Biomacromolecules 2003, 4, 1727-1732
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Characterization of Gelatine and Acid Soluble Collagen by Size Exclusion Chromatography Coupled with Multi Angle Light Scattering (SEC-MALS) Michael Meyer* and Bernd Morgenstern Forschungsinstitut fu¨r Leder und Kunststoffbahnen gGmbH, Meiβner Ring 1-5, D-09599 Freiberg, Germany Received May 20, 2003; Revised Manuscript Received July 23, 2003
Acid soluble collagen (ASC) and a cattle hide gelatine were analyzed by size exclusion chromatography (SEC) coupled with multi angle light scattering (MALS). The SEC system was calibrated with ASC and its cyan bromide cleavage products. The accuracy of calibration was confirmed by MALS by measuring the mass-average molar masses (Mw). ASC acted as a mixture of two polymer standards of Mw ) 90 and 180 kg/mol, respectively. The elution behavior of the gelatine in SEC-MALS was similar to that of ASC. Therefore, the determination of the molar mass distribution of this gelatine was possible either by SEC, using a calibration curve, or by MALS by direct measurement of Mw. According to the scaling law 〈s2〉1/2 ) KMR, R ) 0.78 was determined for the gelatine. This R could reflect a structure in solution, which is more similar to an ellipsoid than to a random coil. 1. Introduction Gelatine is a natural polymer widely used in pharmaceutical, cosmetic, photographic, and food industries. It is obtained by denaturation and partial hydrolysis of fibrous collagen. Collagen is the most abundant structural protein of animals and by far the main organic component of skin and bone of vertebrates. However, collagen is standing for a family of proteins with 21 different types described to date. Skin and bones, the raw materials for gelatine manufacture, mainly consist of type I collagen and a small fraction of type III collagen. Each type I collagen molecule consists of three polypeptide chains (R chains). These chains are twisted into left-handed helices. In a central part, the helices form a right-handed super-triple helix (1014 amino acids), which is stabilized almost exclusively by hydrogen bonds. The end sections of the R chains (telopeptides) are formed by 16 amino acids at the N end and 25 at the C end. The rodlike tripel-helical collagen molecules are arranged in a parallel but staggered orientation to form fibrils. In tissue, the polypeptide chains are covalently cross-linked with others of their own and other collagen molecules. Therefore, the bulk of collageneous tissue of skin and bone is not soluble in water at 50 °C. The degree of cross-linking and the nature of the cross-links change with the age of an animal. The cross linking degree increases with increasing age, and the solubility decreases. Nondenatured soluble collagen molecules can be obtained in low yield by neutral or acidic extraction of the skin of young animals, e.g., calf. This collagen disintegrates during thermal denaturation at temperatures T > 40 °C to a mixture * To whom correspondence should be addressed. E-mail: michael.meyer@ filkfreiberg.de.
of R chains, dimeric β components, in which two R chains are covalently cross-linked, and trimeric γ components with three cross-linked R chains.1 The molar mass of purified R chains is close to 90 kg/ mol, whereas that of the β and γ components amounts to around 180 and 300 kg/mol, respectively.2,3 To obtain gelatine as a water soluble protein from cattle hide, the raw material has to be exposed to strong alkali at ambient temperatures for several weeks. After a washing step, the material is extracted with water in several extraction steps at different temperatures between 50 °C and 95 °C. The most important property of gelatine is its ability to form stiff gels at about 30 °C, when cooling a hot gelatine solution with sufficient high concentration (>1%). In contrast to soluble collagen, gelatines show a broad molar mass distribution in solution. The molar mass distribution of gelatines was first determined by fractionation and measuring their mass-average molar masses by light scattering. The fractions were prepared by coacervation, precipitation, or fractionated dissolution. The obtained results suggested that denatured gelatines show a molar mass distribution from oligo-peptides of several hundred g/mol up to particles with molar masses of several million g/mol.3-5 However, only up to seven fractions could be prepared by these kinds of fractionation. Furthermore, the samples were not only fractionated by molar mass but also according to other physicochemical parameters such as hydrophilic/ hydrophobic properties and charge. The reason for this is the unequal distribution of different amino acids along the polypeptide chains of collagen. The collagen chains are cleaved partly randomly during gelatine manufacture, however. In the gelatine industry, SEC is a method routinely used for process control and product development. The broad molecular mass distribution of gelatines was also confirmed
