Studies on the chemical nature of elastin fluorescence.

Two fluorescent fractions were found in total acid hydrolysate of elastin. The fraction with higher chromatography mobility in isopropyl alcohol/conc. ammonia/water (9 : 1 : 2) was purified by multiple preparative paper chromatography in the same solvent system, and by gel chromatography on Sephadex G-25, ion-exchange chromatography on phosphocellulose and another gel chromatography on Sephadex G-10. The purified material was chromatographically homogeneous, had an ultraviolet absorption maximum at 315 nm and exhibited a strong 320/405 nm fluorescence. 1H- and 13C-NMR spectra were in good agreement with those published previously [6] for pyridinoline, a lysine derived fluorescent compound in collagen. The major part of the fluorescent material present in acid hydrolysate of elastin was always contaminated, even after complex purification procedures. It is concluded that elastin contains several fluorophores, one of which is a cross-linking tricarboxylic amino acid with a pyridinium ring having very probably the structure of 3-(2-amino-2-carboxyethyl)-1-(5-amino-5-carboxy-2-hydroxy-pentyl)-4-(3-amino-3- carboxypropyl)-5-hydroxypyridinium. The position of 2-amino-2-carboxyethyl and 3-amino-3-carboxypropyl residues has not been definitely established and can be interchanged.

The presence of gamma-carboxyglutamic acid-containing protein in atheromatous aortae.

It has been established that a gamma-carboxyglutamic acid-containing protein is present in rat aortae after long term atherogenic diet administration. A similar protein was proven to be present in turkey tibial tendons that are predisposed to undergo physiological calcification. The molecular weight and amino acid composition of both proteins were identical. They contained six glutamic acid residues per molecule, three of which were gamma-carboxylated. The proteins studied were also identical in their N-terminal sequence over six residues. This sequence was fully coincident with that published for osteocalcin (Price, P.A., Poser, J.W. and Raman, N. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 3374--3375). In the region corresponding to residues 20--26 in osteocalcin, a single replacement of valine for isoleucine was found in turkey tendon protein. From the physiological point of view it should be mentioned that the level of the gamma-carboxyglutamic acid containing protein in atherogenic diet fet rat aortae exceeds that found normally in bone or in tissues predisposed for physiological calcification.

Altered ratio of collagen chains in bone of a patient with non-lethal osteogenesis imperfecta.

Bone from a patient with osteogenesis imperfecta contained type III collagen which was absent in control bone. The ratio of alpha 1(I)/alpha 2(I) in type I collagen of patient's bone was increased (2.9 vs. 2.3 +/- 0.2 in controls) and the ratio of dimers beta 11/beta 12/beta 22 was altered due to the increased beta 22 content. No abnormality was observed in collagen from the patient's skin. The altered composition of collagen in bone, but the normal composition in skin suggests that the disease in the patient is due to impaired regulation of the synthesis of collagens in bone, rather than by a mutation in one of the two type I collagen genes. Unlike in skin, all the type III collagen in patient's bone was pepsin-soluble indicating an inability of the bone to incorporate type III collagen into mature highly cross-linked extracellular matrix.

Quantitative changes in insoluble collagen during ontogeny in rodents (collagen type I and type III).

It has been proved in three rodent species that in the insoluble collagen fraction which accumulates in skin collagen with age of the two categories of collagen present (collagen type I and collagen type III), their proportion alters in favour of collagen type I with the advancing age. Since it has also been shown that collagen type I is less resistant towards proteolytic cleavage than is collagen type III its accumulation can be explained either by rapidly advancing cross-linking of this collagen type or more likely by different proteosynthesis. The second alternative is preferred since a step-wise polymerization of collagen type III was also observed. No information revealing to what extent the lysine derived cross-links can combine both collagen types is at present available. On the basis of this information the rapid decrease in insoluble collagen in very early ontogeny (rats below 8 weeks of age) is explained.

Age- and feeding-dependent production of carbonyl compounds in hypoxic heart. The role of carbonyls produced in connective tissue modification.

