Study on Collagen Maturity and Stability: Investigating the Relationship, Notas de estudo de Engenharia de Produção

Baixe Study on Collagen Maturity and Stability: Investigating the Relationship e outras Notas de estudo em PDF para Engenharia de Produção, somente na Docsity! Biochem. J. (1984) 222, 663-668 663 Printed in Great Britain Reconstituted collagen fibrils Fibrillar and molecular stability of the collagen upon maturation in vitro Carl Christian DANIELSEN Department of Connective Tissue Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark (Received 19 March 1984/Accepted 29 May 1984) During the maturation in vitro of reconstituted collagen fibrils prepared from rat skin, the mechanical and thermal stability of collagen increased and the pepsin- solubility decreased. At the same time a larger fraction of the pepsin-soluble collagen attained a lower molecular thermal stability that resulted in a biphasic thermal transition of the soluble collagen. Type-I collagen, with a similar biphasic thermal transition, was isolated from acid-insoluble rat skin collagen. Reconstituted collagen fibrils attain increasing mechanical and thermal stability during matura- tion in vitro when incubated in air at 37°C (Danielsen, 1981a,b). These changes in stability are similar to those occurring in collagenous tissues during aging in vivo. Usually the 'helix-to-random coil' transition upon heating of soluble collagen shows a symmetri- cal transition curve, but some preparations of acid- soluble rat skin collagen have a distinct skewness in the denaturation profile (Danielsen, 1982a). The acid-soluble collagen is supposed to represent the more mature and cross-linked part of fibrillar collagen that is extractable by neutral-salt solutions and dilute acids (Robins, 1980). The changed denaturation profile for this collagen fraction may reflect a conformation or structural change of the collagen molecule during age-related stabilization of the fibrils. Therefore an investigation of the relationship between changes in the denaturation profile of the molecular collagen and the extent of stabilization of reconstituted collagen fibrils that were matured in vitro was performed. Materials and methods Materials Pepsin (crystallized and freeze-dried) was pur- chased from Sigma Chemical Co. The DEAE- cellulose ion-exchanger used was Whatman DE- 52. Reconstitution and maturation of collagen fibrils Collagen fibrils were reconstituted and matured in accordance with a previously described proce- dure (Danielsen, 1981a). Briefly, a stock prepara- tion of purified acid-soluble collagen was obtained from the dorsal skin of 60-day-old male Wistar rats. The collagen was reconstituted into fibrils by gradual heating of neutral solutions of the collagen. The collagen fibrils were dried to membranes within 11 days after aggregation. The membranes were then cut into 4mm-wide strips appropriate for mechanical testing. Eight groups of strips were matured for different time periods (11-150 days after aggregation) by incubation in air at 37°C. The maturation was stopped by transferring the strips to liquid N2. Immediately after the completion of the aggregation, a portion of collagen fibrils was precipitated by centrifugation and stored in liquid N2 until the analyses were performed. Mechanical testing and determination of thermal stability of the fibrils The mechanical strength of the collagen mem- branes that were matured for different time periods after aggregation was determined in accordance with the previously described proce- dures (Danielsen, 1981a). The thermal stability of the collagen membranes was determined as the area shrinkage without tension during heating (AST) and the shrinkage temperature (Ts) was calculated as the temperature for 50% of this area shrinkage (Danielsen, 1981b). Solubilization and isolation of collagen Samples of the stock preparation of acid-soluble collagen and of the reconstituted collagen fibrils that were matured for 0-150 days were incubated with stirring in 0.5M-acetic acid at 1 :10 pepsin/col- Vol. 222 C. C. Danielsen lagen weight ratio at 4°C for I week. After the incubation the suspensions were centrifuged (50000g for Ah). The resulting supernatants were dialysed against 0.15M-CaCl2/0.05M-Tris/HCl buffer, pH 8, and