10.1021/bm0341531 CCC: $25.00 © 2003 American Chemical Society Published on Web 08/14/2003
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by size exclusion chromatography, even though the samples could often not be separated completely by the columns.6 As SEC is a relative method, the distribution of mass average molar masses of the gelatine samples could not be determined, however. A direct determination of molar masses by SEC might only be done by application of a detector being sensitive to the molar masses, e.g., by a light scattering detector. Moreover, light scattering could also provide information about aggregation phenomena even in the case of minor aggregation tendencies. Furthermore, the detection of the light scattering at different angles in relation to the incident laser beam (multi angle light scattering, MALS), allows the determination of the radius of gyration 〈s2〉1/2. As a result of SEC-MALS one gets the mass-average molar mass Mw and the corresponding 〈s2〉1/2 depending on the elution volume, Ve. The determination of the structure is possible according to the scaling law7 〈s2〉1/2 ) KMR by plotting log 〈s2〉1/2 vs l g M. R was calculated theoretically for different shapes of macromolecules. It amounts to 0.33 for hard spheres, 0.5 to 0.6 for random coils, and unity for rigid rods. SEC-MALS had already been used to characterize synthetic polymers as well as polysaccharides, DNA,8 some purified proteins, and protein mixtures.9,10 The analysis of gelatine was tried as well.8 The separation of the gelatine samples was not successful, however, because of insufficient denaturation of the gelatines. In the case of gelatine and collagen, a suitable combination of eluent and chromatographic column is needed. This eluent should allow a complete denaturation of the sample at room temperature, to analyze a monomolecular sample. Furthermore, a good separation of the sample has to be achieved on the column, concentration measurement must be possible, and the eluent should allow light scattering experiments without producing artifacts. Therefore, the aim of this study was to investigate denatured collagen and the first extract of cattle hide gelatine in solution by SEC-MALS and to find out the molar mass distribution as well as the structure of the gelatine molecules in solution. 2. Experimental Section 2.1. Materials. One gelatine sample, acid soluble collagen (ASC), and its BrCN-fragments were investigated. As gelatine, a first extract from a cattle hide stock was used, which was pretreated with alkaline (type B). This commercially available gelatine showed a gel strength of 271 bloom grams and a viscosity of 4.71 mPas (6.67%; 60 °C). It was supplied by Deutsche Gelatine Fabriken Stoess AG, Eberbach. ASC was prepared from extensively washed calf skin by extraction with 0.1 m acetic acid at 4 °C for 2 days under stirring. The supernatant was filtered through a glass fiber filter. The protein was precipitated by addition of a cooled solution of sodium chloride (25%), adjusting the final concentration to 5% NaCl. After centrifugation, the precipitate was redissolved in 0.1 m acetic acid and desalted on a MicroSpin column (Amersham Pharmacia Biotech, Germany). Finally, the collagen was freeze-dried.
Meyer and Morgenstern
Fragments of ASC with defined molar masses were obtained by specific cleavage with cyan bromide (BrCN).11 A total of 5 mg of ASC were weighed in a vial and suspended in 750 µL of 70% formic acid. After warming up to 30 °C, 10 µL of a solution of 7.5 mg of BrCN in 100 µL of acetonitrile were added. Then the vial was incubated at 30 °C for 4 h. Afterward 300 µL of distilled water were added, and the residue of BrCN was separated by desalting on a MicroSpin column with 0.1 M acetic acid as eluent. The BrCN fraction was treated with a solution of concentrated sodium hydroxide. The protein fraction was freeze-dried. The solvent and eluent respectively used for SEC-MALS was an aqueous buffer system containing 50 mmol/L tris(hydroxymethyl)aminomethane (Tris) and 1 mol/L calcium chloride. This high concentration of calcium chloride was necessary to suppress gelling and aggregation of the gelatine at room temperature. A pH of 7.5 was obtained by addition of diluted hydrochloric acid. The eluent was filtered through 0.025 µm cellulose nitrate filters (Schleicher & Schuell, Germany) before use. All chemicals used were of analytical grade. 