The concentration of reactive lipid metabolites (malondialdehyde, formaldehyde, acetaldehyde and acetone) was assayed in rat heart reperfusates after 30 min ischemia in animals of different age and kept on different feeding regimes. It was revealed that there is no difference in the concentration of reactive carbonyl compounds in reperfusates from animals of different age, but the amount of released carbonyl compounds is much lower in animals kept on 50% restricted diet. If tail tendons from young (3 months) rats are incubated in the reperfusate, their solubility after CNBr treatment is decreased so that this material resembles tendons from old animals. Also the amino acid composition of the insoluble residue cannot be distinguished from that obtained from rat tail tendons of 24- or 29-month-old rats. The results prove the ability of carbonyl containing lipid metabolites to create a CNBr-insoluble core in connective tissue.

Changes in pyridinoline content of elastin during ontogeny.

It has been shown that, contrary to desmosine and isodesmosine, the content of pyridinoline increases in elastin preparations obtained from rat and bovine aortae and from bovine ligmentum nuchae throughout the whole lifespan. The molar proportion of this cross-linking amino acid is within the range of 0.6 - 2.5 leucine equivalents per 100 amino acid residues.

Change with age of UV absorbance and fluorescence of collagen and accumulation of epsilon-hexosyllysine in collagen from Wistar rats living on different food restriction regimes.

Accumulation of glycation products (as revealed by the thiobarbituric test and hexosyllysine assay) and the pigmented products (350 nm UV absorbance and 370ex/440em nm fluorescence) in aortal and skin collagen was investigated under the conditions of different nutritional regimes. Four groups of animals were tested: (1) ad libitum fed controls, (2) animals which were food restricted throughout their whole life (50% food intake), (3) animals fed ad libitum during their first year of life and then food restricted and (4) animals food restricted when young and fed ad libitum from the age of 1 year onwards. It was shown that all food-restricted animals showed lower levels of glycation and pigmentation products in collagen preparations from skin and aorta. The lowest accumulation was observed in group 4 which exhibited the longest 50% survival (29.4 months, as compared with 18.3 months in normally-fed controls). Of particular interest is the fact that in this group the decreased rate of accumulation of the glycated and pigmented products was preserved even after 1 year of life, i.e., when the animals had a free access to food. Though not directly supporting the glycation theory of aging (Cerami, 1985), our data are indicative of the involvement of glucose metabolism in the ageing process. Correlation between the levels of glycated and pigmented products in aortal and skin collagen as well as the correlation between the rate of accumulation of these products and 50% survival was impossible to establish. Nevertheless, each time that food restriction was imposed on the animals it always resulted in decreased accumulation of glycated and pigmented products and increased 50% survival. Possible mechanisms for this process are discussed.

The effect of low protein-high dextrin diet and subsequent food restriction upon life prolongation in Fischer 344 male rats.

Fischer 344 rats fed low protein-high dextrin diet exhibit a higher median (but not 10th percentile) survival as compared to controls. The effect of this diet appears already if the diet is administered between 6 weeks and 6 months of age; after this treatment median survival of experimental animals is increased by 96 days while the 10th percentile is not different from standard diet-fed controls. Further treatment of animals with the same diet has minimum effect as animals that lived on this regimen throughout the whole life exhibited a median lifespan increase by 120 days and increase in the 10th percentile by 41 days. However, if such animals at the age of 6 months are transferred to a restricted (60%) food intake regimen (control diet, not carbohydrate enriched) a further increase in median and 10th percentile lifespan prolongation can be observed reaching +328 and +396 days respectively as compared to controls. The effects of this early feeding (6 weeks to 6 months) with a low protein-high carbohydrate diet available ad libitum and the food restricted regimen (standard diet 60% controls) fed from the age 6 months onwards are additive, the final results being identical to those obtained if the animals were kept on the 60% food restricted intake throughout the whole life. The fact that animals fed the low protein-high carbohydrate diet and those kept on 60% standard diet food restriction had different survival though they were equal in daily (identical) protein intake is emphasized.

Increased glycation and pigmentation of collagen in aged and young parabiotic rats and mice.

