Chemical alterations of hyaluronic acid and dermatan sulfate detected in aging human skin by infrared spectroscopy.

Age-mediated deacetylation of hyaluronic acid and dermatan sulfate, and shift of sulfate ester configuration were indicated by infrared spectroscopy. Hyaluronic acid and the three dermatan sulfates (DS18, DS28 and DS35), sequentially precipitated from adult skin with 18%, 28% and 35% ethanol, were analyzed at varying ages. At age 75 years, loss of infrared bands in the 1,650-1,600 cm-1 region, at 1,380 cm-1 and 1,320 cm-1 and appearance of a band at 1,560 cm-1 were characteristic of hyaluronic acid and DS35; moreover, in DS28 and DS35 the intensities of the bands at 840 cm-1 and 860 cm-1 were, respectively, decreased and increased. A low intensity band in the 805-785 cm-1 region was observed in the spectra of DS18 (19-35 years), DS28 (70-80 years) and DS35 (all ages). It intensified in DS28 of the 80-year-olds. In the 75 +/- 5-year-old group, ninhydrin-positive material of hyaluronic acid and DS35 increased, while reducing GlcNAc of hyaluronic acid decreased. The data demonstrated hyaluronic acid and DS35 deacetylation and suggested a decrease of equatorial sulfates with infrared band at 840 cm-1 and an increase of axial sulfates with band at 860 cm-1 in DS28 and DS35 of the 75 +/- 5-year-old set. Equatorial Equatorial sulfates with band in the 805 +/- 785 cm-1 region apparently decreased in DS18 after 35 years and increased in DS28 of the oldest group.

Dietary vitamin E reverses the effects of ultraviolet light irradiation on rat skin glycosaminoglycans.

Chondroitin 4-sulfate (Ch 4-S), three dermatan sulfates (DS18, DS45a, DS45b) and hyaluronic acid (HA) were the major glycosaminoglycans (GAG) isolated from the skin of 4 groups of albino rats. The yields from Group 1 (control) were: Ch 4-S, 0.015%; HA, 0.028% and DS (total), 0.098% (w/w). Traces of heparin were detected only in rats irradiated with ultraviolet (UV) light (Group II), in the GAG pool isolated with 45% ethanol. Yields increased by at least 28% (w/w) in Group II, but decreased, except HA's, also by at least 28%, below the level of the control, in irradiated rats that also ingested vitamin E (Group III). The sulfate composition of these GAG determined by infrared spectroscopy was as follows: approx. 17% (w/w) for DS18, 21-30% for DS45a, 21-35% for DS45b and 26-44% for Ch 4-S. A 60-70% (mol/mol) N-acetylation of hexosamine in DS45 was estimated by Fourier transform 1H-NMR spectroscopy; the IdUA composition of this DS was 30-46% (mol/mol), and the uronic acid/hexosamine ratio ranged from 2.50:1 to 1.6:1. The data show UV light irradiation of rat skin to result in an abnormally elevated production of the major GAG and oversulfation of Ch 4-S and DS. These effects are reversed, except for the sulfation of DS45b, when the irradiated animals also ingest vitamin E.

A tightly bound protein-porphyrin complex isolated from the plasma of a patient with variegate porphyria.

Free acid porphyrins were isolated from plasma of a patient with variegate porphyria. Part of the total porphyrin content--which included protoporphyrin IX, harderoporphyrin and uroporphyrin in a molar ratio of 1.2:1:0.5 and traces of pentacarboxylic porphyrin--was extractable with ethyl acetate/acetic acid as described previously [1]. Unextractable porphyrins remained in the precipitate formed after mixing the lower liquid layer and precipitate from the extraction procedure (Fig. 1, [1]) with excess ethyl acetate/acetic acid. A portion of this precipitate was hydrolyzed in 8 mol/l HCl; its porphyrins were extracted with N-butanol and analyzed by high pressure liquid chromatography; another portion was chromatographed on Sephadex G-150 with 1 mol/l MgCl2, and the major porphyrin-protein pool was hydrolyzed in 8 mol/l HCl, reacted separately with AgNO3 and Ag2SO4, and subjected to cellulose acetate and polyacrylamide-gel electrophoresis. The results support the hypothesis that a dicarboxylic porphyrin, a major portion of which was unextractable by standard procedures [1] and which appeared to be covalently bound to a protein of approximately 68 000 mol. wt. that moved with human serum albumin during cellulose acetate electrophoresis, is the preponderant porphyrin in this plasma.

