High-throughput estimation of specific activities of enzyme/mutants in cell lysates through immunoturbidimetric assay of proteins

Abstract

High-throughput estimation of specific activities of an enzyme and its mutants in a group (enzyme/mutants) in cell lysates via high-throughput assay of their activities and separate immunoturbidimetric assay (ITA) of their proteins was proposed. Pseudomonas aeruginosa arylsulfatase (PAAS) and Bacillus fastidious uricase (BFU) served as two models. ITA employed 0.75 mg of antisera against PAAS or BFU as the reference in 96-well microplates to measure the difference of extinction at 340 and 700 nm. According to the calibration curve, ITA quantified the reference from 0.40 to about 2.4 μg. The consistency among the abundance of enzyme/mutants through ITA of proteins in cell lysates prepared under the same conditions supported their consistent immunological reactivity to the antisera. Specific activities of PAAS/mutants or BFU/mutants in cell lysates through ITA of proteins showed excellent proportionality to those carefully determined after purification. Receiver-operating-characteristic (ROC) analysis of specific activities through ITA of proteins gave a higher area-under-curve than those for ROC analyses of other activity indices, which allowed the recognition of a PAAS/mutant of 50% higher activity after cell amplification in high-throughput mode. Therefore, ITA of enzyme/mutants as proteins is promising to estimate their specific activities in cell lysates in high-throughput mode for quantitative comparison.