Changes of non-enzymatic collagen glycosylation were followed in 2- and 24-month-old rats and mice and in parabiotic animals of the same age. With advancing age increased glycation of collagen was observed in both old male Wistar rats and white mice. Further it was demonstrated that both aortal and skin collagen of young animals is rapidly non-enzymatically glycosylated in the common milieu created by parabiotic animals and the proportion of non-enzymatically incorporated glucose approaches in the young counterparts the level found in old individuals. Similar trends as with non-enzymatic glycosylation were found with a fluorescent (370/440 nm) product present in both categories of collagen preparations. This fluorescence was higher in old animals and was considerably increased in the young counterpart of the parabiotic couple 6 weeks after operation. The nature of the fluorescent product appears different from pyridinoline and remains to be elucidated.

Altered expression of collagen phenotype in osteoarthrosis.

Immunofluorescence studies have shown the presence of collagen type III in addition to types I and II in osteoarthrotic cartilage. The presence of collagen type III was verified biochemically: collagen was isolated and purified from the pronase digest of osteoarthrotic cartilage. Mixtures of different collagen types were fractionated first by two different DEAE-cellulose runs and then the collagen polymers were isolated by molecular sieve chromatography. Afterwards the collagenous material was reduced, Alkylated, and rechromatographed on an agarose column. Three major peaks corresponding to gamma-, beta- and alpha-chains were observed. Amino acid analyses and the CNBr peptide pattern indicated the identity of the alpha peak as alpha 1 (III). Additional staining of control and disease cartilages for fibronectin revealed the presence of this protein in the diseased tissue.

Cartilage collagen in osteoarthrosis.

Pronase digestion of osteoarthrotic cartilage collagen indicated that this type of pathological collagenous structure is more susceptible to proteolytic degradation than human controls or bovine and calf articular cartilage. There were some differences between individual patients in the collagen susceptibility to pronase cleavage. Also, different zones of the same diseased femoral head exhibited varied susceptibility. It is suggested that changes in collagen stability are reflected in cartilage permeability and in this way also in alterations in the supply of nutrients.

Glycation of collagen in hypertriglyceridemic rats.

Nonenzymatic collagen glycation and modification with lipid derived metabolites was studied in rat skin and tail tendon collagen of control and hypertriglyceridemic (HTG) rats. Age-dependent changes typical for lipid and sugar derived adducts were evaluated by measuring fluorescence of these collagens at wavelengths typical for sugar (335/385 and 370/440 nm) and lipid derived adducts (356/460 and 390/460 nm). In addition pentosidine assay (corresponding to the fluorescence parameters 335/385 nm) was performed as well. It was found that pentosidine concentration as well as fluorescence intensities in skin collagen was the same for control and HTG rats and significantly increased with age. On the other hand, no significant age-dependent changes in fluorescence intensities were observed in tail tendon collagen. Pentosidine concentration in tail tendon collagen was much lower than that in skin and it was decreased in young HTG rats compared to control ones. It increased with age, more distinctly in HTG rats than in their control counterparts, in such a way that at the age of 19 months the pentosidine levels were undistinguishible in both rat strains. Possible mechanisms underlying these results are discussed.

Separation of proteins and peptides by capillary electrophoresis in acid buffers containing high concentrations of surfactants.

Separations of proteins at acid pH in the presence of a high concentration of surfactant [sodium laurylsulfate (SDS), 50 mmol/l] was investigated. The purpose of using high concentrations of SDS as background electrolyte modifier was threefold: First, the surfactant exerts a washing effect upon the capillary wall thus preventing binding of analytes and possible clogging of the capillary. Second, it was revealed that even under very acid conditions (below pH 3) the surfactant is capable of forming associates with protein analytes which still bear considerable negative charge and can be separated on this basis. Third, the system can be applied not only for protein mixtures sufficiently soluble in neutral to alkaline media (leukocyte lysates, standard proteins), but it can be used also with proteins, that are under such conditions virtually insoluble and their solubilization is possible in acid buffers only (eggshell proteins or collagen CNBr fragments). The result was that adsorption to the capillary wall was minimized and the analytes were separated as negatively charged associates with high efficiency. With collagen fragments partition was possible on the affinity differences of the peptides to the surfactant micelles and inner wall of the capillary. Theoretical plate counts approaching 100,000 were easily achieved even with proteins which under the more conventional operation conditions exhibit considerable sticking to the capillary wall. The other feature of this system is that the associates move very rapidly to the anode. Owing to the low pH, endoosmotic flow is negligible, and therefore the system has to be operated at reversed polarity.

