Go to the main menu
Skip to content
Go to bottom
REFERENCE LINKING PLATFORM OF KOREA S&T JOURNALS
> Journal Vol & Issue
Journal Basic Information
Journal DOI :
Korean Society for Biochemistry and Molecular Biology
Editor in Chief :
Volume & Issues
Volume 37, Issue 6 - Nov 2004
Volume 37, Issue 5 - Sep 2004
Volume 37, Issue 4 - Jul 2004
Volume 37, Issue 3 - May 2004
Volume 37, Issue 2 - Mar 2004
Volume 37, Issue 1 - Jan 2004
Selecting the target year
The Mitochondrial Tricarboxylate Carrier of Silver Eel: Chemical Modification by Sulfhydryl Reagents
Capobianco, Loredana ; Impagnatiello, Tecla ; Ferramosca, Alessandra ; Zara, Vincenzo ;
BMB Reports , volume 37, issue 5, 2004, Pages 515~521
DOI : 10.5483/BMBRep.2004.37.5.515
The tricarboxylate (or citrate) carrier was purified from eel liver mitochondria and functionally reconstituted into liposomes. Incubation of the proteoliposomes with various sulfhydryl reagents led to inhibition of the reconstituted citrate transport activity. Preincubation of the proteoliposomes with reversible SH reagents, such as mercurials and methanethiosulfonates, protected the eel liver tricarboxylate carrier against inactivation by the irreversible reagent N-(1-pyrenyl)maleimide (PM). Citrate and L-malate, two substrates of the tricarboxylate carrier, protected the protein against inactivation by sulfhydryl reagents and decreased the fluorescent PM bound to the purified protein. These results suggest that the eel liver tricarboxylate carrier requires a single population of free cysteine(s) in order to manifest catalytic activity. The reactive cysteine(s) is most probably located at or near the substrate binding site of the carrier protein.
Combined Cytogenetic and Molecular Analyses for the Diagnosis of Prader-Willi/Angelman Syndromes
Borelina, Daniel ; Engel, Nora ; Esperante, Sebastian ; Ferreiro, Veronica ; Ferrer, Marcela ; Torrado, Maria ; Goldschmidt, Ernesto ; Francipane, Liliana ; Szijan, Irene ;
BMB Reports , volume 37, issue 5, 2004, Pages 522~526
DOI : 10.5483/BMBRep.2004.37.5.522
Prader-Willi (PWS) and Angelman (AS) are syndromes of developmental impairment that result from the loss of expression of imprinted genes in the paternal (PWS) or maternal (AS) 15q11-q13 chromosome. Diagnosis on a clinical basis is difficult in newborns and young infants; thus, a suitable molecular test capable of revealing chromosomal abnormalities is required. We used a variety of cytogenetic and molecular approaches, such as, chromosome G banding, fluorescent in situ hybridization, a DNA methylation test, and a set of chromosome 15 DNA polymorphisms to characterize a cohort of 27 PWS patients and 24 suspected AS patients. Molecular analysis enabled the reliable diagnosis of 14 PWS and 7 AS patients, and their classification into four groups: (A) 6 of these 14 PWS subjects (44%) had deletions of paternal 15q11-q13; (B) 4 of the 7 AS patients had deletions of maternal 15q11-q13; (C) one PWS patient (8%) had a maternal uniparental disomy (UPD) of chromosome 15; (D) the remaining reliably diagnoses of 7 PWS and 3 AS cases showed abnormal methylation patterns of 15q11-q13 chromosome, but none of the alterations shown by the above groups, although they may have harbored deletions undetected by the markers used. This study highlights the importance of using a combination of cytogenetic and molecular tests for a reliable diagnosis of PWS or AS, and for the identification of genetic alterations.
