Some scientific publications
Department of Clinical Veterinary Science, University of Bristol, Collagen Research Group, Langford, Bristol BS40 5DU, U. K. chris.miles@bristol.ac.uk
The mechanism that renders collagen molecules more stable when precipitated as fibers than the same molecules in solution is controversial. According to the
polymer-melting mechanism the presence of a solvent depresses the melting point of the polymer due to a thermodynamic mechanism resembling the depression of the freezing point of a solvent due to the presence of a solute. On the other hand, according to the polymer-in-a-box-mechanism, the change in configurational entropy of the collagen molecule on denaturation is reduced by its confinement by surrounding molecules in the fiber. Both mechanisms predict an approximately linear increase in the reciprocal of the denaturation temperature with the volume fraction (epsilon) of solvent, but the polymer-melting mechanism predicts that the slope is inversely proportional to the molecular mass of the solvent (M), whereas the polymer-in-a-box mechanism predicts a slope that is independent of M. Differential scanning calorimetry was used to measure the denaturation temperature of collagen in different concentrations of ethylene glycol (M = 62) and the slope found to be (7.29 +/- 0.37) x 10(-4) K(-1), compared with (7.31 +/- 0.42) x 10(-4) K(-1) for water (M = 18). This behavior was consistent with the polymer-in-a-box mechanism but conflicts with the polymer-melting mechanism. Calorimetry showed that the enthalpy of denaturation of collagen fibers in ethylene glycol was high, varied only slowly within the glycol volume fraction range 0.2 to 1, and fell rapidly at low epsilon. That this was caused by the disruption of a network of hydrogen-bonded glycol molecules surrounding the collagen is the most likely explanation.
A. Natishvili Institute of Experimental Morphology, Georgian Academy of Science, Tbilisi, Republic of Georgia. burj@kheta.ge
Recent data concerning the thermostability and the primary structure of type IV collagens, some invertebrate collagens, and for the stability of synthetic collagen-like polypeptides, show that our earlier analysis of the phylogenetic change of thermostability has some shortcomings. The results of the analysis were corrected and it has been shown that the dependence of denaturation temperature Td on 4-hydroxyproline content is hyperbolic and the total Gly-Pro-Hyp sequence content is a main, but not exclusive, factor influencing the change of collagen thermostability. It appears possible that the same mechanism underlies the thermostability of fibril-forming collagens of all animal life, ranging from Antarctic ice fish to at least one annelid (Alvinella pompejana) living at very high temperatures at the bottom of the ocean near thermal vents.thermal vents.
C A Miles, T V Burjanadze, A J Bailey
Division of Molecular and Cellular Biology, University of Bristol Langford, U.K.
This paper shows that the position and shape of the denaturation endothem of collagen fibrils are governed by the kinetics of an irreversible rate process. This was proved by measuring the rate of denaturation in rat tail tendons held isothermally at different temperatures, thereby determining rate constant characteristics such as the activation enthalpy and entropy and predicting endotherm position and shape therefrom. Comparison with actual scanning results showed good correspondence. Isothermal measurements of the rate of collagen denaturation, measured continuously using a calorimetric method, were used to determine rate constants for collagen denaturation in tendons immersed in water and 0.5 M acetic acid. The temperature dependence of the rate constants were fitted to the three rate process models, previously examined theoretically: the D and z formulation, the Arrhenius equation and the absolute rate theory. For example, in water the activation enthalpy was 0.518 (+/- 0.016) Mj mol-1 and the activation entropy 1.485 (+/- 0.049) kj mol-1 K-1, while in acetic acid the corresponding figures were 1.306 (+/- 0.099) Mj mol-1 and 4.142 (+/- 0.323) kj mol-1 K-1. These characteristics are discussed in terms of the thermal activation of a region of the molecule, the co-operative unit. The ratio of the activation enthalpy to the calorimetry enthalpy of denaturation indicated a co-operative unit that was 66 (+/- 5) residues long when fibrils were swollen in acetic and the collagen molecules acted essentially independently. On the other hand the intact fibrils in water gave a co-operative unit of 26 (+/- 1) residues long. The reason for the reduction in size of the co-operative unit is that it is surrounded, and therefore stabilized by other molecules in the fibre. It is interesting to note that the suggested co-operative unit lies almost entirely within the "gap" zone of the collagen fibril in its quarter-staggered arrangement of molecules. We believe that the co-operative unit would be represented by a domain that is free of stabilising hydroxyproline residues. Indeed such a domain exists near the C terminus of the triple helix from Gly877 to Pro941, i.e. 65 residues. In acetic acid, activation is similar to that of collagen molecules in solution. All the inter alpha-chain hydrogen bonds in the co-operative unit are broken and the separate chains in this short region are free to flail around under the action of thermal collisions relatively unimpeded by intermolecular interactions. (ABSTRACT TRUNCATED AT 400 WORDS).