Glycosaminoglycans of skin and urine in pseudoxanthoma elasticum: evidence for chondroitin 6-sulfate alteration.

Abnormally elevated hyaluronic acid and dermatan sulfate were isolated from lesional skin of a patient with severe pseudoxanthoma elasticum. These glycosaminoglycans (estimated as uronic acid) surpassed the normal controls by 33.6 and 4.8 magnitudes, respectively. Urine chondroitin 6-sulfate of the same patient moved faster by electrophoresis on cellulose acetate than its counterpart isolated from urine of another four patients or from the normal controls. Hyaluronic acid and chondroitin 6-sulfate exceeded their counterparts in normal urine by 2- to 10- and 4- to 18-folds, respectively. These increments correlated with the pseudoxanthoma elasticum severity in three of the five patients studied. The data showed: the first conclusive evidence that dermatan sulfate increased in lesional skin of a patient, with severe pseudoxanthoma elasticum, who also had considerably augmented HA, alteration of the same patient's urine chondroitin 6-sulfate, and diversified urine glycosaminoglycans in pseudoxanthoma elasticum.

Evidence for structural changes in dermatan sulfate and hyaluronic acid with aging.

Three dermatan sulfates (DS18, DS28, and DS35) were isolated from women's skin of ages 19 +/- 2.5, 35 +/- 3.5, 47 +/- 1.7, 60 +/- 0.8, and 75 +/- 5 years. They sequentially precipitated with 18, 28, and 35% ethanol. Their sulfate content was: 23.5, 25.3, and 29.0% (w/w) for DS18 at ages 19-35, 47, and 60 years, respectively; 29.0, 24.0, and 18.8% for DS28; and 18.0, 20.0, and 20.6% for DS35 at ages 19-47, 60, and 75 years, respectively. Both DS18 and DS28 decreased, respectively, from 0.030% (of wet-skin weight) to traces at age 75, and from 0.020 to 0.010% at 60 years. At age 75, DS28 apparently increased by 30%. The DS35 values (traces-0.006%) had no age-related trend. Hyaluronic acid (HA) precipitated with 45% ethanol, was 0.030% of skin-weight at ages 19-47, and decreased to 0.015 and 0.007% at 60 and 75 years, respectively. Its electrophoretic mobility was slower at age 47. In the oldest group, i.r. spectra of HA and DS35 displayed no bands at 1650-1600, 1380, and 1320 cm-1, and a new band at 1560 cm-1. Moreover, ninhydrin-positive material of HA and DS35 increased by 75 and 95%, respectively, and the reducing GlcNAc content of HA decreased. These data showed three chemically different dermatan sulfates (two of which were preponderant) and N-deacetylation of HA and DS35 of the oldest group. After age 47, total DS and HA considerably decreased, DS18 and DS35 were oversulfated, and DS28 became undersulfated with aging.

Human skin dermatan sulfate with sulfated and unsulfated non-reducing ends.

The non-reducing ends of the preponderant dermatan sulfates of adult human skin (DS18 and DS28) can have D-GalNAc, D-GlcA and L-1doA. D-GlcA of DS18 and D-GalNAc of both DS18 and DS28 are sulfated.

Partial characterization of dermatan sulfate by proton nuclear magnetic resonance spectroscopy.