Construction and Differential Screening of a cDNA Library Specific to Osmotic Stress of Haloxylon ammodendron Seedlings
Jiang, Xiao-Cheng ; Guo, Xin-Hong ; Pan, Xiao-Ling ; Song, Song-Quan ;
BMB Reports , volume 37, issue 5, 2004, Pages 527~532
DOI : 10.5483/BMBRep.2004.37.5.527
A subtracted cDNA library specific to osmotic stress of Haloxylon ammodendron (Mey.) Bge seedlings was constructed by suppression subtractive hybridization (SSH) and T/A cloning. SSH was performed between two groups of H. ammodendron seedlings, one was cultivated in Hoagland (H) solution as a driver and the other group was treated with osmotic stress of the Hoagland solution by the addition of 400 mM mannitol (M), as a tester. The library consisted of about 400 recombinant clones, with the average size being of 500 bp, ranging from 300 bp to 1500 bp. Using a PCR-select differential screening kit, 100 recombinant clones were randomly chosen from the subtracted cDNA library and hybridized with forward,reverse subtracted and unsubtracted probes for two rounds. As a result, 21 positive clones specific to osmotic stress were obtained and some of them were verified by Northern blot analysis. The sequencing analysis of 6 positive clones and the following homology comparison to GenBank [blastx] non-redundant databases characterized that two sequences obtained in this experiment may contribute to novel drought-related genes.
Substrate Ground State Binding Energy Concentration Is Realized as Transition State Stabilization in Physiological Enzyme Catalysis
Britt, Billy Mark ;
BMB Reports , volume 37, issue 5, 2004, Pages 533~537
DOI : 10.5483/BMBRep.2004.37.5.533
Previously published kinetic data on the interactions of seventeen different enzymes with their physiological substrates are re-examined in order to understand the connection between ground state binding energy and transition state stabilization of the enzyme-catalyzed reactions. When the substrate ground state binding energies are normalized by the substrate molar volumes, binding of the substrate to the enzyme active site may be thought of as an energy concentration interaction; that is, binding of the substrate ground state brings in a certain concentration of energy. When kinetic data of the enzyme/substrate interactions are analyzed from this point of view, the following relationships are discovered: 1) smaller substrates possess more binding energy concentrations than do larger substrates with the effect dropping off exponentially, 2) larger enzymes (relative to substrate size) bind both the ground and transition states more tightly than smaller enzymes, and 3) high substrate ground state binding energy concentration is associated with greater reaction transition state stabilization. It is proposed that these observations are inconsistent with the conventional (Haldane) view of enzyme catalysis and are better reconciled with the shifting specificity model for enzyme catalysis.
Isolation and Molecular Characterization of a New CRT Binding Factor Gene from Capsella bursa-pastoris
Wang, Xinglong ; Liu, Li ; Liu, Sixiu ; Sun, Xiaoqing ; Deng, Zhongxiang ; Pi, Yan ; Sun, Xiaofen ; Tang, Kexuan ;
BMB Reports , volume 37, issue 5, 2004, Pages 538~545
DOI : 10.5483/BMBRep.2004.37.5.538
A new CRT binding factor (CBF) gene designated Cbcbf25 was cloned from Capsella bursa-pastoris, a wild grass, by the rapid amplification of cDNA ends (RACE). The full-length cDNA of Cbcbf25 was 898 bp with a 669 bp open reading frame (ORF) encoding a putative DRE/CRT (LTRE)-binding protein of 223 amino acids. The predicted CbCBF25 protein contained a potential nuclear localization signal (NLS) in its N-terminal region followed by an AP2 DNA-binding motif and a possible acidic activation domain in the C-terminal region. Bioinformatic analysis revealed that Cbcbf25 has a high level of similarity with other CBF genes like cbf1, cbf2, and cbf3 from Arabidopsis thaliana, and Bncbf5, Bncbf7, Bncbf16, and Bncbf17 from Brassica napus. A cold acclimation assay showed that Cbcbf25 was expressed immediately after cold triggering, but this expression was transient, suggesting that it concerns cold acclimation. Our study implies that Cbcbf25 is an analogue of other CBF genes and may participate in cold-response, by for example, controlling the expression of cold-regulated genes or increasing the freezing tolerance of plants.