2.2. SEC Measurements. The SEC measurements were carried out at room temperature, using a chromatographic system consisting of a PU-980 pump and a UV-975-detector (Jasco Corp., Japan) and a Dawn DSP MALS detector (Wyatt Technology Corp., USA) equipped with a K5 cell and a HeNe laser (λ ) 632.8 nm). For concentration detection, the absorbance was measured at 214 nm. The extinction coefficient of both samples was ) 1.20. For chromatographic separations, a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech, Germany) was used at an eluent flow rate of 0.4 mL/min. For sample preparation, 50 mg of dry gelatine or ASC were rehydrated for 1 h in 10 mL of buffer at 10 °C and then dissolved at 60 °C. The warm solution was filtered through 0.2 µm cellulose acetate membrane filters. A total of 20 µL of the filtered solution were injected. 2.3. Data Evaluation. The data were collected and analyzed by using the ASTRA SEC-software (version 4.20, Wyatt Technology Corp., USA). The calculation of massaverage molar mass Mw and gyration radius 〈s2〉1/2 was carried out according to the eqs 1 and 2 for each sliced section of the scattering chromatogram by plotting Kc/Rθ versus sin2 (θ/2) (Zimm plots)12 Rθ )
(Iθ(P) - Iθ(S))rD2 I0V0
(1)
where Rθ is the Raleigh ratio at the angle θ, Iθ is the scattering intensity of the polymer solution (P) and the solvent (S) at θ, I0 is the intensity of the incident radiation, rD is the distance between detector and scattering volume, and V0 is the scattering volume; and
(
( ))
1 16π2 2 θ Kc ) 1+ 〈s 〉z sin + 2A2c 2 R θ Mw 2 3λ
(2)
where K is the optical constant, Mw is the mass-average molar mass, c is the concentration of the polymer in solution, λ is
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Characterization of gelatine by SEC-MALS
Figure 1. Mass-average molar masses calculated from the concentration determined by absorbance measurement (A214nm) and from the scattering intensity I90°: R chains 90 kg/mol; β components 180 kg/mol; γ components 300 kg/mol; p particles with Mw . 500 kg/ mol. (-) absorbance A214nm; (O) scattering signal I90; (b) Mw.
the wavelength of laser light; 〈s2〉z1/2 is the z-average radius of gyration, and A2 is the second virial coefficient; and K)
4π2n02 ∂n 2 N λ 4 ∂c A 0
( )
(3)
where n0 is the refractive index of the solvent, λ0 is the vacuum wavelength of laser light, NA is the Avogadro constant, and (∂n/∂c) is the refractive index increment. The values were fitted by a first-order polynomial. Polynomials of higher order were also tried but gave poorer fitting results. An extrapolation to c f 0 was not necessary, because the concentration in each slice was very low (150 mAU ≈ 10 µg/mL) and the influence of the second virial coefficient could be neglected.13 〈s2〉1/2 was calculated from the slope of the straights for molecules with sufficient size in the range of 2〈s2〉1/2 > λ/20 (Mw ≈ 100 000 g/mol). The values for refractive index increments δn/δc were measured with a DR1 differential refractometer (SLS Systemtechnik, Germany) equipped with a He-Ne laser (λ ) 632.8 nm). δn/δc of the gelatine was found to be 0.190 cm3/g at 20 °C. The value is in good agreement with other values for different gelatines.14 This refractive index was used for ASC, too. Generally, proteins in aqueous solvents show low variation of the refractive index increment. δn/δc values in a range from 0.185 to 0.195 cm3/g were reported.15 In addition, from eqs 1 and 2, it follows that Iθ;P ∼ cMw. This means that very high molar masses of around several million g/mol lead to high scattering intensities even at low concentrations of the polymer.16 3. Results and Discussion ASC and four fragments resulting from a cleavage of ASC by bromine cyanide served as molar mass standards to determine the linear range of the SEC column used and the Ve-M relationship. The chromatograms of ASC obtained by detection with UV and MALS are depicted in Figure 1. Both chromatograms show two distinct peaks at elution volumes Ve of 10.0 and 12.2 mL, respectively. The peak at 12.2 mL can be assigned to the R chains (monomers) and the peak at 10.0 mL to the β components (dimers). A slight shoulder is
Figure 2. SE chromatogram of ASC and its BrCN fragments on a Superose 6 column. For molar masses and elution volumes see Table 1. (p) High-molecular component. Table 1. Molar Mass of the Components of ASC (R, β, and γ) and Their Specific Fragments after Cleavage by BrCN (CB) component/CB fragment
M (g/mol)
Ve (mL)
γ β R CB 3-5 CB 3-7 CB 7/8 CB 6
300 000 180 000 90 000 61 000 38 000 25 000 19 000
9.0 9.9 12.0 13.5 14.4 15.6 16.3
Figure 3. Calibration curve calculated from the peaks of ASC and its BrCN fragments, log(M) ) 3.86-0.157 Ve.