re-centrifuged (1 h), and NaCl was added to give 4M. The precipitated collagen was dissolved with 5mM-acetic acid and centri- fuged (50000g for 1 h). The solubility of a sample was defined by the amount of hydroxyproline in the resulting supernatant divided by the total amount of hydroxyproline in the supernatant and the pooled precipitates. A collagen membrane that was prepared and matured for 85 days, as described above, was subjected to more extensive pepsin digestion in 0.5M-acetic acid at 4°C for 4 days. Pepsin was added to give a pepsin/collagen weight ratio of 1: 5 at the start of the incubation and added again after 2 days' incubation to give the same weight ratio. The insoluble residue resulting from the extrac- tion of 40g (wet wt.) of rat skin with 0.5M-acetic acid from the 60-day-old rats (Danielsen, 1981a) was re-homogenized in 100 ml of 0.5M-acetic acid, combined with 100mg of pepsin and incubated with stirring at 4°C for 1 week. The incubation mixture was then centrifuged (500OOg for 1 h) and the resulting supernatant dialysed against 0.15M- CaCl2/0.05M-Tris/HCl buffer, pH7.5. After re- centrifugation (50000g for 1 h), the collagen in the supernatant was precipitated by the addition of NaCl to give 4M and dissolved in 5mM-acetic acid. Thereafter the collagen solution was diluted 1:1 with a 0.4M-NaCl/O.lM-Tris/HCl buffer, pH7.4, and chromatographed on a DEAE-cellulose col- umn by the procedure of Miller (1971). The break- through fractions were pooled, dialysed against 5mM-acetic acid and diluted 1:1 with 2M- NaCl/0. 1 M-Tris/HCl buffer, pH 7.4, and, after adjustment of pH to 7.4 with 1M-NaOH, the collagen was fractionated by the method based on that of Chung & Miller (1974) by sequential addition of NaCl to give 1.7M, 2.5M and 4M. The 4M-NaCl-precipitated fraction was subjected to DEAE-cellulose chromatography by the procedure of Bentz et al. (1978). The collagen was dissolved in 2M-urea/20mM-NaCl/0.05M-Tris/HCl buffer, pH 8.6, and applied to a column that was equili- brated with the same buffer at 15°C. The absorbed collagen was eluted with 100mM-NaCl. Sodium dodecyl sulphate / polyacrylamide - gel electrophoresis Polyacrylamide-gel electrophoresis was carried out in 5% (w/v) acrylamide gels in the presence of sodium dodecyl sulphate at 6mA/tube for 6 h at room temperature according to a previously de- scribed procedure (Danielsen, 1982b) based on that of Furthmayr & Timpl (1971). Absorbance temperature transitions Duplicate determinations of the thermal stabil- ity of molecular collagen and production of smoothed denaturation profiles were performed by the procedures described in detail previously (Danielsen, 1982a). Briefly, the 'melting' of colla- gen was measured by recording the absorption difference at 227nm between identical sample and reference collagen solutions (0.10-0.25mg/ml in 5mM-acetic acid) during gradual heating of the sample (0.24°C/min). For comparative purposes, the reproduced denaturation profiles (the first derivative of the absorbance versus temperature) were normalized to an area of one unit by dividing the first derivative by the total transition absorp- tion change. The temperature for each successive 5% absorption change in the total transition absorption change was calculated. The fraction of collagen that 'melted' below a certain temperature was calculated from these data by interpolation. The 'melting' temperature (Tm) was defined as the temperature at which 50% of the transition absorption change had occurred. Results and discussion The stability of the reconstituted collagen fibrils increased during the maturation (Fig. 1). The mechanical stiffness (and strength) of the collagen membranes increased 3-fold from the 11th to the 150th day of maturation. The area shrinkage without tension during heating and the fraction of 80 r 0 to E 2= 60 2- . 20 0.- r__ 0Z4 r: - ua E 20 ._ x ce 7- 0 i4, I 50 100 Maturation time (days) 150 100 wD75 0 5 0 as A: C S.2 25 1-- To Fig. 1. Stabilization of reconstituted collagen fibrils upon maturation The collagen fibrils were incubated at 37°C in air and after various times removed for determination of mechanical strength (maximum stiffness, 0), percentage area shrinkage during heating (AST, A) and solubility by limited peptic digestion (%, 0). (Vertical bars indicate + S.E.M.) 1984 664 I "'--I I I Stability of collagen on maturation in vitro (ii) 2 4 6 I 0 Migration distance (cm) 2 4 6 Fig. 5. