Binding of β-Lactolobulin to Pectins Varying in their Overall and Local Charge Density

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The formation of complexes between proteins and polysaccharides is of great importance for many food systems like foams, emulsions, acidified milk drinks, and so on. The complex formation between beta-lactoglobulin (beta-lg) and pectins with a well-defined physicochemical fine structure has been studied to elucidate the influence of overall charge and local charge density of pectin on the complex formation. Binding isotherms of beta-lg to pectin are constructed using fluorescence anisotropy, which is shown to be an excellent technique for this purpose, as it is fast and requires low sample volumes. From the binding isotherms the maximal adsorbed amount, binding constant (k(obs)) and the cooperativity of binding are obtained at different ionic strengths. The Hill model is used to fit the binding isotherms and is shown to be preferable over a Langmuir fit. At pH 4.25, k(obs) shows a maximum at an ionic strength of 10 mM when using a low methyl esterified pectin (LMP) due to the balance of attractive and repulsive electrostatic forces between beta-lg and pectin and beta-lg neighbors. For two high methyl esterified pectins, one with a blockwise distribution of methyl esters (HMP(B)) and one with a random distribution (HMP(R)), this ionic strength maximum is absent and k(obs) decreases with increasing ionic strength. k(obs) is found to be largest for LMP and HMP(B) and considerably lower for HMP(R). A positive cooperativity is observed for both LMP (above an ionic strength of 45 mM) and HMP(R) (above an ionic strength of 15 mM) but not for HMP(B). Positive cooperativity is thought to be caused by a rearrangement of the pectin helix structure caused by binding of beta-lg, thus creating new or binding sites with a higher affinity. To attain strong binding of beta-lg to pectin it is preferable to use a pectin with a blockwise distribution of methyl esters. When complex formation takes place in high ionic strength media an LMP gives the best results, while at low ionic strength a high methyl esterified pectin with blockwise distribution may give better results, due to reduced electrostatic repulsion between both pectin and beta-lg and beta-lg neighbors.