Microarrays for the Detection of HBV and HDV
Sun, Zhaohui ; Zheng, Wenling ; Zhang, Bao ; Shi, Rong ; Ma, Wenli ;
BMB Reports , volume 37, issue 5, 2004, Pages 546~551
DOI : 10.5483/BMBRep.2004.37.5.546
The increasing pace of development in molecular biology during the last decade has had a direct effect on mass testing and diagnostic applications, including blood screening. We report the model Microarray that has been developed for Hepatitis B virus (HBV) and Hepatitis D virus (HDV) detection. The specific primer pairs of PCR were designed using the Primer Premier 5.00 program according to the conserved regions of HBV and HDV. PCR fragments were purified and cloned into pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The DNA microarray was prepared by robotically spotting PCR products onto the surface of glass slides. Sequences were aligned, and the results obtained showed that the products of PCR amplification were the required specific gene fragments of HBV, and HDV. Samples were labeled by Restriction Display PCR (RD-PCR). Gene chip hybridizing signals showed that the specificity and sensitivity required for HBV and HDV detection were satisfied. Using PCR amplified products to construct gene chips for the simultaneous clinical diagnosis of HBV and HDV resulted in a quick, simple, and effective method. We conclude that the DNA microarray assay system might be useful as a diagnostic technique in the clinical laboratory. Further applications of RD-PCR for the sample labeling could speed up microarray multi-virus detection.
Association between the Angiotensin-converting Enzyme Gene Insertion/Deletion Polymorphism and Essential Hypertension in Young Pakistani Patients
Ismail, Muhammad ; Akhtar, Naveed ; Nasir, Muhammad ; Firasat, Sadaf ; Ayub, Qasim ; Khaliq, Shagufta ;
BMB Reports , volume 37, issue 5, 2004, Pages 552~555
DOI : 10.5483/BMBRep.2004.37.5.552
Several studies have demonstrated the importance of angiotensin-converting enzyme (ACE) insertion (I)/deletion (D) polymorphisms in the pathogenesis of hypertension. This study sought to determine the association between the ACE I/D polymorphism and essential hypertension in young Pakistanis. The frequency of the ACE I/D polymorphism was established by a comparative cross-sectional survey of Pakistani patients suffering from essential hypertension and ethnically matched normotensive controls. Samples were collected from tertiary care hospitals in northern Pakistan. Hypertensive individuals were defined as those with a systolic blood pressure > 140 mmHg and/or diastolic blood pressure > 90 mmHg on three separate occasions, or those currently receiving one, or more, anti-hypertensive agents. DNA samples obtained from hypertensive (n=211) and normotensive (n=108) individuals were typed by PCR. The frequency of the ACE I/I genotype was significantly higher in hypertensive patients, aged 20-40 years, than in normotensive controls of the same age group (
= 4.0, P = 0.041). Whereas no overall significant differences were observed between the I/I, I/D and D/D ACE genotypes (One way ANOVA, F=0.672; P=0.413). The association between the ACE I/I genotype and essential hypertension in individuals aged
40 years suggests that ACE has a role in early onset essential hypertension in Pakistan.
Neutralizing Chimeric Mouse-human Antibodies against Burkholderia pseudomallei Protease: Expression, Purification and Characterization
Chan, Shzu-Wei ; Ong, Guan-Im ; Nathan, Sheila ;
BMB Reports , volume 37, issue 5, 2004, Pages 556~564
DOI : 10.5483/BMBRep.2004.37.5.556
A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2% glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.
Solution Structure of Human Orexin-A: Regulator of Appetite and Wakefulness
Kim, Hai-Young ; Hong, Eun-Mi ; Kim, Jae-Il ; Lee, Weon-Tae ;
BMB Reports , volume 37, issue 5, 2004, Pages 565~573
DOI : 10.5483/BMBRep.2004.37.5.565
Orexin-A and orexin-B (hypocretin-1 and hypocretin-2, respectively) are important hypothalamic neuro-peptides, which are encoded by a single mRNA transcript and stimulate food intake as well as regulate wakefulness. Here we determined the solution structure of orexin-A by NMR spectroscopy and by simulated-annealing calculation. The structural features of orexin-A involve two
-helices, with the hydrophobic residues disposed to on one side of helix, and hydrophilic residues to the other. A hydrophilic turn induced by two disulfide bonds provides the key difference between orexin-A and -B. With previous mutagenic studies, the derived structure of orexin-A provides us with a structure-functional view for novel drug design.
