MaintainsPhenol-chloroform Extraction Protects Pyridine Nucleotide from Enzymatic Degradation during Preparations and Dissociates Protein-bound NADHIt has long been known that most of the NAD+ exists in free solution form while most of the NADH is 3PO site protein bound [27]. In order to accurately measure the total amount of NADH, the extraction method should be able to denature protein and release NADH. Denaturing protein will also reduce enzymatic degradation of pyridine nucleotides. We compared three extraction 1676428 methods: phenol chloroform extraction [28], chloroform-only extraction and 6 M guanidine-HCl. Guanidine-HCl extraction is a strong denaturing reagent which is capable of breaking protein secondary structure and has been used in assaying other metabolites [29,30,31]. We found guanidine extraction interfered with ADH activity (Fig. 4a) and is not suitable for this enzymatic recycling assay. For reasons unknown, phenol-chloroform 
treated samples also give a more linear kinetic curve than that of chloroform-only treated samples (Fig. 4a and 4b). We 
compared the redox ratio (NAD+/NADH) obtained from the same fly tissue homogenate: untreated control, chloroformonly treated and phenol-chloroform treated (see Table 2). Phenolchloroform treatment gives the lowest value, suggesting NADH recovery in this reaction is the highest among all three. Also noticeable is that 25837696 the amount of pyridine nucleotides detected following phenol-chloroform extraction is the highest among three treatments.Measuring Redox Ratio by a Coupled Cycling AssayFigure 2. Comparison of reaction kinetics of NADPx, NADx assay with and without hydrazine. A: Standard curve of NADPx assay. B: Standard curve of NADx assay without hydrazine. C: Standard curve of NADx assay with hydrazine. D: reaction kinetic of NADPx assay. E: NADx assay kinetic, showing additional hydrazine increases Vmean. F: the increase of Vmean by addition of hydrazine is dependent on the concentration of NAD+. All assays were performed in duplicates and hydrazine concentration is 0.02 . doi:10.1371/journal.pone.0047584.gFigure 3. The optimal concentration of hydrazine is around 0.02 . A: In a reaction containing 25 mM NAD+, Vmean increases with hydrazine concentration log-linearly up to 0.02 . B: The rate of absorbance increase in no NAD+ blank control is affected by hydrazine concentration higher than 0.02 . doi:10.1371/journal.pone.0047584.gMeasuring Redox Ratio by a Coupled Cycling BTZ-043 AssayTable 1. Slopes between the rate of absorbance increase (Vmean) and the concentration of NAD+ (1024?OD?min21?mM21) in either homogenate or homogenization buffer.NAD+ diluted in: fly homogenateTable 2. Phenol-Chloroform extraction is efficient for extracting NADH.(mmol/g protein) Treatment 30 min, 65uC 30 min, 65uC, 30 min, 65uC, homogenization buffer 30 min, 65uC, 30 min, 65uC, 0.01 M H+ Slope 2.820 0.01 M OH2 0.027 15.875 0.01 M OH2 0.104 0.01 M H+ 14.r0.793, ** 0.009, N.S. 0.965, *** 0.244, N.S. 0.997, ***Extraction Method NAD+ Control 5.NADH Compared 1.Redox Ratio Compared 5.114 Compared60.125 to Control Chloroform 4.959 ***60.087 to Control 60.419 to Control 0.915 N.S. 4.871 N.S.60.095 p,0.001 Phenol7.159 Chloroform ***60.090 p = 0.192 1.869 ***60.516 p = 0.717 3.820 **N.S.: non-significant; **, p,0.01; ***, p,0.001. Fly homogenate is generated by homogenizing 15 male D. melanogaster adults in 250 ul homogenization buffer. Particles were removed by 5 min 160006g centrifugation. doi:10.1371/journal.pone.0047584.t60.119.MaintainsPhenol-chloroform Extraction Protects Pyridine Nucleotide from Enzymatic Degradation during Preparations and Dissociates Protein-bound NADHIt has long been known that most of the NAD+ exists in free solution form while most of the NADH is protein bound [27]. In order to accurately measure the total amount of NADH, the extraction method should be able to denature protein and release NADH. Denaturing protein will also reduce enzymatic degradation of pyridine nucleotides. We compared three extraction 1676428 methods: phenol chloroform extraction [28], chloroform-only extraction and 6 M guanidine-HCl. Guanidine-HCl extraction is a strong denaturing reagent which is capable of breaking protein secondary structure and has been used in assaying other metabolites [29,30,31]. We found guanidine extraction interfered with ADH activity (Fig. 4a) and is not suitable for this enzymatic recycling assay. For reasons unknown, phenol-chloroform treated samples also give a more linear kinetic curve than that of chloroform-only treated samples (Fig. 4a and 4b). We compared the redox ratio (NAD+/NADH) obtained from the same fly tissue homogenate: untreated control, chloroformonly treated and phenol-chloroform treated (see Table 2). Phenolchloroform treatment gives the lowest value, suggesting NADH recovery in this reaction is the highest among all three. Also noticeable is that 25837696 the amount of pyridine nucleotides detected following phenol-chloroform extraction is the highest among three treatments.Measuring Redox Ratio by a Coupled Cycling AssayFigure 2. Comparison of reaction kinetics of NADPx, NADx assay with and without hydrazine. A: Standard curve of NADPx assay. B: Standard curve of NADx assay without hydrazine. C: Standard curve of NADx assay with hydrazine. D: reaction kinetic of NADPx assay. E: NADx assay kinetic, showing additional hydrazine increases Vmean. F: the increase of Vmean by addition of hydrazine is dependent on the concentration of NAD+. All assays were performed in duplicates and hydrazine concentration is 0.02 . doi:10.1371/journal.pone.0047584.gFigure 3. The optimal concentration of hydrazine is around 0.02 . A: In a reaction containing 25 mM NAD+, Vmean increases with hydrazine concentration log-linearly up to 0.02 . B: The rate of absorbance increase in no NAD+ blank control is affected by hydrazine concentration higher than 0.02 . doi:10.1371/journal.pone.0047584.gMeasuring Redox Ratio by a Coupled Cycling AssayTable 1. Slopes between the rate of absorbance increase (Vmean) and the concentration of NAD+ (1024?OD?min21?mM21) in either homogenate or homogenization buffer.NAD+ diluted in: fly homogenateTable 2. Phenol-Chloroform extraction is efficient for extracting NADH.(mmol/g protein) Treatment 30 min, 65uC 30 min, 65uC, 30 min, 65uC, homogenization buffer 30 min, 65uC, 30 min, 65uC, 0.01 M H+ Slope 2.820 0.01 M OH2 0.027 15.875 0.01 M OH2 0.104 0.01 M H+ 14.r0.793, ** 0.009, N.S. 0.965, *** 0.244, N.S. 0.997, ***Extraction Method NAD+ Control 5.NADH Compared 1.Redox Ratio Compared 5.114 Compared60.125 to Control Chloroform 4.959 ***60.087 to Control 60.419 to Control 0.915 N.S. 4.871 N.S.60.095 p,0.001 Phenol7.159 Chloroform ***60.090 p = 0.192 1.869 ***60.516 p = 0.717 3.820 **N.S.: non-significant; **, p,0.01; ***, p,0.001. Fly homogenate is generated by homogenizing 15 male D. melanogaster adults in 250 ul homogenization buffer. Particles were removed by 5 min 160006g centrifugation. doi:10.1371/journal.pone.0047584.t60.119.