Gelatin is a heterogeneous protein with a broad molecular weight profile (MWP). Addition of a non-solvent to gelatin solutions causes progressive desolvation, coacervation and precipitation of the polymer. This behaviour is highly dependent on pH and ionic strength. At pH’s close to the isoelectric point (IEP) of the protein, which for B-type gelatins also corresponds to the isoionic point, the gelatin molecules carry a reduced net charge; the electrical double layer around each molecule inhibits aggregation poorly, and coacervation and precipitation occur more readily. Added salt reduces aggregation at these pH’s, possibly by reducing intramolecular electrostatic attractive forces and causing unfolding of the molecule into a more soluble configuration. The objective of this work was to attempt to investigate in greater depth the role of intramolecular electrostatic interactions in the desolvation behaviour of B-type gelatin solutions at pH’s close to the IEP.
Unbuffered gelatin solutions were prepared by heating aqueous suspensions of undissolved lime-cured gelatin from bovine skin (Type B), of bloom strength 225, to 40ºC with stirring for 20 minutes, and the pH adjusted to pH 5. The gelatin solutions were then incubated at 25ºC, 37ºC, 45ºC or 55ºC for 1.5 hours and mixed with ethanol/water mixtures that had been similarly incubated. The final solutions contained 0.2% w/w gelatin and ethanol concentrations from 0% to 75% w/w. Similar mixtures containing 0.1% w/v sodium chloride were also prepared. The mixtures were incubated at the chosen temperature for a further 20 minutes and the turbidity measured by % transmittance using a Shimadzu 160 UV/Vis spectrophotometer operated at 600nm. The data obtained was subjected to nonlinear regression analysis and the parameter V₅₀ (the ethanol concentration at the % transmittance midway between the initial and final values) was used to monitor the effects of the experimental conditions on the phase behaviour of gelatin in solution, lower V₅₀ values being indicative of a greater sensitivity to desolvation. The gelatin solutions incubated at 37ºC, with and without added sodium chloride, were also subjected to HPLC using an Ultrahydrogel Linear size exclusion column at flow rate of 0.3 mL/min, with a UV/Vis detector set at 205nm. The chromatograms were subdivided into fractions, and the % composition of the various fractions with and without added sodium chloride compared.
The V₅₀ values of the gelatin solutions were sensitive to changes in temperature (F = 12.06, p<0.05), both in the presence and absence of added salt. However, the effect of added NaCl dramatically altered the behaviour of gelatin solutions towards ethanol, with the solutions becoming less sensitive to increasing ethanol concentration in the presence of salt, and exhibiting greater solubility of gelatin in ethanol. Nevertheless, the size exclusion HPLC chromatograms showed that the % compositions of the various gelatin fractions in the presence and absence of salt at 37ºC were statistically identical (t = 3.69×10^-3, p>0.05; r = 0.977, p<0.01). Furthermore, the effect of added NaCl on the desolvation behaviour was observed to be greatest at 25ºC (V50 = 33.8% ± 0.2% without salt vs. 42.6% ± 0.2% in the presence of salt), the effect decreasing with increasing temperature such that at 55ºC there was practically no difference in the V₅₀ values obtained in the presence (52.5% ± 0.1%) and absence (51.5% ± 0.1%) of added salt.
Studies have shown that factors affecting the MWP of gelatin in solution affect the phase behaviour of a desolvating agent such as ethanol. Thus, increasing temperature causes a shift in the MWP to lower molecular weights. The latter fractions, in accordance with the Flory-Huggins theory of polymer solutions, exhibit a higher critical value for solvent-polymer interaction parameter, c, and are therefore less sensitive to the effect of added desolvating agent, as exhibited by the increased V₅₀ values at higher temperatures. However, the effect of sodium chloride on the desolvation behaviour does not appear to be due to a shifting of the gelatin MWP, as shown by the identical molecular weight composition of gelatin solutions in the presence and absence of NaCl.
The increased resistance of gelatin to desolvation in the presence of salt is therefore more likely to be explained in terms of the effect of electrostatic forces on the net energy-distance relationship for noncovalent interactions. At the isoionic point of B-type gelatins, the pH of the solution is close to the IEP of the protein and the net charge on the gelatin molecules is reduced or absent. However, charged groups still exist along the molecules, resulting in intramolecular electrostatic attractive forces that cause the molecules to fold. Addition of salt provides an electrostatic shielding effect between the oppositely charged groups, resulting in a reduction in these forces and hence an increase in molecular extension, yielding a more soluble entity than the coiled structure. Moreover, the zwitterionic decoupling that is caused by the addition of salt would expose more charged groups to the solvent, increasing the hydrophilic nature of the polymer.
This hypothesis is further supported by observing the effect of increasing temperature on the difference in desolvation behaviour in the presence and absence of added salt. The shift in the MWP to lower molecular weights at higher temperatures is probably achieved through the disruption of covalent cross-links in the gelatin structure and of intramolecular noncovalent interactions, including electrostatic interactions. Thus at higher temperatures, the role of electrostatic interactions in the higher structure of gelatin is probably significantly reduced, and added salt would have little or no effect in causing a change in the configuration of the polypeptide. This further confirms the importance of electrostatic interactions in determining the helical structure of gelatin at low temperatures, and in accounting for the role of salt in altering the desolvation behaviour of gelatin at these temperatures.
We have therefore concluded that electrostatic interactions play an important role in determining the higher structure of gelatin. The disruption of these interactions, either by increase in temperature or by added salt, results in an altered configuration which has a significant effect on the interaction of the protein with surrounding solvent and non-solvent molecules.