Gelatin gels in deuterium oxide
Introduction
When gelatin forms a gel, an extended network of protein molecules is formed in which the cross-linkages (junction zones) are regions where the gelatin has reverted to the triple helix structure of collagen (Clark and Ross-Murphy, 1987, Johnston-Banks, 1990, te Nijenhuis, 1997). These junction zone structures can be visualised using atomic force microscopy (Mackie et al., 1998). Electron microscopy (Hermansson & Langton, 1994) and dynamic light scattering have been used to investigate the gelation process and the network that is formed (Djabourov, Grillon, & Leblond, 1995). The formation and stabilisation of the collagen fold intimately involves the solvent water molecules (Traub & Piez, 1971). The process appears to involve both direct formation of intermolecular hydrogen bonds between –NH groups on the backbone of one chain and –CO groups on the backbone of another and a mutual stabilisation of the collagen structure and the three-dimensional hydrogen bonded structure of the neighbouring water molecules (Traub & Piez, 1971).
The solvent isotope effect, exchange of D2O for H2O, is a powerful means of deriving information about molecular interactions in aqueous solution—particularly those involving the formation of hydrogen bonds (Eagland, 1975) and interactions of the solvent water molecules (Privalov & Tiktopulo, 1970). Exchange of D2O for H2O increases the thermodynamic stability of tertiary and quaternary structures in proteins generally (Harrington & von Hippel, 1961). But very little is known about gelatin gels in D2O other than that the melting temperature is higher than in H2O and that the rate of mutarotation (reflecting the rate of formation of the collagen fold) is greater in D2O than in H2O (Naryshkima, Izmailova, & Dolinnyi, 1985). We have therefore prepared gelatin gels in D2O and measured the absolute shear modulus (G) and the melting temperature (Tm) and enthalpy of melting (ΔHm). The results confirmed that Tm and the rate of gelation are higher in D2O than in H2O, reflecting the greater stability of the collagen fold in D2O.
Section snippets
Materials
The gelatin was a commercial preparation (Davis Gelatine, Sydney, Australia) containing 124 g/kg moisture. Its amino acid composition has been published previously (Oakenfull and Scott, 1986, Oakenfull and Scott, 1986). Its intrinsic viscosity (measured at 38 °C in 0.2 M acetate buffer, pH 4.80) was 23.6 ml/g. The viscosity average molecular weight calculated (Pouradier & Venet, 1950) from this value was 49 300. The deuterium oxide was obtained from the Australian Atomic Energy Commission. Its
Shear modulus
In Fig. 2 we show results of measurements of shear modulus of dilute gels over a range of concentrations of gelatin in H2O and D2O. These results show the usual proportionality of shear modulus to the square of the concentration of gelatin (Mitchell, 1976). They also show that, for equal concentrations of gelatin, D2O as a solvent produces stronger gels than H2O. Gels formed in D2O had higher shear modulus than in H2O; the minimum concentration that would form a gel was lower in D2O than in H2O.
The size and thermodynamic stability of junction zones
The shear modulus data in Fig. 2 can be used to calculate the size and thermodynamic stability of junction zones, using the theory developed by Oakenfull (1984). From the theory of rubber elasticity, the following equation can be derived which relates G to the weight concentration (c) of the polymer:where M is the number average molecular weight of the polymer, MJ the number average molecular weight of the junction zones and the quantity [J] is effectively the ‘molar
Conclusions
Gelatin gels formed in D2O are more rigid, have higher melting temperatures and form more rapidly than gels of the same concentration formed in H2O. The junction zones in the gel network consist of regions of reformed collagen triple helix structure and this structure is more stable in D2O than in H2O. Consequently, in D2O the junction zones are smaller, but more numerous, producing a tighter network, and hence a more rigid gel. The higher melting temperature of the gel increases the
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