Reversible adsorption of proteins at the oil/water interface I. Preferential adsorption of proteins at charged oil/water interfaces

Abstract

The behaviour of positively and negatively charged oil-in-water emulsions, stabilized with hexadecyl trimethyl ammonium bromide and sodium hexadecyl sulphate respectively in the presence of protein solutions has been studied. Under certain conditions proteins will adsorb to a charged oil/water interface. When finely dispersed oil-in-water emulsion was used to provide this oil/water interface, adsorption of protein resulted in flocculation of the oil droplets. Flocculation of emulsion on the addition of protein is pH conditioned and occurred on the acid side of the isoelectric point of the protein with negatively charged and on the alkaline side with positively charged oil globules. No flocculation occurred on the alkaline side of the isoelectric point with a negative emulsion or the acid side with a positive emulsion. The amount of protein required to cause maximum clarification of the subnatant fluid corresponded with that needed to give a firmly gelled protein monolayer at the interface, namely, $2\cdot 5$ mg. of protein/sq.m. of interfacial area. With that amount of protein the flocculated oil globules remained discrete and no coalescence or liberation of free oil occurred. If only 1 mg. of protein/sq.m. of interfacial area was added, flocculation was followed by rapid coalescence of oil globules and liberation of free oil. If smaller amounts still were used, no visible change in the dispersion of the oil droplets could be seen macroscopically. With greater amounts than $2\cdot 5$ mg./sp.m. of interfacial area, up to ten times the monolayer concentration was adsorbed to the interface. Sodium chloride affected the flocculation range, and instead of the clear-cut change-over between the positive and negative interfaces at the isoelectric point of the protein, overlapping occurred. 5% sodium chloride shifted the flocculation point about 1 unit of pH. The addition of sodium chloride also altered the point of maximum clarification. Thus with haemoglobin the maximum clarification point was shifted from $2\cdot 5$ to $1\cdot 7$ mg./sq.m. of interfacial area by the addition of 1% sodium chloride. The adsorption of protein on to charged oil/water interfaces was reversible. This was best demonstrated with haemoglobin. Thus, haemoglobin was adsorbed at pH $5\cdot 0$ to a negative emulsion-the red floccules were washed and transferred to a buffer at pH 10. The haemoglobin was released and the emulsion was redispersed. The effect of adsorption and desorption on the structure of the protein molecule has been studied with haemoglobin. By solubility and colour tests it was shown that the haemoglobin molecule was changed to parahaematin by adsorption and subsequent desorption from a charged oil/water interface. Molecular weight and shape determinations were carried out on the desorbed protein. Two proteins have been separated by this adsorption mechanism. This was demonstrated on a mixture of albumin and haemoglobin. Some applications of the flocculation technique are indicated and the significance of the phenomena described are discussed.