Multicomponent analysis by off-line combination of synchronous fluorescence spectroscopy and capillary electrophoresis of collagen glycation adducts.

Capillary electrophoresis separation and synchronous fluorescence spectral detection was used off-line to reveal the nature of fluorescent adducts formed in vivo in the collagen molecule and their distribution in the molecule. It was shown that by using the delta lamda in the area of the Stokes shift for the analyzed entities (approximately 10 nm for pentosidine, 4,5(E)-epoxy-2(E)-heptenal and 4,5(E)-epoxy-2(E)-decenal lysine adducts) a distinct profile of spectral bands can be obtained allowing for differentiation of the several entities involved. In combination with capillary electrophoretic separation of the CNBr peptides the location of individual adducts was possible: while pentosidine (and, perhaps, pentosidine related compounds K1-K4) is found in the large alpha 1(I)CB6 and alpha 2(I)CB3.5 peptides along with a complete set of the other fluorescent adducts, low-molecular-mass peptides originating from the terminal region of the molecule are devoid of any fluorescence. All other parts of the molecule possess synchronous fluorescence profiles corresponding to the intact molecule except that they are devoid of pentosidine. The results indicate random distribution of fluorescent adducts in the collagen molecule and, in a broader context, indicate the usefulness of multicomponent analysis by means of combining synchronous luminescence spectra and capillary electrophoresis.

Capillary electrophoresis of hair proteins modified by alcohol intake in laboratory rats.

A capillary zone electrophoretic method was used to obtain profiles of solubilized rat hair keratin proteins. The same methodology was used to reveal the presence of additional protein peaks in alcohol-consuming rats. Two types of separation were investigated. Alkali-solubilized keratins from hair of rats treated for 5 weeks with 5% ethanol and 2 weeks with 10% ethanol (instead of drinking water) and from controls were analysed. Whereas under alkaline conditions (pH 9.2, 50 mM borate) an additional fraction of "low-sulphur" keratins with the highest anodic mobility of this keratin category was shown in alcohol-treated animals, acid electrophoresis carried out at pH 3.5 in phosphate buffer (50 mM) revealed the presence of two sharp peaks absent in the controls. These findings were confirmed by two-dimensional separations of carboxymethylated keratin samples. An attempt was made to identify further one of the newly occurring fractions in alcohol-consuming animals. It was revealed that the tryptic hydrolysate of "low-sulphur" proteins obtained from alcohol-consuming animals contained a peptide not found in controls.

Open-tubular electrochromatography of organic phosphates on a sapphyrin-modified capillary.

Sapphyrin coating of the inner wall of the capillary results in a distinct interaction of the phosphate residue-possessing compounds as proven by a seven-membered model mixture of nucleoside mono- and diphosphates and ATP. Modification of the inner surface of the capillary not only alters the endoosmotic flow (as would have been expected) but brings about an electrochromatographic effect based on the interaction of tested phosphate moiety-bearing solutes with the immobilized sapphyrin layer. Elution of the sample can be achieved by using either 25 mM borate-acetate buffer in which monophosphates are not only separated from each other, but also selectively separated from di- and triphosphates (ATP). With the other two buffer systems tested, i.e. borate-phosphate and Tris-HCl, better selectivity (though smaller interaction with the capillary coating) was observed. The coating is relatively stable (can be used for 20 subsequent runs at least), simple to materialize, and in spite of a strong UV absorbancy of sapphyrin at the wavelength used (254 nm), decreases the limit of detection by no more than one order of magnitude as compared to the untreated capillary. Resolution factors (calculated to the preceding peak) are in most cases better in the electrochromatographic separation mode as compared to the separation in the untreated capillary, which reflects both the decrease in the electroosmotic flow and the interaction with the capillary wall coating.

