular dichroism and absorption spectra were determined for digitonin extracts of three rhodopsins: cattle, grass frog, and pigeon; and three porphyropsins: channel catfish, bluegill sunfish, and redear sunfish. A comparison of these spectra shows the following: (1) Porphyropsins, like rhodopsins, exhibit two positive CD peaks in the spectral region 321–700 nm: an α peak at about 520 nm and a small β peak at about 355 nm. These peaks substantially diminish upon bleaching. (2) In the CD spectra the α peaks of the porphyropsins are larger than the α peaks of the rhodopsins, while the β peaks are smaller than those of the rhodopsins. This is just the opposite of the corresponding relationship between the peaks in the absorption spectra. (3) The maxima of these peaks in the CD spectra of rhodopsins and porphyropsins are consistently blue-shifted from the corresponding maxima in absorption spectra. (4) Some of the visual pigments show additional positive CD peaks in the spectral region 250–320 nm. In all the visual pigments studied, the CD spectra in this region decrease on bleaching. No reciprocal relationship is observed between any of the CD bands in the visible and near ultraviolet region of the spectrum.     

Hyaluronic acid of human skin and post-burn scar: heterogeneity in primary structure and molecular weight.

Hyaluronic acid (HA) was isolated from the dermis and epidermis of normal human skin and from normal and hypertrophic scar tissue, and the molecular properties of this polysaccharide were studied by circular dichroism (CD) and high performance liquid chromatography. The molecular weights of HA of normal skin and post-burn scar tissue range from 62,000 to 180,000. Hexosamine analysis showed no galactosamine contamination and 0.37 to 2.2 w/w% of protein in the HA sample. Uronic acid analysis suggests a heterogeneous distribution of glucuronic and iduronic acids. The CD profiles of these samples are similar, indicating no significant conformational variations among them. These data suggest that the variation in the molecular properties of HA between skin and scar tissue may be due to diversity of embryonic origin between epidermis HA and dermis HA, and to the diversity of the wound-healing process between normal scar HA and hypertrophic scar HA.     

Effect of formaldehyde on the circular dichroism of chicken erythrocyte chromatin.

Formaldehyde (HCHO) fixation of chicken erythrocyte chromatin produces a marked decrease in its positive circular dichroism (CD), above 260 nm, and the appearance of s small negative ellipticity around 295 nm. The ultraviolet spectrum of chromatin is unaffected, nor does HCHO produce any changes in the uv or CD spectra of chicken erythrocyte DNA. The extent of the circular dichroism transition from the native chromatin to the suppressed spectrum is dependent on the concentration of HCHO and salt concentration. The kinetics of the reactions are complex, implicating at least two reactive species. Studies of the reaction of HCHO with chromatin in ethylene glycol and CD measurements of aqueous chromatin solution with added glutaraldehyde preclude simple dehydration and general cross-linking effects as causes of the CD changes observed. The results are interpreted as indicating a conformational change of the DNA in chromatin caused by histone-DNA or histone-histone cross-linking.     

Evidence for Z-form RNA by vacuum UV circular dichroism.

Circular dichroism (CD) spectra in the vacuum UV region for different conformations of poly d(G-C) X poly d(G-C) and poly r(G-C) X poly r(G-C) are very characteristic. The CD of the RNA in the A-form (6 M NaClO4 and 22 degrees C) is very similar to that of the DNA in 80% alcohol where it is believed to be in the A-form. With the exception of the longest wavelength transition, the CD of the RNA in 6 M NaClO4 at 46 degrees C is similar to the CD of the DNA under conditions where it is believed to be in the Z-form (2 M NaClO4). This substantiates that poly r(G-C) X poly r(G-C) assumes a left-handed Z-conformation in 6 M NaClO4 above 35 degrees C. CD spectra for the left-handed Z-forms of both the RNA and DNA are characterized by an intense negative peak at 190-195 nm, a crossover at about 184 nm, and an intense positive peak below 180 nm. The right-handed A- and B-forms of RNA and DNA all have an intense positive peak in their CD spectra near 186 nm. The large difference in CD in the range 185-195 nm for right- and left-handed conformations of nucleic acids can be used to identify the sense of helix winding.     

Effect of ethanol on the activity and conformation of Penaeus penicillatus acid phosphatase.