Molecular Cloning, Sequencing, and Expression of a Fibrinolytic Serine-protease Gene from the Earthworm Lumbricus rubellus
Cho, Il-Hwan ; Choi, Eui-Sung ; Lee, Hyung-Hoan ;
BMB Reports , volume 37, issue 5, 2004, Pages 574~581
DOI : 10.5483/BMBRep.2004.37.5.574
The full-length cDNA of the lumbrokinase fraction 6 (F6) protease gene of Lumbricus rubellus was amplified using an mRNA template, sequenced and expressed in E. coli cells. The F6 protease gene consisted of pro- and mature sequences by gene sequence analysis, and the protease was translated and modified into active mature polypeptide by N-terminal amino acid sequence analysis of the F6 protease. The pro-region of F6 protease consisted of the 44 residues from methionine-1 to lysine-44, and the mature polypeptide sequence (239 amino acid residues and one stop codon; 720 bp) started from isoleucine-45 and continued to the terminal residue. F6 protease gene clones having pro-mature sequence and mature sequence produced inclusion bodies in E. coli cells. When inclusion bodies were orally administrated rats, generated thrombus weight in the rat' venous was reduced by approximately 60% versus controls. When the inclusion bodies were solubilized in pepsin and/or trypsin solutions, the solubilized enzymes showed hemolytic activity in vitro. It was concluded the F6 protease has hemolytic activity, and that it is composed of pro- and mature regions.
Genetic Polymorphism of Glutathione S-transferase P1 and Breast Cancer Risk
Kim, Sook-Un ; Lee, Kyoung-Mu ; Park, Sue-Kyung ; Yoo, Keun-Young ; Noh, Dong-Young ; Choe, Kook-Jin ; Ahn, Sei-Hyun ; Hirvonen, Ari ; Kang, Dae-Hee ;
BMB Reports , volume 37, issue 5, 2004, Pages 582~585
DOI : 10.5483/BMBRep.2004.37.5.582
To evaluate the potential association between the GSTP1 genotype and the development of breast cancer, a hospital based case-control study was conducted on Korean women. The study population consisted of 171 histologically confirmed incident breast cancer cases and 171 age-matched controls with no present or previous history of cancer. PCR-RFLP was used for the GSTP1 genotyping and statistical evaluations were performed using an unconditional logistic regression model. Postmenopausal women with the GSTP1 Val allele were found to have a reduced risk of breast cancer (OR = 0.3, 95% CI = 0.10 - 0.74). A significant interaction was observed between the GSTP1 genotype and alcohol consumption (p for interaction = 0.01); compared with never-drinking women with Ile/Ile genotype, ever-drinking women with the GSTP1 Val allele had almost a three-fold risk of breast cancer (OR = 2.9, 95% CI = 1.05-7.85), whereas never-drinking women with Val allele had half this risk (OR = 0.5, 95% CI = 0.27-0.93). Our findings suggest that the GSTP1 polymorphism influences individual susceptibility to breast cancer in the Korean women and this effect may be modified by alcohol consumption.
Unfolding of Ervatamin C in the Presence of Organic Solvents: Sequential Transitions of the Protein in the O-state
Sundd, Monica ; Kundu, Suman ; Dubey, Vikash Kumar ; Jagannadham, Medicherla V. ;
BMB Reports , volume 37, issue 5, 2004, Pages 586~596
DOI : 10.5483/BMBRep.2004.37.5.586
The folding of ervatamin C was investigated in the presence of various fluorinated and non-fluorinated organic solvents. The differences in the unfolding of the protein in the presence of various organic solvents and the stabilities of O-states were interpreted. At pH 2.0, non-fluorinated alkyl alcohols induced a switch from the native
-helix to a
-sheet, contrary to the
-helix conversion observed for many proteins. The magnitude of ellipticity at 215 nm, used as a measure of
-content, was found to be dependent on the concentration of the alcohol. Under similar conditions of pH, fluorinated alcohol enhanced the intrinsic a-helicity of the protein molecule, whereas the addition of acetonitrile reduced the helical content. Ervatamin C exhibited high stability towards GuHCl induced unfolding in different O-states. Whereas the thermal unfolding of O-states was non-cooperative, contrary to the cooperativity seen in the absence of the organic solvents under similar conditions. Moreover, the differential scanning calorimetry endotherms of the protein acquired at pH 2.0 were deconvoluted into two distinct peaks, suggesting two cooperative transitions. With increase in pH, the shape of the thermogram changed markedly to exhibit a major and a minor transition. The appearance of two distinct peaks in the DSC together with the non-cooperative thermal transition of the protein in O-states indicates that the molecular structure of ervatamin C consists of two domains with different stabilities.
