Conformational studies of bovine alkaline phosphatase in hydrophobic interaction and size-exclusion chromatography with linear diode array and low-angle laser light scattering detection.
Alkaline phosphatase has been studied in hydrophobic interaction chromatography (HIC), using a bonded C1-ether phase on a silica gel support, together with an aqueous salt gradient. Its behavior under various gradient elution conditions has demonstrated good chromatographic performance and retention of enzymatic activity under aqueous conditions. It has now been studied using linear photodiode array (LDA) spectroscopy in combination with low-angle laser light scattering (LALLS) in gradient elution HIC. HIC-LALLS permitted the use of routine salt gradients for collection of molecular weight information, despite small changes in the baseline, via computerized baseline subtraction. Size-exclusion chromatography (SEC)-LALLS measurements, under various isocratic conditions, meant to mimic HIC elution, have indicated the presence of monomer/dimer, dimer/trimer, or mainly trimer. aggregates of alkaline phosphatase can also be detected under salt gradient HIC conditions, but at lower levels relative to the monomer. This paper also describes the behavior of alkaline phosphatase when detected using LDA under various chromatographic, temperature, and concentration (injected) conditions. The results indicate a facile equilibrium of at least two monomeric forms of alkaline phosphatase of the same molecular weight, which change relative populations as a function of operational conditions. Most interesting is the suggestion that alkaline phosphatase undergoes rapid conformational interconversions on the chromatographic detection time scales, and that these interconverting conformations, concentration dependent, produce a novel dual wavelength ratioing, viz., a pseudo-Gaussian peak mimicking the chromatographic elution profile at either wavelength. The reasons for these observations and their possible use in future high-performance liquid chromatographic biopolymer studies are discussed and described.
Krull
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IS
Department of Chemistry, Barnett Institute, Northeastern University, Boston, MA 02115.
Stuting
H H
HH
Krzysko
S C
SC
D000469:Alkaline Phosphatase / Q000032:analysis*; D000818:Animals; D001419:Bacteria / Q000201:enzymology; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003124:Colorimetry; D007413:Intestinal Mucosa / Q000201:enzymology; D007422:Intestines / Q000382:microbiology; D007834:Lasers; D011487:Protein Conformation; D012542:Scattering, Radiation; D012997:Solvents; D013056:Spectrophotometry, Ultraviolet
D012997:Solvents; D000469:Alkaline Phosphatase