Protein behavior at the water/methylene chloride interface.
The objective of this study was to investigate the behaviors of proteins at the water/methylene chloride interface to better understand denaturing effects of emulsification upon proteins. Ribonuclease A (RNase) and human serum albumin (HSA) were used as model proteins throughout this study. Their behaviors at the interface were studied in terms of protein recovery after emulsification, interfacial protein aggregation, and dynamic interfacial tension. This study demonstrated that protein instability during emulsification was traced to consequences of the adsorption and conformational rearrangements of proteins at the water/methylene chloride interface. Compared to HSA, RNase was much more vulnerable to the interface-induced aggregation reactions that led to formation of water-insoluble aggregates upon emulsification. Even though HSA was almost completely recovered from the emulsified aqueous phase, the protein underwent dimerization and oligomerization reactions to some extent. The results also demonstrated that the extent of interfacial RNase aggregation was affected by its aqueous concentration and the presence of HSA. Interestingly, RNase stability during emulsification was almost achieved by dissolving an adequate quantity of HSA in the RNase solution. HSA seemed to compete for the interface site and to effectively keep RNase out the interface, minimizing the likelihood of the interface-induced RNase aggregation. These results indicated that competitive adsorption modes of proteins could be used to stabilize a protein of interest against the denaturing effects of emulsification.
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Department of Pharmaceutical Sciences, The University of Tennessee, College of Pharmacy, Room 214, 26 South Dunlap Street, Memphis, Tennessee 38163, USA. hsah@utmem1.utmem.edu
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008752:Methylene Chloride / Q000737:chemistry*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D012260:Ribonucleases / Q000737:chemistry; D012709:Serum Albumin / Q000737:chemistry; D012997:Solvents; D013499:Surface Properties; D014867:Water
D011506:Proteins; D012709:Serum Albumin; D012997:Solvents; D014867:Water; D008752:Methylene Chloride; D012260:Ribonucleases