A Mutagenic Study of β-1,4-Galactosyltransferases from Neisseria meningitidis
Park, Jae-Eun ; Do, Su-Il ; Lee, Ki-Sung ; Lee, Sang-Soo ;
BMB Reports , volume 37, issue 5, 2004, Pages 597~602
DOI : 10.5483/BMBRep.2004.37.5.597
N-terminal His-tagged recombinant
-1,4-galactosyltransferase from Neisseria meningitidis was expressed and purified to homogeneity by column chromatography using Ni-NTA resin. Mutations were introduced to investigate the roles of, Ser68, His69, Glu88, Asp90, and Tyr156, which are components of a highly conserved region in recombinant
-1,4 galactosyltransferase. Also, the functions of three other cysteine residues, Cys65, Cys139, and Cys205, were investigated using site-directed mutagenesis to determine the location of the disulfide bond and the role of the sulfhydryl groups. Purified mutant galactosyltransferases, His69Phe, Glu88Gln and Asp90Asn completely shut down wild-type galactosyltransferase activity (1-3%). Also, Ser68Ala showed much lower activity than wild-type galactosyltransferase (19%). However, only the substitution of Tyr156Phe resulted in a slight reduction in galactosyltransferase activity (90%). The enzyme was found to remain active when the cysteine residues at positions 139 and 205 were replaced separately with serine. However, enzyme reactivity was found to be markedly reduced when Cys65 was replaced with serine (27%). These results indicate that conserved amino acids such as Cys65, Ser68, His69, Glu88, and Asp90 may be involved in the binding of substrates or in the catalysis of the galactosyltransferase reaction.
Effects of Chlorpromazine·HCl on the Structural Parameters of Bovine Brain Membranes
Jang, Hye-Ock ; Jeong, Dong-Keun ; Ahn, Shin-Ho ; Yoon, Chang-Dae ; Jeong, Soo-Cheol ; Jin, Seong-Deok ; Yun, Il ;
BMB Reports , volume 37, issue 5, 2004, Pages 603~611
DOI : 10.5483/BMBRep.2004.37.5.603
Fluorescence probes located in different membrane regions were used to evaluate the effects of chlorpromazine HCl on structural parameters (transbilayer lateral mobility, annular lipid fluidity, protein distribution, and lipid bilayer thickness) of synaptosomal plasma membrane vesicles (SPMVs) isolated from bovine cerebral cortex. The experimental procedure was based on the selective quenching of 1,3-di(1-pyrenyl)propane (Py-3-Py) by trinitrophenyl groups, radiationless energy transfer from the tryptophan of membrane proteins to Py-3-Py, and energy transfer from Py-3-Py monomers to 1-anilinonaphthalene-8-sulfonic acid (ANS). In this study, chlorpromazine HCl decreased the lateral mobility of Py-3-Py in a concentration dependent-manner, showed a greater ordering effect on the inner monolayer than on the outer monolayer, decreased annular lipid fluidity in a dose dependent-manner, and contracted the membrane lipid bilayer. Furthermore, the drug was found to have a clustering effect on membrane proteins.
The Gene Expression Profile of Cyst Epithelial Cells in Autosomal Dominant Polycystic Kidney Disease Patients
Lee, Jae-Eun ; Park, Min-Ha ; Park, Jong-Hoon ;
BMB Reports , volume 37, issue 5, 2004, Pages 612~617
DOI : 10.5483/BMBRep.2004.37.5.612
Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by the formation of fluid-filled cysts in the kidney and progressive renal failure. Other manifestations of ADPKD include the formation of cysts in other organs (liver, pancreas, and spleen), hypertension, cardiac defects, and cerebral aneurysms. The loss of function of the polycystin -1 and -2 results in the formation of epithelium-lined cysts, a process that depends on initial epithelial proliferation. cDNA microarrays powerfully monitor gene expression and have led to the discoveries of pathways regulating complex biological processes. We undertook to profile the gene expression patterns of epithelial cells derived from the cysts of ADPKD patients using the cDNA microarray technique. Candidate genes that were differently expressed in cyst tissues were identified. 19 genes were up-regulated, and 6 down-regulated. Semi-quantitative RT-PCR results were consistent with the microarray findings. To distinguish between normal and epithelial cells, we used the hierarchical method. The results obtained may provide a molecular basis for understanding the biological meaning of cytogenesis.