The effect of ethanol on the activity of Penaeus penicillatus acid phosphatase has been studied. The results show that ethanol significantly inhibits enzyme activity as a non-competitive inhibitor, with Ki 8.75%. The conformational changes of the enzyme molecule induced by ethanol were followed using fluorescence emission, ultraviolet difference and circular dichroism (CD) spectra. Increasing the ethanol concentration caused the fluorescence emission intensity of the enzyme to increase. The ultraviolet difference spectra of the enzyme denatured with ethanol had two negative peaks at 220 and 278 nm, and a positive peak at 240 nm. Increasing the ethanol concentration produced a small shoulder peak at 287 nm in addition to the increases in the negative magnitudes of the 220 and 278 nm peaks. The changes of the fluorescence and ultraviolet difference spectra reflected the changes of the microenvironments of the tryptophan and tyrosine residues of the enzyme. The CD spectrum changes of the enzyme show that the secondary structure of the enzyme also changed. The results suggest that ethanol is a non-competitive inhibitor and the conformational integrity of the enzyme is essential for its activity.     

Novel circular dichroism spectroscopic approach for detection of ligand binding of proteins: Avidin as example

Various globular proteins having low or no α-helical content exhibit atypical far-ultraviolet (UV) circular dichroism (CD) spectra due to aromatic-aromatic residue and aromatic residue-peptide bond exciton coupling interactions. As a representative example of such proteins, far-UV CD spectra of chicken avidin were recorded before and after the addition of different small molecules. Intensity increase-decrease and/or wavelength shift of the positive CD peak of avidin at 228 nm were observed in the presence of various drugs, dyes, and natural compounds. The results were interpreted by exciton interactions between the aromatic residues of the biotin binding site and the substances bound to it. This novel, fast, microgram (μg) scale approach can be applied for detection of ligand binding of additional proteins displaying avidin-like far-UV CD spectra.     

Synthesis and physical properties of (hydroxyproline-proline-glycine)10: Hydroxyproline in the X-position decreases the melting temperature of the collagen triple helix

The collagen-like polytripeptide (hydroxyproline-proline-glycine)₁₀ was synthesized with a solid-phase procedure. Analytical ultracentrifugation indicated that the peptide in aqueous solution at 6 °C had a molecular weight of 2550, the expected size of a single chain. The peptide had a relatively small negative optical rotation at 578 nm, and it did not show a thermal transition as is seen with collagen or collagen-like polytripeptides which form triple helices. At low temperatures in aqueous solution, the circular dichroism spectrum was similar to that of triple-helical collagen and collagen-like peptides in that there was a positive peak at 224 nm and a negative peak at 200 nm. The amplitudes of the peaks, however, were considerably less than the peaks obtained with triple-helix proteins and peptides. Since (proline-proline-glycine)₁₀ was triple helical under the same conditions, the results demonstrated that hydroxyproline in the X-position of the repeating -glycine-X-Y- sequences decreases rather than increases, the thermal stability of the triple helix. This positional specificity cannot be explained by any of the current models for the structure of the triple helix or any of the current proposals for how hydroxyproline stabilizes the structure.     

UV damage of collagen: insights from model collagen peptides

Fibrils of Type I collagen in the skin are exposed to ultraviolet (UV) light and there have been claims that collagen photo-degradation leads to wrinkles and may contribute to skin cancers. To understand the effects of UV radiation on collagen, Type I collagen solutions were exposed to the UV-C wavelength of 254 nm for defined lengths of time at 4°C. Circular dichroism (CD) experiments show that irradiation of collagen leads to high loss of triple helical content with a new lower thermal stability peak and SDS-gel electrophoresis indicates breakdown of collagen chains. To better define the effects of UV radiation on the collagen triple-helix, the studies were extended to peptides which model the collagen sequence and conformation. CD studies showed irradiation for days led to lower magnitudes of the triple-helix maximum at 225 nm and lower thermal stabilities for two peptides containing multiple Gly-Pro-Hyp triplets. In contrast, the highest radiation exposure led to little change in the T(m) values of (Gly-Pro-Pro)(10) and (Ala-Hyp-Gly)(10) , although (Gly-Pro-Pro)(10) did show a significant decrease in triple helix intensity. Mass spectroscopy indicated preferential cleavage sites within the peptides, and identification of some of the most susceptible sites of cleavage. The effect of radiation on these well defined peptides gives insight into the sequence and conformational specificity of photo-degradation of collagen.     

