Colorimetric endpoint assay for enzyme-catalyzed iodide ion release for high-throughput screening in microtiter plates

• Published in: Archives of biochemistry and biophysics Vol. 464; no. 2; pp. 284 - 287
• Main Authors: Kurtovic, Sanela, Jansson, Ronnie, Mannervik, Bengt
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-08-2007
• Subjects: alkane; Biochemistry; Biokemi; cell lysate; Chemistry; colorimetry; Colorimetry - methods; cost; Dehalogenation; Enzyme Activation; enzyme activity; enzyme analysis; enzyme engineering; enzyme specificity; enzyme substrate; Glutathione transferase; Glutathione Transferase - chemistry; glutathione transferase A1; haloolefin; high throughput screening; human; Iodide; iodine; Iodine - chemistry; Iodoalkane; Kemi; Microchemistry; Microchemistry - instrumentation; Microchemistry - methods; microtiter plate assay; mutant protein; NATURAL SCIENCES; NATURVETENSKAP; nonhuman; priority journal; protein expression; Screening; Titrimetry; Titrimetry - instrumentation; Titrimetry - methods; Iodide; Screening; Iodoalkane; Dehalogenation; Glutathione transferase
• ISSN: 0003-9861; 1096-0384; 1096-0384
• DOI: 10.1016/j.abb.2007.04.009

Efforts are being made to engineer enzymes with enhanced activities against haloalkanes, a toxicologically important class of compounds widely used and frequently occurring in the environment. Here we describe a facile, inexpensive, and robust method for the screening of libraries of mutated enzymes with iodoalkane substrates. Iodide formed in the enzymatic reaction is oxidized to iodine, which in the presence of starch gives blue color that can be measured at 610nm or scored with the human eye. The assay can be performed with enzymes in crude cell lysates in 96-wells microtiter plates. Expression clones of several glutathione transferases showed diverse activities with different iodoalkanes, and a mutant library of human glutathione transferase A1-1 expressed variants with enhanced substrate selectivities.