Micropreparation of tissue collagenase fragments of type I collagen in the form of surfactant-peptide complexes and their identification by capillary electrophoresis and partial sequencing.

Combination of standard approaches like pepsin digestion and slab gel electrophoresis with capillary separations allows a relatively easy identification of in vivo occurring collagen fragments. Capillary electrophoresis can be done either in 25 mM phosphate buffer (pH 2.5) or in a 25 mM phosphate buffer (pH 4.5) made 0.1% with respect to sodium dodecyl sulfate (SDS). While in the first case peptides move to the cathode in a molecular mass dependent manner, in the second case they move towards anode (also in a molecular mass dependent manner). The profiles obtained by the two approaches resemble mirror images with low molecular mass peptides moving first in the acid background electrolyte while they move last in the presence of SDS. It is proposed that in the capillary electrophoretic separation at pH 2.5 the separation mechanism involves the interaction of the individual peptides with the capillary wall while in the second case (pH 4.5) the leading mechanism of separation involves the interaction of the analytes with the micellar phase. For micellar phase separation the system must be run at reversed polarity. Capillary electrophoretic separation in the pH 2.5 buffer is considerably affected by the presence of SDS in the previous steps of peptide preparation. If the peptides are obtained from SDS slab gel electrophoresis, their movement in the capillary electrophoresis step is about three times faster that the movement of corresponding peptides which have not been complexed with SDS.

Interaction of surfactants with homologous series of peptides studied by reversed-phase thin-layer chromatography.

The relative strength of interaction between anionic (SDS) and nonionic surfactant (octaethoxylated oleyl alcohol, GEN) and homologous series of peptides was determined by reversed-phase thin-layer chromatography (RP-TLC) carried out on alumina layers impregnated with paraffin oil. The relative strength of interaction was calculated and was correlated with the physicochemical parameters of peptides. It was established that each peptide interacted with both surfactants and with their mixture (1:1, m/m). The relative strength of interaction depended on the number of amino acid units in the peptide, side chain bulk and electronic properties and hydrophobicity of the amino acids. The impact of individual parameters highly depended on the character of surfactant. The data prove that the retention order of peptides can be modified by adding different surfactants and surfactant mixtures to the mobile phase resulting in improved separation.

Mixtures of nonionic and anionic surfactants: interactions with low-molecular-mass homopeptides.

The interaction between low molecular-mass homopeptides and mixtures of nonionic and anionic surfactants has been assessed by using reversed-phase thin-layer chromatography. The relative strength of interaction for mixtures of sodium dodecylsulfate and tridecylalcohol diglycolate (GNX) at the molar ratios of 8:2, 6:4, 4:6 and 2:8 has been calculated and its relationship with the physicochemical parameters (number of amino acid units, hydrophobicity, side chain bulkiness, electronic characteristics) of peptides has been computed by stepwise regression analysis. Each peptide interacted with each surfactant mixture the strength of interaction markedly depending on both the character of the peptide and the composition of the surfactant mixture. The hydrophobicity and electronic properties of the amino acid units exerted the highest influence on the strength of interaction at the highest concentration of the nonionic surfactant (GNX) whereas the number of amino acid units in the peptide molecule and the bulkiness of the amino acid side chain governed the strength of interaction at the lowest concentration of GNX.

Determination of apparent binding constants of drugs by capillary electrophoresis using beta-cyclodextrin as ligand and three different linear plotting methods.

Capillary electrophoretic estimation of apparent binding constants (Kapp) for naproxen, salbutamol, indomethacine and procaine with beta-cyclodextrin is presented. While with naproxen and indomethacine this approach was straightforward and gave well compatible results by three different linearization plots (double reciprocal, x reciprocal and y reciprocal), with salbutamol a higher value than reported for the electromigration estimation of this magnitude was obtained (a fourfold increase). This difference is ascribed to the fact that the measurements were done in the acid region (while the reported values were obtained at higher pH values). As a matter of fact the values of Kapp, reported in this communication for salbutamol comply better with the value of Kapp (69.3) obtained by the solubility method.