recognized at approximately 9 mL in front of the β components. It corresponds to the γ components (trimers). The corresponding mass-average molar masses calculated by the ASTRA software amount to 90, 180, and 305 kg/ mol. These values are in satisfactory agreement with theoretical values for the R, β, and γ components, respectively. Therefore, ASC behaves like a mixture of two polymer standards (the γ component is neglected). This observation is confirmed by different steps in the plot of log (Mw) vs Ve. Figure 2 shows the UV signal of the analyzed mixture of ASC and its BrCN fragments. Table 1 summarizes the molar masses of these fragments as well as those of the ASC components and the estimated corresponding elution volumes. Locations of the peaks of all fragments were exactly determined by separating the peaks by preparative SEC, purification with IEC, and rechromatography by SEC. The molar masses of the fragments were calculated by using the sequence of type I calf collagen.2 Figure 3 shows that the used column separates the investigated polypeptides according to their hydrodynamic size. The calibration curve reads log(M) ) 3.86-0.157 Ve (Ve in ml, M in g/mol) in the range of Ve from 9 to 16.5 mL.
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Figure 4. Absorbance, A214nm, of cattle hide gelatine. Peaks are observed at 12 mL (R chains of ASC) and at 10 mL (β component of ASC). Further peaks at lower Ve cannot be correlated to known cleavage products. About 8% of the gelatine are not separated (Mw > 500 kg/mol) and are eluted in the void volume. (b) Gelatine; (-) ASC; (p) high-molecular component.
Furthermore, the chromatograms of ASC showed an additional peak of the scattering signal at 7.5 mL (Figure 1), whereas no peak could be observed in the UV signal at this Ve. This scattering peak is close to the exclusion limit because the void volume of the column amounts to approximately 7.9 mL. It corresponds qualitatively to particles with very high molar masses, which are not separated by the used column. The fraction of these particles is very low, because only a slight shoulder can be observed in the UV signal at 7.8 mL. The reason for this dominating scattering peak is the proportionality of the scattering intensity to concentration and molar mass. The particles and molecules corresponding to the exclusion peak are too small to be separated by a 200 nm filter during preparation of the samples. However, they show molar masses by far higher than the major components of ASC. The UV signal of the gelatine sample (Figure 4) represents qualitatively the molar mass distribution in the linear range of the calibration curve. This molar mass distribution seems to be very broad. A distinct peak at Ve ≈ 12.1 mL could be observed, which corresponds to the R chains. Furthermore, the chromatogram shows a maximum at approximately 10 mL (β component) and additional peaks at Ve > 13 mL. The latter are a result of decomposition of collagen leading to fragments with molar masses less than 90 kg/mol. They cannot be correlated to known specific cleavage products. However, the sample is not completely separated by the column, especially in the range of high molar masses. Therefore, a high UV signal is measured in the void volume. The scattering signal of the gelatine sample shows a distinct peak at Ve ) 7.8 mL (Figure 5). This peak again corresponds to the void volume and reflects the exclusion limit of the used column. In contrast to ASC, the scattering peak of the gelatine sample in the void volume is split into a double peak. The first one shows the same elution volume as the scattering peak found in the void volume for ASC, whereas the second one correlates with the high UV signal of the gelatine sample in the exclusion limit. This splitting can be explained by a bimodal molar mass distribution. The higher molecular component as well as parts of the lower molecular component are not separated by the column. This result confirms other early investigations, where in several
Meyer and Morgenstern
Figure 5. Scattering signal I90° of the gelatine sample shows a splitting of the peak in the void volume. The first part of the peak (p) corresponds to the very high-molecular component found in the scattering signal of ASC. The second part corresponds to the UV peak in the void volume of the gelatine. This second part can be explained with molar masses being only a few above the exclusion limit. (-) absorbance, A214nm, gelatine; (- - - ) scattering signal, I90°, ASC; (b) scattering signal, I90°, gelatine.