The degree of galactosamine N-acetylation, iduronic acid composition, and total uronic acid/hexosamine ratios of the three dermatan sulfates of human skin, DS18, DS28, and DS35 (M. O. Longas et al. (1987) Carbohydr. Res. 159, 127-136), were determined by Fourier transform, proton nuclear magnetic resonance (FT 1H NMR) spectroscopy. Analysis of DS of varying ages was conducted at 400 MHz and 60 degrees C. Chemical shifts for H-1, H-2, H-4, and H-5 of L-IdUA were independent of those for the respective protons of D-GalNAc and D-GlcUA. The resonance intensities of H-1 and acetamido methyl protons of D-GalNac did not display the expected 1:3 ratios. Therefore, their integration values were employed to estimate the percentage N-acetylation (N-CH3/3 H-1) which was corroborated chemically. The L-IdUA content, relative to total uronic acid, was calculated from signal intensities of H-1 of L-IdUA and D-GlcUA and ascertained by quantitative chemical methods. Total uronic acid/hexosamine ratios were determined from both 1H NMR spectroscopy and chemical analyses. The data show the following N-acetylation (N-CH3/3 H-1) of galactosamine in DS:DS18, 61-72% between 17 and 60 years, unaffected by senescence; DS28, 78-86% with no age-related trend; DS35, 101% at 19 years. Furthermore, in all ages investigated, the percentage (wt/wt) L-IdUA relative to total uronic acid was 42-44% for DS18 and 37-40% for DS28. At age 19 years, DS35 had a 29% (wt/wt) L-IdUA. The total uronic acid/hexosamine ratios for DS18 and DS28 varied from 1.40:1.0 to 1.70:1.0 irrespective of age.

Immunoelectron microscopy of proteodermatan sulfate in human mid-dermis.

Proteodermatan sulfate was isolated from the skin of human, female breast in 6-M urea and proteolytic inhibitors at 70 degrees C and purified on Sephacryl S-200. It was composed of 55% protein and 45% dermatan sulfate, displayed one protein and carbohydrate-stainable band on agarose-polyacrylamide gels, yielded dermatan sulfate after digestion by papain, and its calculated E0.1% 1 cm, 280 nm was 16.2. Its mucopolysaccharide portion was digested by chondroitinase ABC but not by chondroitinase AC. This proteoglycan was used to immunize rabbits. Double diffusion of antiserum against the antigen or its core protein resulted in one precipitation band. Antiserum did not cross-react with bovine collagen type I, human fibronectin, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate or the chondroitin sulfates by double diffusion. The antiserum titer determined by radioimmunoassay was 1:16,000. This assay was not affected by a 40-fold excess of dermatan sulfate. Purified IgG molecules were apparently associated with collagen in human breast mid-dermis as demonstrated by indirect immunoelectron microscopy with ferritin-labeled goat antirabbit IgG. The results indicate that rabbit anti-human, anti-proteodermatan sulfate IgG is highly specific for the core protein of dermatan sulfate and confirm the hypothesis that in vivo proteodermatan sulfate is closely associated with collagen.

Sulfate composition of glycosaminoglycans determined by infrared spectroscopy.

Anhydrous sodium sulfate (Na2SO4) was analyzed at varying concentrations by infrared (ir) spectroscopy. A standard curve was obtained from a linear plot of sulfate (SO2-(4] concentration vs the weight of the ir band area of S = O stretching. Standard chondroitin 4-sulfate, chondroitin 6-sulfate, heparan sulfate, heparin, keratan sulfates, and various dermatan sulfates isolated from human and rat skins were also studied by ir spectroscopy. The spectrum of every glycosaminoglycan (GAG) displayed an ir band around 1230 cm-1 which originated from S = O stretching of sulfate esters. Therefore, the weight of the latter band was employed to quantify sulfate, by using the standard curve indicated above. Sulfate was also estimated quantitatively by the gelatin/BaCl2 method of K.S. Dodgson and R.G. Price (Biochem. J. 1962, 84, 106-110). The sulfate composition determined by ir spectroscopy ranged from 8.5 to 22.1% (w/w), and agreed closely with the values obtained chemically. In the ir spectroscopy method, sulfate was determined using the polymer forms of the GAGs. After analysis, these heteropolysaccharides were recovered unaffected in a yield greater than 95%. The data show that the infrared spectroscopy technique, in addition to being sensitive and reliable, is much more economical than the chemical procedures currently employed to quantify GAG sulfate.

Evidence that a reducible xylosyl-lysine is the protein linkage of dermatan sulfate.