The retention and ultrastructural appearances of various extracellular matrix molecules incorporated into three-dimensional hydrated collagen lattices

Artificial extracellular matrices composed of collagen, glycosaminoglycans (GAG), proteoglycans (PG), plasma fibronectin (FN), and a hyaluronate-binding protein (HABP) have been prepared that morphologically resemble embryonic extracellular matrices in vivo at the light and electron microscope level. The effect of each of the above matrix molecules on the structure and "self-assembly" of these artificial matrices was delineated. (1) Matrix components assembled in vitro morphologically resemble their counterparts in vivo, for the most part. Scanning and transmission electron microscopy indicate that under our assembly and fixation conditions, collagen forms striated fibrils that are 125 nm in diameter, FN forms 30- to 60-nm granules, chondroitin sulfate proteoglycan (CSPG) forms 27- to 37-nm granules, chondroitin sulfate (CS) assembles into 100- to 250-nm spheres, and hyaluronate (HA) appears either as granular mats when fixed with cetylpyridinium chloride (CPC) or as 1.5- to 3-nm microfibrils when preserved with ruthenium red plus tannic acid. These molecules are known to assume the same configurations in embryonic matrices when the same preservation techniques are used with the exception of FN, which generally forms fibrillar arrays. (2) Addition of various matrix molecules can radically change the appearance of the collage gels. HA greatly expands the volume of the gel and increases the space between collagen fibrils. CSPG at low concentrations (less than 1 mg/ml) and CS at high concentrations (greater than 20 mg/ml) bundle the collagen fibrils into twisted ropes. (3) A variety of assays were used to examine binding between various matrix components and retention of these components in the hydrated collagen lattices. These assays included solid-phase binding assays, negative staining of spread mixtures of matrix components, cryostat sections of unfixed mixtures of matrix components, and retention of radiolabeled matrix molecules in fixed and washed gels. A number of these binding interactions may play a role in the assembly and stabilization of the matrix. (a) HA, CSPG, and FN bind to collagen. CS appears to only weakly bind to collagen, if at all. (b) FN promotes the increased retention of HA, CSPG, and to a very small degrees, CS, in collagen gels. Conversely, the GAG increase the retention of 3H-FN in the gels. Furthermore, FN binds to HA, CS, and CSPG as demonstrated by solid surface binding assays and morphological criteria. The increased retention of GAG and CSPG by the addition of FN may be due to both stabilization of binding to the collagen and trapping of matrix complexes within the gel. (c) HA binds to both CS and CSPG.(ABSTRACT TRUNCATED AT 400 WORDS)     

The binding of copper ions to glycine-rich proteins (GRPs) from Cicer arietinum

Cicer arietinum GRP1 and GRP2 are rich in glycine interposed with histidine and tyrosine. In order to study whether or not these proteins bind Cu(2+), circular dichroism (CD) and nuclear magnetic resonance (NMR) were measured for three synthetic peptides corresponding to sections of the protein's sequences including 1, N(1)Y(2)G(3)H(4)G(5)G(6)G(7)N(8)Y(9)G(10)N(11), where all peptides were chemically blocked with an acetyl group at the N-terminus and an -NH(2) group at the C-terminus. The visible CD spectra for 1 showed a positive peak near 590 nm not at pH 6.0 but pH 7.4 in the presence of copper ions. The Cu(2+) binding induced a drastic change in the far-UV CD spectra, showing the occurrence of large conformation changes. In the 2D TOCSY NMR spectra at pH 7.4, the addition of small amounts of CuSO(4) caused a significant broadening of proton resonances of not only His4 but also Gly5, Asn8 and Asn11. CD titration experiment suggested that NYGHGGGNYGN including one repeat unit comprises the fundamental Cu(2+) binding unit.     

Detection of vibron phenylalanine bands in circular dichroism spectra for collagen

For the first time the individual positive circular dichroism peaks at 268 nm, 262 nm and 256 nm have been found for collagen. These peaks are due to the vibronic nature of a weak pi-pi* phenylic chromophore transition of phenylalanine and the unique structural organization of collagen where water molecules are an essential element of the triple helix.     