Biopolymers. 2000 May ;53 (6):523-8 10775067 Cit:19
A. Natishvili Institute of Experimental Morphology, Georgian Academy of Science, Tbilisi, Republic of Georgia. burj@kheta.ge
Recent data concerning the thermostability and the primary structure of type IV collagens, some invertebrate collagens, and for the stability of synthetic collagen-like polypeptides, show that our earlier analysis of the phylogenetic change of thermostability has some shortcomings. The results of the analysis were corrected and it has been shown that the dependence of denaturation temperature Td on 4-hydroxyproline content is hyperbolic and the total Gly-Pro-Hyp sequence content is a main, but not exclusive, factor influencing the change of collagen thermostability. It appears possible that the same mechanism underlies the thermostability of fibril-forming collagens of all animal life, ranging from Antarctic ice fish to at least one annelid (Alvinella pompejana) living at very high temperatures at the bottom of the ocean near thermal vents.
A. N. Natishvili Institute of Experimental Morphology, Academy of Sciences of Georgia, Tbilisi, USSR.
A solution of the problem of topology of a hydrogen bond net in a triple helix of collagen is suggested on the basis of an analysis of thermodynamic data on denaturation of phylogenetically different collagen [T. V. Burjanadze (1982), Vol. 21, pp. 1489-1501; T. V. Burjanadze, E. I. Tiktopulo, and P. L. Privalov (1987), Dokl. Akad. Nauk. USSR, Vol. 293, pp. 720-724] as well as on the earlier evaluation of the energy of the OH group of the 4-hydroxyproline bond [A. R. Ward and P. Mason (1973), Journal of Molecular Biology, Vol. 29, pp. 431-435]. It is shown that only the water-bridged collagen structure [G. N. Ramachandran and R. Chandrasekharan (1968), Biopolymers, Vol. 6, pp. 1649-1661; G. N. Ramachandran, M. Bansal, and R. S. Bhatnagar (1973), Biochimica Biophysica Acta, Vol. 322, pp. 166-171; M. Bansal, C. Ramakrishnan, and G. N. Ramachandran (1975), Proceedings of the Indian Academy of Sciences, Vol. 82, pp. 152-164] can explain both the change of thermostability upon proline hydroxylation [J. Rosenbloom, M. Harsch, and S. Jimenez (1973), Archives of Biochemistry and Biophysics, Vol. 158, pp. 478-484] and its phylogenetic change [T. V. Burjanadze (1982)].
Department of Clinical Veterinary Science, University of Bristol, Collagen Research Group, Langford, Bristol BS40 5DU, United Kingdom. chris.miles@bristol.ac.uk
The mechanism that renders collagen molecules more stable when precipitated as fibers than the same molecules in solution is controversial. According to the polymer-melting mechanism the presence of a solvent depresses the melting point of the polymer due to a thermodynamic mechanism resembling the depression of the freezing point of a solvent due to the presence of a solute. On the other hand, according to the polymer-in-a-box mechanism, the change in configurational entropy of the collagen molecule on denaturation is reduced by its confinement by surrounding molecules in the fiber. Both mechanisms predict an approximately linear increase in the reciprocal of the denaturation temperature with the volume fraction (epsilon) of solvent, but the polymer-melting mechanism predicts that the slope is inversely proportional to the molecular mass of the solvent (M), whereas the polymer-in-a-box mechanism predicts a slope that is independent of M. Differential scanning calorimetry was used to measure the denaturation temperature of collagen in different concentrations of ethylene glycol (M = 62) and the slope found to be (7.29 +/- 0.37) x 10(-4) K(-1), compared with (7.31 +/- 0.42) x 10(-4) K(-1) for water (M = 18). This behavior was consistent with the polymer-in-a-box mechanism but conflicts with the polymer-melting mechanism. Calorimetry showed that the enthalpy of denaturation of collagen fibers in ethylene glycol was high, varied only slowly within the glycol volume fraction range 0.2 to 1, and fell rapidly at low epsilon. That this was caused by the disruption of a network of hydrogen-bonded glycol molecules surrounding the collagen is the most likely explanation.
A. N. Natishvili Institute of Experimental Morphology, Academy of Sciences of Georgia, Tbilisi, USSR.
A study has been done of the effect of neutral salts (NaCl and CaCl2) on the mechanism of type I collagen triple helix folding and unfolding in concentrated acetic acid solutions (2-8.8 M). It is shown that in these conditions, thermoabsorption and secondary structure change in heated solutions proceed in two consecutive stages. Salts exert a different destabilizing effect on different sites of the macromolecule, promoting the detection of a thermostable domain. The presence of a thermostable domain permits one to carry out reversible denaturation of collagen and to study the mechanism of the triple helix folding. Proceeding from the mechanism of the triple helix folding, an assumption has been made on the localization of the thermostable domain and its biological role.
A. Natishvili Institute of Experimental Morphology, Georgian Academy of Science, Tbilisi, Republic of Georgia.