Figure 6. Consistent curves of the mass-average molar masses Mw of gelatine and ASC from 8 to 12 mL. At Ve > 15 mL, the scattering intensity is too low to calculate Mw. (b) ASC; (O) gelatine.
gelatine samples very high molecular components were also described.3,4 The fraction of the gelatine molecules with Mw > 300 kg/ mol was approximately 8%, estimated on the basis of the peak area. The observed very large molecules of the gelatine sample could be molecules of incompletely disintegrated collagen fibrils. This is unlikely, however, because an extended incubation of the samples at 50 °C for several hours before injection led to the same chromatographic behavior. Therefore, the particles with high molar masses seem to be covalently cross-linked collagen-derived molecules, which are not cleaved during gelatine production. Figure 6 shows the correlation of Mw and Ve. The massaverage molar masses were calculated from scattering intensity. In the linear separation range between elution volumes from 8 to 12 mL the curves of gelatine and ASC are consistent. Therefore, the gelatine molecules were separated according to their molar masses in the same way as the molecules of ASC. At Ve > 15 mL, the scattering intensity of small gelatine molecules (Mw e 20 kg/mol) was too low to calculate Mw with sufficient accuracy. The gyration radii are depicted versus the elution volume in Figure 7. The evaluated elution for 〈s2〉1/2 had to be limited to a range from 8 to 11 mL. Values of 〈s2〉1/2 < 15 nm were ignored, because of their uncertain calculation. Radii of gyration of approximately 22 nm were obtained for the β component of ASC, and ones of approximately 20 nm were obtained for the R chains. Especially 〈s2〉1/2 of the R-chains
Characterization of gelatine by SEC-MALS
Figure 7. Gyration radii 〈s2〉1/2 of the gelatine sample that were calculated from 8 to 11 mL. They decrease from 40 nm at 8 mL to about 15 nm at 11 mL. The radii of R chains and β components of ASC were found to be 20 and 22 nm, respectively. The values show great variation. (b) 〈s2〉1/2 gelatine; (O) 〈s2〉1/2 ASC; (-) absorbance, A214 nm, ASC.
Figure 8. Double logarithmic plot of gyration radii 〈s2〉1/2 versus massaverage molar masses Mw enables to get hints about the molecular structure of the gelatine in solution. The slope was calculated with R ) 0.78. According to the scaling law 〈s2〉1/2 ) KMwR, this R can be explained by an ellipsoidal structure. (b) linear separation range; (O) void volume.
shows a great variation, however. The radii of the gelatine molecules decreased from approximately 40 nm at 8 mL to 15 nm at 11 mL. In the past, only a few investigations concerning the molecular structure of gelatine molecules and their molar mass distributions in solution were carried out. In an early investigation,17 results of light scattering measurements were reported for fractions with a different molar mass distribution of an ossein gelatine dissolved in an aqueous solution of 2 m KSCN at room temperature. By comparing the values of Mw and 〈s2〉1/2 of the fractions with values of synthetic polymers the authors concluded that the ideal chain model applies to the conformation of gelatine molecules. Pezron et al.18 confirmed this model in principle by combined measurements of static light scattering and neutron scattering (0.1 M NaCl in water; 40 °C). The authors found a wormlike chain structure of the gelatine with a persistence length of 2 nm. Herning et al.19 investigated the same sample by static and dynamic light scattering and concluded an ideal chain conformation. To determine the structure of gelatine molecules, 〈s2〉1/2 of our gelatine sample was plotted double-logarithmically versus Mw (Figure 8). The values were then fitted by a straight line in the range from 150 to 500 kg/mol. The slope of this straight line was used to evaluate the structure according to the above-mentioned scaling law. For the gelatine R ) 0.78 and K ) 0.002 was determined. Helmstedt20 measured Mw and 〈s2〉1/2 of eight different gelatines in an aqueous solution of 0.5 m KSCN at room
