Hydrophobic character of surface regions and total hydrophobicity of four variants of chromosomal class C β-lactamase from Pseudomonas aeruginosa are identical. : Chromatographic comparison of the hydrophobic character of the variants and the effect of focusing buffer composition on the separation of the variants by chromatofocusing with internal and external pH gradients

Abstract

The hydrophobic character of class C β-lactamase molecular variants from Pseudomonas aeruginosa was compared by hydrophobic interaction chromatography and reversed-phase liquid chromatography, respectively. Separation of the variants by hydrophobic interaction chromatography was not achieved by modifying salt and pH of mobile phases. Reversed-phase liquid chromatography of the variants resulted in almost identical retention times. The results showed that the hydrophobic character of surface regions as well as total hydrophobicity of the variants are identical. The resolving power of external, internal and gradient chromatofocusing of the variants on strong and weak anion exchangers using low-molecular-mass buffers was compared to that of commercial ampholytes and showed no difference in separation pattern of the variants. Comparisons of variant isoelectric point (p I) values determined by chromatofocusing and isoelectric focusing showed that p I values determined by gradient chromatofocusing were most similar to the p I values determined by isolectric focusing.