The Activities of Antioxidant Enzymes in Response to Oxidative Stresses and Hormones in Paraquat-tolerant Rehmannia glutinosa Plants
Choi, Dong-Geun ; Yoo, Nam-Hee ; Yu, Chang-Yeon ; De Los Reyes, Benildo ; Yun, Song-Joong ;
BMB Reports , volume 37, issue 5, 2004, Pages 618~624
DOI : 10.5483/BMBRep.2004.37.5.618
All members of R. glutinosa show the unique characteristic of intrinsic tolerance to paraquat (PQ). Antioxidant enzymes have been proposed to be the primary mechanism of PQ resistance in several plant species. Therefore, the antioxidant enzyme systems of R. glutinosa were evaluated by comparatively analyzing cellular antioxidant enzyme levels, and their responses of oxidative stresses and hormones. The levels of ascorbate peroxidase (APX), glutathione reductase (GR), non-specific peroxidase (POX), and superoxide dismutase (SOD) were 7.3-, 4.9-, 2.7- and 1.6-fold higher in PQ-tolerant R. glutinosa than in PQ-susceptible soybeans. However, the activity of catalase (CAT) was about 12-fold higher in the soybeans. The activities of antioxidant enzymes reduced after PQ treatment in the two species, with the exception of POX and SOD in R. glutinosa, which increased by about 40%. Interestingly, the activities of APX, SOD and POX in R. glutinosa, relative to those in soybeans, were further increased by 49, 67 and 93% after PQ treatment. The considerably higher intrinsic levels, and increases in the relative activities of antioxidant enzymes in R. glutinosa under oxidative stress support the possible role of these enzymes in the PQ tolerance of R. glutinosa. However, the relatively lower levels of SOD versus PQ tolerance, and the mixed responses of antioxidant enzymes to stresses and hormones, suggest a possible alternative mechanism(s) for PQ tolerance in R. glutinosa.
Presence of Proboscipedia and Caudal Gene Homologues in a Bivalve Mollusc
Carpintero, Pablo ; Pazos, Antonio Juan ; Abad, Marcelina ; Sanchez, Jose Luis ; De La Luz Perez-Paralle, Maria ;
BMB Reports , volume 37, issue 5, 2004, Pages 625~628
DOI : 10.5483/BMBRep.2004.37.5.625
Homeobox genes encode a family of transcription factors that have essential roles in regulating the development of eukaryotes. Although they have been extensively studied in different phyla, relatively little is known about homeobox-containing genes and their function in molluscs. In this study, we used a polymerase chain reaction to investigate homeobox genes in the bivalve mollusc Pecten maximus. Four different homeobox sequences were identified; two were homologues of the non-Hox cluster gene caudal and the two remaining sequences had a significant homology to the ANT-C gene proboscipedia. These sequences represent the first cad and pb homologues isolated from a member of the class Bivalvia, phylum Mollusca.
A Continuous Spectrophotometric Assay for NADPH-cytochrome P450 Reductase Activity Using 1,1-Diphenyl-2-Picrylhydrazyl
Yim, Sung-Kun ; Yun, Su-Jung ; Yun, Chul-Ho ;
BMB Reports , volume 37, issue 5, 2004, Pages 629~633
DOI : 10.5483/BMBRep.2004.37.5.629
NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450, and catalyzes the one-electron reduction of many drugs and foreign compounds. Various forms of spectrophotometric titration have been performed to investigate the electron-accepting properties of CPR, particularly, to examine its ability to reduce cytochrome c and ferricyanide. In this study, the reduction of 1,1-diphenyl-2-picrylhydrazyl (DPPH) by CPR was assessed as a means of monitoring CPR activity. The principle advantage of DPPH is that its reduction can be assayed directly in the reaction medium by a continuous spectrophotometry. Thus, electrons released from NADPH by CPR were transferred to DPPH, and DPPH reduction was then followed spectrophotometrically by measuring
reduction. Optimal assay concentrations of DPPH, CPR, potassium phosphate buffer, and NADPH were first established. DPPH reduction activity was found to depend upon the strength of the buffer used, which was optimal at 100 mM potassium phosphate and pH 7.6. The extinction coefficient of DPPH was
. DPPH reduction followed classical Michaelis-Menten kinetics (
). This method uses readily available materials, and has the additional advantages of being rapid and inexpensive.