Native purification of biomolecules with temperature-mediated hydrophobic modulation liquid chromatography

The high-resolution purification of native enzymes is impeded by the limita tions in the mobile-phase choices required for conventional hydrophobic sep arations such as in reverse phase chromatography. To avoid problems associa ted with varying the composition of the mobile phase, we developed a statio nary 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. Visual ization of the temperature-dependent hy drophobic interaction with the chro matographic matrix was performed with fluorescence microscopy of CY3-ATP. A mino acids adsorbed to the column according to their known hydrophobicities , confirming the hydrophobic nature of their interaction with the matrix. B iomolecules were separated by modulating the hydrophobicity of the column m atrix with slight adjustments to the running temperature between 22 and 37 degrees C without changing the mobile phase. Freedom in the choice of a mob ile phase for both the loading and the elution of samples provides great pr actical advantages by eliminating the need for buffer-exchange steps and al lowing more native conditions for purifying delicate enzymes, such as myosi n. (C) 2000 Academic Press.