Dermatan sulfate, purified by standard methods, displayed one spot at the position of the standard after two-dimensional cellulose acetate electrophoresis and was 99% in GalN, 21.5% in sulfate, and 0.6% in protein; Gal and Xyl (2:1) were the only neutral sugars detected. Its glucuronic acid/iduronic acid ratio was 0.15 and its Mr was approximately equal to 16,000. On reaction with 0.4 M NaOH, its reducing group(s) determined as Glc increased by 71% with concomitant separation of protein and polysaccharide and no alteration of the amino acids; when this reaction was repeated in the presence of 0.3 M NaBH4, only 31% of Lys was detected by standard amino acid analysis and none by TLC of the dansylated amino acids; alkaline cleavage in these conditions yielded only 30% of the original Xyl, xylitol, and a ninhydrin-positive substance different from GalN, which had the retention time of xylitol on Affi-Gel 601 and was also obtained from reduction of dermatan sulfate with a 400-eq excess of Na3BH4 in water under conditions that did not cleave the dermatan sulfate-protein bond. The data indicate that a reducible xylosyl-lysine is the protein linkage of dermatan sulfate from calf ligamentum nuchae.

The covalent nature of the human antithrombin III--thrombin bond.

1. Cleavage of the human antithrombin III--thrombin complex with [14C]methoxyamine hydrochloride results in inactive thrombin and 14C-labelled antithrombin III. 2. Discontinuous polyacrylamide-gel electrophoresis of the reduced dissociation fragments of the complex in the presence of sodium dodecyl sulphate reveals two antithrombin III bands that do not resolve during electrophoresis without reduction. The heavy band has the electrophoretic mobility of the native protein. The light band has an apparent mol.wt. that is approx. 4000 less than the molecular weight of native antithrombin III. 3. Treatment of the cleavage products of the complex with carboxypeptidase B yields 1 mumol of arginine, a new C-terminal amino acid, per mumol of thrombin dissociated. The results indicate that during formation of the antithrombin III--thrombin complex, the inhibitor is cleaved at an arginine--X bond; this arginine residue forms a carboxylic ester with the enzyme, while the excised polypeptide remains bound through a disulphide bridge(s).

Sequential hydrolysis of hyaluronate by beta-glucuronidase and beta-N-acetylhexosaminidase.

1. Hyaluronate extracted from rooster comb was digested by a mixture of beta-N-acetylhexosaminidase and beta-glucuronidase with simultaneous dialysis for 96 h. 2. The produjct, yielding 99.6% of a mixture of mono- and oligo-saccharides, was purified by gel chromatography and analysed for glucuronic acid, N-acetylglucosamine and other sugars. 3. The oligosaccharide portion was chromatographed on DEAE-cellulose, and the effluent fractions were analysed for glucuronic acid and N-acetylglucosamine, reduced with NaBH4, digested by beta-N-acetylhexosaminidase and subjected to acid hydrolysis and glucosamine determination. 4. GlcNAc-GlcA-GlcNAc, GlcA-GlcA-GlcNAc and GlcA-GlcA-GlcA-GlcNAc were the oligosaccharides obtained, which resulted from the transferase activity of the enzymes and represented 57% of the digestion products. The results demonstrate that this hyaluronate is an unbranched polymer of approximatey equal amounts of glucuronic acid and N-acetylglucosamine. The data also indicate that if this glycosaminoglycan contains any of the neutral sugars for which it was analysed, their concentration must be less than 0.020% of the sum of the known components.

A disulfide bond in antithrombin is required for heparin-accelerated thrombin inactivation.

Heparin accelerates the rate of reaction of antithrombin with thrombin, an effect which is abolished by mild reduction of the antithrombin with dithiothreitol. Reduced antithrombin incorporates 1.7 mol of [14C]acetamide/mol of protein, with cysteine as the only amino acid modified. Tryptic digestion of the reduced and alkylated antithrombin results in the formation of only two labeled peptides. In the absence of heparin, the second order rate constant for the reaction of thrombin with both reduced and native antithrombin is 5.9 to 9.6 x 10(5) M-1 min-1. In the presence of heparin, the rate constant for the reaction between reduced antithrombin and thrombin is 8.3 to 12.2 x 10(5) M-1 min-1, while the rate of reaction between native antithrombin and thrombin is too fast to follow under the conditions used. Reduced antithrombin elutes from a heparin-Sepharose column at 0.5 M NaCl, contrast to 10 M NaCl required for elution of the native protein. The intrinsic tryptophan fluorescence enhancement caused by heparin binding to native antithrombin is not observed with reduced antithrombin. These data indicate that cleavage of one of the three antithrombin disulfide bonds results in reduced affinity for heparin and the loss of heparin-accelerated antithrombin activity and imply that heparin and thrombin bind at different sites on the antithrombin molecule.