Flow injection analysis with diode array absorbance detection and dynamic surface tension detection for studying denaturation and surface activity of globular proteins.
In this article, a multidimensional dynamic surface tension detector (DSTD), in a parallel configuration with a UV-visible diode array absorbance detector, is presented in a novel flow injection analysis (FIA) application to study the effects of chemical denaturants urea, guanidinium hydrochloride (GdmHCl), and guanidinium thyocyanate (GdmSCN) on the surface activity of globular proteins at the liquid-air interface. The DSTD signal is obtained by measuring the changing pressure across the liquid-air interface of 4-mul drops repeatedly forming at the end of a capillary using FIA. The sensitivity and selectivity of the DSTD signal is related to the surface-active protein concentration in aqueous solution combined with the thermodynamics and kinetics of protein interaction at a liquid-air drop interface. Rapid on-line calibration and measurement of dynamic surface tension is applied, with the surface tension converted into surface pressure results. Continuous surface tension measurement throughout the entire drop growth is achieved, providing insight into kinetic behavior of protein interactive processes at the liquid-air drop interface. Specifically, chemical denaturation of 12 commercial globular proteins-chicken egg albumin, bovine serum albumin, human serum albumin, alpha-lactalbumin (alpha-Lac), myoglobin, cytochrome c, hemoglobin, carbonic anhydrase, alpha-chymotrypsinogen A, beta-lactoglobulin (beta-LG), lysozyme, and glyceraldehyde-3-phosphate-dehydrogenase-is studied in terms of surface pressure (i.e., surface activity) after treatment with increasing concentrations of urea, GdmHCl, and GdmSCN in the 0-8, 0-6, and 0-5 M ranges, respectively. For several of these proteins, the spectroscopic absorbance changes are monitored simultaneously to provide additional information prior to drop formation. Results show that surface pressure of proteins generally increases as the denaturant concentration increases and that effectiveness is GdmSCN > GdmHCl > urea. Protein unfolding curves obtained by plotting surface pressure as a function of denaturant concentration are presented and compared with respect to unfolding curves obtained by using UV absorbance and literature data. Kinetic information relative to the protein adsorption to the air-liquid interface of two proteins, alpha-Lac and beta-LG (chosen as representative proteins for comparison), denatured by the three denaturants is also studied and discussed.
Bramanti
Emilia
E
CNR-Institute for Chemical and Physical Processes (IPCF), Laboratory of Instrumental Analytical Chemistry, 56124 Pisa, Italy. emilia@ipcf.cnr.it
Allegrini
Chiara
C
Onor
Massimo
M
Raspi
Giorgio
G
Skogerboe
Kristen J
KJ
Synovec
Robert E
RE
D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D017022:Flow Injection Analysis / Q000379:methods*; D007700:Kinetics; D011489:Protein Denaturation; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet; D013500:Surface Tension; D013816:Thermodynamics