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis ofsoluble collagens isolatedfrom reconstituted and native fibrils (i) Acetic acid-soluble collagen. (ii) Pepsin-solubilized collagen from reconstituted collagen fibrils incubated at 370C in atmospheric air for 85 days. (iii)-(v) Pepsin-solubilized acetic acid-insoluble collagen from skin that was precipitated by 4M-NaCl (iii) and that on subjection to DEAE-cellulose chromatography by the procedure of Bentz et al. (1978) was separated in collagen unadsorbed (iv) and adsorbed (v) on the ion-exchanger. The pepsin-solubilized acetic acid-insoluble skin collagen that was precipitated by 4M-NaCl during the sequential precipitation by 1.7M-, 2.5M- and 4M-NaCl constituted 7% of the solubilized collagen and contained in addition to type-I collagen an electrophoretic band with mobility similar to that of a type-V collagen chain (Brown & Weiss, 1979) (Fig. 5iii). The latter collagen type was adsorbed on the ion-exchanger during the DEAE-cellulose chromatography by the procedure of Bentz et al. (1978) (Fig. 5v). The denaturation profile of the resulting type-I collagen fraction (Fig. 5iv) was similar to that of the collagen isolated from the collagen fibrils matured in vitro (Fig. 4). This similarity suggests that a modification of collagen is occurring both in vitro and in vivo. Attempts to isolate collagen with a biphasic thermal transition from skin gave the highest yield for peptic digests of the acetic acid-insoluble fraction. They have been unsuccessful for neutral- Vol. 222 0 667 668 C. C. Danielsen salt extracts. This indicates that the changed denaturation pattern is associated with the least- soluble fractions of skin collagen. A diminished molecular stability of the acid-insoluble collagen constituting the oldest fraction of collagen in skin may have implications for the enzymic degrada- tion of the collagen during the collagen turnover in vivo. However, the relationship between the changed denaturation pattern of the molecular collagen and the changing mechanical and thermal stability of collagenous tissues during aging re- mains to be elucidated. The conformational or structural changes un- derlying the decreased thermal stability of a fraction of the collagen are unknown. As charged amino acid residues contribute to the molecular stability (Dick & Nordwig, 1966; Rauterberg & Kuhn, 1968), a blockade or exposure of charged residues could affect the thermal stability of the collagen. The local relaxations of the triple-helical structure, as revealed by the proteolytic-probe technique (Ryhanen et al., 1983) may also result in collagen molecules with locally confined defects in the triple-helical structure if the relaxations of the helix are not fully reversible. This work was supported by grants from the Danish Medical Research Council (J. nos. 12-2227 and 12-3932). References Bentz, H., Bachinger, H. P., Glanville, R. & Kuhn, K. (1978) Eur. J. Biochem. 92, 563-567 Brown, R. A. & Weiss, J. B. (1979) FEBS Lett. 106, 71- 75 Chung, E. & Miller, E. J. (1974) Science 183, 1200-1201 Danielsen, C. C. (1981a) Connect. Tissue Res. 9, 51-57 Danielsen, C. C. (1981b) Mech. Ageing Dev. 15, 269-278 Danielsen, C. C. (1982a) Collagen Relat. Res. 2, 143-150 Danielsen, C. C. (1982b) Biochem. J. 203, 323-326 Dick, Y. P. & Nordwig, A. (1966) Arch. Biochem. Biophys. 117, 466-468 Furthmayr, H. & Timpl, R. (1971) Anal. Biochem. 41, 510-516 Miller, E. J. (1971) Biochemistry 10, 1652-1659 Rauterberg, J. & Kuhn, K. (1968) Hoppe-Seyler's Z. Physiol. Chem. 349, 611-622 Robins, S. P. (1980) in Biology ofCollagen (Viidik, A. & Vuust, J., eds.), pp. 135-151, Academic Press, Lon- don, New York, Toronto, Sydney and San Francisco Robins, S. P. & Bailey, A. J. (1977) Biochim. Biophys. Acta 492, 408-414 Ryhanen, L., Zaragoza, E. J. & Uitto, J. (1983) Arch. Biochem. Biophys. 223, 562-571 Viidik, A. & Busted, N. (1977) in Fifth Symposium on Basic Research in Gerontology (Schmidt, U. J., Briischke, G., Lang, E., Viidik, A., Platt, D., Frolkis, V. V. & Schulz, F. H., eds.), pp. 493-502, Perimed Verlag D. Straube, Erlangen Vogel, H. G. (1978) Connect. Tissue Res. 6, 161-166 1984