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temperature without fractionation and determined a value of R ) 0.58. These gelatines were not separated by chromatography. A recalculation by neglecting one of these pairs of values led to a value of R ) 0.65. R between 0.6 and unity can be explained as an expanded random coil structure as well as a deformed, ellipsoidal structure. In the native state, the collagen chains and the triple helices are arranged in a strictly parallel manner. Therefore, collagen fibrils show a typical periodic cross striation of 67 nm in TEM pictures.1 The collagen molecules are crosslinked intra-triplehelically and between different triple helices. If the collagen of tissue, such as skin, is denatured by agents such as calcium chloride, the covalent cross-links remain stable and try to stabilize the parallel arrangement of the collagen molecules. Therefore, in TEM pictures, this denaturation leads to a shortening of the periodicity of this cross striation.21 In gelatine, which is produced from collagen raw material, some of the covalent cross-links must be conserved during production, because high molecular particles are found in gelatines as well as high contents of molecules with molar masses higher than 100 kg/mol, the molar mass of the R chain. Therefore, the ellipsoidal structure seems to be more likely than the expanded random coil structure, if the raw material structure of collagen is partly conserved in gelatine. Summary Molar mass distribution of cattle hide gelatine and acid soluble collagen was studied by SEC-MALS measurements. The separation was carried out on a Superose 6 column, using an aqueous buffer system containing 1 M CaCl2 and 50 mmol/L Tris. The samples were completely denatured by this solvent at room temperature. Acid soluble collagen (ASC) and its BrCN fragments were shown to be applicable as standard substances to calibrate the column. ASC showed the expected distribution of R chains and β and γ components, with a good separation of the R chains and the β components. The cattle hide gelatine gave a very broad molar mass distribution with several maxima. Two of these maxima could be assigned to R chains and β components of ASC. The structure of the gelatine molecules in solution was calculated to be more similar to ellipsoids than to random coils. Both samples contained a particle fraction with very high molar mass values, however. This high-molecular component were eluted in the void volume of the column. In the case of ASC the particles could only be detected by light scattering, but not by UV absorption, because of its low concentration. Acknowledgment. We thank Prof. K.-F. Arndt for helpful discussions and his comprehensive support of this work and R. Mu¨hlbach for her help during laboratory work. This work was supported by the Ministry of Economy of the Federal Republic of Germany and the DGF Stoess AG, Germany. References and Notes (1) Ku¨hn, K. In Structure and Function of Collagen Types; Mayne, R., Burgeson, R. E., Eds.; Academic Press: New York, 1987
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(2) Piez, K. A. In Treatise on Collagen; Academic Press: New York, 1967. (3) Boedtker, H.; Doty, P. J. Am. Chem. Soc. 1956, 78, 4267. (4) Veis, A.; Cohen, J. J. Polym. Sci. 1957, 26, 113. (5) Scholtan, W.; Lange, H.; Rosenkranz, H.; Moll, F. Colloid Polym. Sci. 1974, 252, 949. (6) Quanten, E. Proceedings of the 7th IAG Conference 1999; Brussels 2000; p 79. (7) De Gennes, P. G. Scaling Concepts in Polymer Physics; Cornell University Press: Ithaca, NY, 1979. (8) Lloyd, L. L.; Kennedy, J. F.; Knill, C. J. In Light Scattering Principles and DeVelopment; Brown, W., Ed.; Clarendon Press: Oxford, 1996. (9) Folta-Stogniew, E.; Williams, K. R. J. Biomol. Technol. 1999, 10, 51. (10) Lucey, J. A.; Srinivasan, M.; Singh, H.; Munro, P. A. J. Agric. Food Chem. 2000, 48, 1610. (11) Bornstein, P.; Piez, K. A. Science 1965, 148, 1653.
Meyer and Morgenstern (12) Zimm, B. H. J. Chem. Phys 1948, 16, 1099. (13) Prochazka, O.; Kratochvil, P. J. Appl. Polym. Sci. 1986, 31, 919. (14) Veis, A. The macromolecular chemistry of gelatine; Academic Press: New York, 1964. (15) Huglin, M. B. Light scattering from polymer solutions; Academic Press: New York, 1972. (16) Wyatt, P. J. Anal. Chim. Acta 1993, 272, 1. (17) Boedtker, H.; Doty, P. J. Phys. Chem. B 1954, 58, 968. (18) Pezron, I.; Djabourov, M.; Leblond, J. Polymer 1991, 32, 3201. (19) Herning, T.; Djabourov, M.; Leblond, J.; Takerkart, G. Polymer 1991, 32, 3211. (20) Helmstedt, M. Habilitationsschrift; University of Leipzig: Leipzig, Germany, 1994. (21) Nimni, M. E.; Harkness, R. D. In Collagen, Vol. 1, Biochemistry; Nimni, M. E., Ed.; CRC Press: Boca Raton, FL, 1988.
BM0341531
