Effects of aqueous phase composition upon protein destabilization at water/organic solvent interface.
The objective of this study was to evaluate the effects of an aqueous phase composition upon the destabilization of lactoglobulin toward emulsification. Eight different buffers were used to make aqueous lactoglobulin solutions at pH 5.6-9. Each buffer concentration varied from 20 to 60, 100, and 150 mM. After emulsification of the aqueous solutions in methylene chloride, the aqueous contents of lactoglobulin were determined by a native SEC-HPLC assay. The properties of lactoglobulin precipitates detected at the water/methylene chloride interface were assessed by SDS-PAGE experiments. Dynamic interfacial tension was also monitored to examine the interfacial adsorption of lactoglobulin. When dissolved in plain water, only 41.1+/-1.4% of lactoglobulin was recovered after emulsification. The remaining lactoglobulin became aggregated at the interface. A series of SDS-PAGE experiments demonstrated that its aggregation was driven by intermolecular covalent linkages and hydrophobic interactions. Interestingly, both the buffer type and its concentration considerably influenced the degree of lactoglobulin recovery. For instance, at 150 mM buffer concentrations, percent recovery of lactoglobulin was as low as 27.3+/-1.7%. In contrast, the degree of its recovery increased up to 89.4+/-3.9% at a 20 mM buffer concentration. The dynamic interfacial tension study substantiated that at an optimal concentration buffer species helped lactoglobulin molecules to better withstand interfacial destabilization. Therefore, choosing a suitable buffer and its concentration should deserve special attention, since they could affect the interfacial stability of a protein of interest during emulsification.
Sah
Hongkee
H
Department of Pharmaceutical Sciences, College of Pharmacy, Catholic University of Daegu, Gyeongsan City, Gyeongbuk 712-702, South Korea. hsah@cu.ac.kr
Bahl
Yogita
Y
D000327:Adsorption; D011232:Chemical Precipitation; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004655:Emulsions / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007782:Lactoglobulins / Q000032:analysis / Q000737:chemistry*; D008752:Methylene Chloride / Q000737:chemistry*; D012997:Solvents / Q000737:chemistry; D014867:Water / Q000737:chemistry*
D004655:Emulsions; D007782:Lactoglobulins; D012997:Solvents; D014867:Water; D008752:Methylene Chloride