An acyl-CoA dehydrogenase microplate activity assay using recombinant porcine electron transfer flavoprotein

Abstract

Acyl-CoA dehydrogenases (ACADs) play key roles in the mitochondrial catabolism of fatty acids and branched-chain amino acids. All nine characterized ACAD enzymes use electron transfer flavoprotein (ETF) as their redox partner. The gold standard for measuring ACAD activity is the anaerobic ETF fluorescence reduction assay, which follows the decrease of pig ETF fluorescence as it accepts electrons from an ACAD in vitro. Although first described 35 years ago, the assay has not been widely used due to the need to maintain an anaerobic assay environment and to purify ETF from pig liver mitochondria. Here, we present a method for expressing recombinant pig ETF in E coli and purifying it to homogeneity. The recombinant protein is virtually pure after one chromatography step, bears higher intrinsic fluorescence than the native enzyme, and provides enhanced activity in the ETF fluorescence reduction assay. Finally, we present a simplified protocol for removing molecular oxygen that allows adaption of the assay to a 96-well plate format. The availability of recombinant pig ETF and the microplate version of the ACAD activity assay will allow wide application of the assay for both basic research and clinical diagnostics.

Keywords: Acyl-CoA dehydrogenase; Electron transfer flavoprotein; Enzyme activity assay; Fatty acid oxidation; Mitochondria.

Figure 1.. Expression and purification of recombinant pig ETF.

A) Brief outline of purification scheme and representative elution profile of recombinant pig ETF from the CM-Sepharose column. B) SDS page of two samples of recombinant pig ETF demonstrating purity. Each lane contains 2.5 μg of protein. The gel was stained with Coomassie blue.

Figure 2.. Recombinant pig ETF is purer and more fluorescent than native pig ETF.

A) Coomasie-stained SDS -page gel showing purity of 2.5 μg of recombinant pig ETF (R) versus native pig ETF (N). Native ETF has two prominent contaminating bands. B) Anti-acetyllysine western blot of 100 ng each of recombinant (R) and native (N) pig ETF proteins. C) Absorbance scans of recombinant and native pig ETF proteins. Shown are the peaks associated with the FAD cofactor, with the total spectra in the inset. D) Three-dimensional fluorescence scanning of recombinant and pig ETF proteins produces similar spectra, but fluorescence is of higher intensity in recombinant pig ETF. E) The fluorescence maxima for both proteins was at Ex₃₇₅/Em₄₈₅; the intensity of the maxima was 60% higher for the recombinant pig ETF.

Figure 3.. Recombinant pig ETF outperforms native ETF in the anaerobic ETF fluorescence reduction assay.

Recombinant human LCAD (A) and VLCAD (B) were assayed for enzymatic activity with both ETF proteins. Bar graphs represent means and standard deviations. *P<0.01.

Figure 4.. Comparison of deoxygenation techniques in the cuvette-based ETF fluorescence reduction assay for LCAD activity.

Triplicate reactions were subjected to oxygen removal with either a combination of argon/vacuum (physical) and glucose oxidase/catalase (enzymatic), just the enzymatic deoxygenation alone, or no deoxygenation. A) Representative ETF fluorescence traces, and B) calculated LCAD activities measured with physical + enzymatic deoxygenation versus enzymatic deoxygenation only. Bar graphs represent means and standard deviations.

Figure 5.. The ETF fluorescence reduction assay in a microplate format.

Activities of recombinant LCAD and MCAD were tested in quadruplicate with the LCAD-specifìc substrate 2,6-dimethylheptanoyl-CoA with recombinant pig ETF in a microplate reader; MCAD is expected to have zero or near-zero activity with this substrate. A) Representative trace of the activities of recombinant MCAD and LCAD against 2,6-dimethylheptanoyl-CoA. After addition of substrate, there is a clear loss of ETF fluorescence for recombinant LCAD but not MCAD. B) Means and standard deviations from quadruplicate reactions of LCAD and MCAD with 2,6-dimethylheptanoyl-CoA. C,D) 40 μg of liver mitochondria extract from wild-type and LCAD−/− mice were assayed with 2,6-dimethylheptanoyl-CoA, using either the anaerobic cuvette-based assay (triplicate assays, panel C) or the microplate assay (quadruplicate assays, panel D). Background activity, calculated from the slope prior to substrate addition, was subtracted from each activity value, which in some cases yielded negative numbers.

Figure 6.. High-performance glass-bottom plates improve the microplate assay.

A) Wild-type mouse liver mitochondria (N=4) were assayed on four different black-walled fluorescence microplates—two plastic (Greiner Bio-one #655090 and Invitrogen #M33089) two glass-bottomed (Cellvis P96–1-N and Cellvis P96–1.5H-N “high performance”). Additional wild-type mouse liver mitochondria (N=8) were then assayed on the four plates; B) the percentage of background subtraction (baseline activity in absence of substrate divided by activity in presence of substrate) and C) the correlation coefficients of each assay were calculated.

Figure 7.. Application of the microplate assay to mouse tissue and VLCAD patient fibroblast lysates.

Mouse heart homogenates—15 μg of total protein per reaction—from wild-type and LCAD−/− mice (N=4) were assayed with 2,6-dimethylheptanoyl-CoA as substrate. Shown are representative activity traces (A) and calculated activity data from N=4 mice (B). For wild-type heart homogenates the average R²=0.85 ± 0.04. C,D) Two VLCAD-deficient patient fibroblast cell lines (PI, P2) were evaluated for VLCAD antigen (C) and palmitoyl-CoA dehydrogenase activity (D). Activity was measured with both the anaerobic cuvette method and the microplate method. Bars represent data (mean, standard deviation) normalized to activity measured in a normal control fibroblast cell line. Unnormalized mean specific activity values appear in italics near each bar. *P<0.05, t-test of normalized data from microplate versus cuvette method.