Influence of Complexing Polyanions on the Thermostability of Basic Proteins

Abstract

Lysozyme (Lyz), chymotrypsinogen (Cht), and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) were used as model proteins capable of forming water‐soluble polyelectrolyte complexes with linear synthetic polyanions. The complex formation with sodium poly(methacrylate) (PMA), sodium poly(acrylate) (PAA), sodium poly(anetholsulfonate) (PAS), and potassium poly(vinylsulfate) (PVS) markedly reduced the temperature of protein denaturation, Tmax, as determined by differential scanning calorimetry (DSC). The effect of sodium poly(styrenesulfonate) (PSS) on Lyz was so drastic that the protein melting peak was not observed at all during DSC measurements. The temperature shift, most pronounced for Lyz, increased upon substitution of the polyanions according to the following series: PMA < PVS < PAA < PAS < PSS. Decomposition of the complexes by addition of either sodium chloride or poly(N‐ethyl‐4‐vinylpyridinium) cation completely restored the initial Tmax of the protein (except for PSS and PAS). The complex formation slightly affected the enzyme activity up to temperatures close to Tmax of the polyanion–protein complex. On further heating, the activity of the complex decreased steeply, whereas the free enzyme maintained a high activity. The data obtained strongly suggest that the protein–polyelectrolyte interactions in solution, while leaving the thermostability and activity of the proteins practically unaffected over a rather wide temperature range, result in the effective denaturation of proteins once a certain critical temperature is achieved. This finding appears to be crucial for further development of immobilized enzymes in biotechnology and essential for understanding mechanisms and principles of the functioning of proteins immobilized on charged matrices in vivo.Temperature dependence of partial heat capacity ΔCp of free Lyz and Lyz in the mixtures with PMA.magnified imageTemperature dependence of partial heat capacity ΔCp of free Lyz and Lyz in the mixtures with PMA.

The dynamics of heat gelation of casein glycomacropeptide – β-lactoglobulin mixtures as affected by interactions in the aqueous phase

“…The transition temperatures of b-lg (Table 1) were decreased by the presence of CMP in the mixed systems. The decrease in denaturation temperature on mixtures of proteins has been ascribed to complex formation by electrostatic interactions (Ivinova, Izumrudov, Muronetz, Galaev, & Mattiasson, 2002;Martinez et al, 2009). Electrostatic interactions have been reported to perturb the structure of myoglobulin and bovine serum albumin causing decreased thermal stability (Imeson, Ledward, & Mitchell, 1977).…”

The effect of heating on β-lactoglobulin–chitosan mixtures as influenced by pH and ionic strength

“…It was suggested that similar destabilisation may occur for other proteins upon interaction with highly charged polymeric compounds or surfaces. However, some studies also show that complexation of the protein with a polysaccharide can preserve the functionality of the protein and prevent denaturation (Burova, Varfolomeeva, Grinberg, Haertle´, & Tolstoguzov, 2002;Ivinova, Izumdurov, Munoretz, Galaev, & Mattiasson, 2003).…”

Complexation between lysozyme and sodium poly(styrenesulfonate): The effect of pH, reactant concentration and titration direction

“…Various experimental methods could be used for studying the formation and properties of such complexes. For example, the influence of complexation between small proteins and negatively charged polyelectrolytes on thermodynamic stability of proteins was investigated by means of differential scanning calorimetry [7]. In that study lysozyme, chymotrypsin and glyceraldehyde-3-phosphate dehydrogenase were used as model proteins, while as negatively charged polyelectrolytes poly(acrylic) acid (PAA), poly(methacrylic) acid (PMA), poly(N-ethyl-4-vinylpyridinium) bromide (PEVP), sodium poly(anetholesulfonate) (PAS), potassium poly(vinylsulfate) (PVS), sodium poly(styrene sulfonate) (PSS) were used.…”

“…[4][5][6]. If instead of a polyelectrolyte a protein is used, the formed complexes are commonly called protein-polyelectrolyte complexes [7][8][9][10][11][12][13][14]. Various experimental methods could be used for studying the formation and properties of such complexes.…”