Native purification of biomolecules with temperature-mediated hydrophobic modulation liquid chromatography.
The high-resolution purification of native enzymes is impeded by the limitations in the mobile-phase choices required for conventional hydrophobic separations such as in reverse-phase chromatography. To avoid problems associated with varying the composition of the mobile phase, we developed a stationary phase with a hydrophobicity that can be modulated by slight variations in temperature to bind and elute biomolecules. This chromatographic matrix was tested on nucleotide analogs, amino acids, and protein samples. Visualization of the temperature-dependent hydrophobic interaction with the chromatographic matrix was performed with fluorescence microscopy of CY3-ATP. Amino acids adsorbed to the column according to their known hydrophobicities, confirming the hydrophobic nature of their interaction with the matrix. Biomolecules were separated by modulating the hydrophobicity of the column matrix with slight adjustments to the running temperature between 22 and 37 degrees C without changing the mobile phase. Freedom in the choice of a mobile phase for both the loading and the elution of samples provides great practical advantages by eliminating the need for buffer-exchange steps and allowing more native conditions for purifying delicate enzymes, such as myosin.
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Department of Biological Sciences, University of North Texas, Denton, Texas 76203, USA.
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D000180:Acrylic Resins / Q000737:chemistry; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D003911:Dextrans; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D015879:Myosin Subfragments / Q000032:analysis / Q000302:isolation & purification; D009218:Myosins / Q000378:metabolism; D009711:Nucleotides / Q000737:chemistry; D011817:Rabbits; D013696:Temperature
D000180:Acrylic Resins; D000596:Amino Acids; D003911:Dextrans; D005456:Fluorescent Dyes; D015879:Myosin Subfragments; D009711:Nucleotides; C052970:poly-N-isopropylacrylamide; C025614:sephadex; D009218:Myosins; D002118:Calcium