Hyaluronic acid inhibits adhesion of hepatic stellate cells in spite of its stimulation of DNA synthesis

Unbalanced accumulation of fibers in extracellular matrix (ECM) results from attachment and activation of hepatic stellate cells (HSCs) during chronic liver diseases, in which the content of hyaluronic acid (HA), a glycosaminoglycan, in ECM changes. No information is available on the effect of HA on adhesion and activation of HSCs although that of collagen (Col) on HSCs was extensively studied. This study investigated the effects of HA with or without Col on adhesion of HSCs or the rate of DNA synthesis. Attachment of primary cultured HSCs was microscopically monitored in the plate simultaneously coated with HA or other ECM components. HA inhibited adhesion of quiescent HSCs at least up to 7 days after seeding, whereas HSCs were adherent to plastic or type I collagen (Col-I), type III collagen (Col-III), type IV collagen (Col-IV) or fibronectin. Both microscopy and alpha-smooth muscle actin immunocytochemistry revealed that the number of HSCs, which had been re-seeded after 15 days of culture, attached to HA-coated area was remarkably lower compared to that of HSCs on Col-I or plastic. Incorporation of HA into Col-I prevented adhesion of activated HSCs to matrix film. The number of HSCs adherent to HA at early times after seeding was minimal and significantly lower than that of the cells adherent to plastic. In contrast, either Col-I or Col-IV increased the number of adherent cells. Attachment of HSCs to plastic was inhibited by soluble HA in culture medium. CD44, the cell surface receptor to which HA binds, was immunochemically detected in HSCs. Adhesion of HSCs to plastic, HA or Col-I was not changed by anti-CD44 antibody. Either HA or Col increased the basal or platelet-derived growth factor-inducible rate of thymidine incorporation into DNA in HSCs. In conclusion, HA inhibits adhesion of quiescent or activated HSCs in spite of its stimulation of DNA synthesis, whereas Col increases HSC attachment and DNA synthesis, and inhibition of HSC adhesion by HA does not involve CD44.     

Circular dichroism of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus

Circular dichroism (CD) spectra of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus exhibit three positive ellipticity bands between 240 and 300 nm (250, 283, and 292 nm), two negative bands at 327 and 480 nm, and a low-intensity positive band at 390 nm. The fractions of helix β-form, and unordered form of the enzyme are 8, 38, and 54%, respectively. The circular dichroic bands at 327 and 480 nm and a part of the positive bands at 292 and 390 nm are associated with enzyme activity. Significant changes in absorption and CD spectra of the enzyme were observed when the temperature of the enzyme preparation was increased to 47°C, coinciding with the sharp decrease in enzyme activity observed at this temperature.     

Deconvolution of the circular dichroism spectra of proteins: the circular dichroism spectra of the antiparallel beta-sheet in proteins.

A recently developed algorithm, called Convex Constraint Analysis (CCA), was successfully applied to determine the circular dichroism (CD) spectra of the pure beta-pleated sheet in globular proteins. On the basis of X-ray diffraction determined secondary structures, the original data set used (Perczel, A., Hollosi, M., Tusnady, G. Fasman, G.D. Convex constraint analysis: A natural deconvolution of circular dichroism curves of proteins, Prot. Eng., 4:669-679, 1991), was improved by the addition of proteins with high beta-pleated sheet content. The analysis yielded CD curves of the pure components of the main secondary structural elements (alpha-helix, antiparallel beta-pleated sheet, beta-turns, and unordered conformation), as well as a curve attributed to the "aromatic contribution" in the wavelength range of 195-240 nm. Upon deconvolution the curves obtained were assigned to various secondary structures. The calculated weights (percentages determining the contributions of each pure component curve in the measured CD spectra of a given protein) were correlated with the X-ray diffraction determined percentages in an assignment procedure and were evaluated. The Pearson product correlation coefficients (R) are significant for all five components. The new pure component curves, which were obtained through deconvolution of the protein CD spectra alone, are promising candidates for determining the percentages of the secondary structural components in globular proteins without the necessity of adopting an X-ray database. The CD spectrum of the CheY protein was interesting because it has the characteristic shape associated with the alpha-helical structure, but upon analysis yielded a considerable amount of beta-sheet in agreement with the X-ray structure.