Leaf protein availability in food: Significance of the binding of phenolic compounds to ribulose-1,5-diphosphate carboxylase

The binding of phenolic compounds to ribulose-1,5-diphosphate carboxylase (RubisCO) is known to give rise to some digestive problems in human beings. In fact, the biological value of protein and hence the Protein Efficiency Ratio and Net Protein Utilization decrease drastically. For this reason the binding of phenolic compounds (e.g. rutin and chlorogenic acid) to ribulose-1,5-diphosphate carboxylase (RubisCO) was studied by means of ultrafiltration techniques in order to better elucidate the nature of this interaction and the factors influencing it in an attempt to limit or avoid it. RubisCO behaviour was also compared with that of Bovine Serum Albumin. A multivariate approach was used to determine the most influencing variables and their effects on binding. A classical binding study with the aim of determining the binding stoichiometry was also carried out. pH was found to be the most important variable affecting the binding of rutin to RubisCO as well as rutin to Bovine Serum Albumin while contact time became relevant when operating in sub-alkaline pH conditions. Classical binding analysis was carried out at pH 7.0 to 7.3 by both direct partition and diafiltration methods. A total number of five binding sites was determined, with two kinds of binding mechanisms, one of which was hydrophobic. The diafiltration method can be considered a useful tool when high affinity interactions are studied; RubisCO protein stability was disturbed by stirring, but this allowed an increased affinity of aggregated RubisCO to chlorogenic acid to be noted. This might have important consequences on RubisCO extraction technology since the most critical phase of phenolic contamination is the crystallization-precipitation step.