Purification Buffer Research Articles

Improved production of class I phosphatidylinositol 4,5-bisphosphate 3-kinase

Phosphatidylinositol 4,5-bisphosphate 3-kinases (PI3K) are a family of kinases whose activity affects pathways needed for basic cell functions. As a result, PI3K is one of the most mutated genes in all human cancers and serves as an ideal therapeutic target for cancer treatment. Expanding on work done by other groups we improved protein yield to produce stable and pure protein using a variety of modifications including improved solubility tag, novel expression modalities, and optimized purification protocol and buffer. By these means, we achieved a 40-fold increase in yield for p110α/p85α and a 3-fold increase in p110α. We also used these protocols to produce comparable constructs of the β and δ isoforms of PI3K. Increased yield enhanced the efficiency of our downstream high throughput drug discovery efforts on the PIK3 family of kinases.

Measuring Minimal Residual Disease in Plasma of Multiple Myeloma Patients By Intact Mass Spectrometry

Multiple Myeloma (MM) is a hematological cancer that is currently treatable but incurable, and is characterized by the accumulation of malignant plasma cells in bone marrow. Clinical studies have shown that post-cancer treatment, the minimal residual disease (MRD) status is a prognostic factor indicative of the extent of disease remission. MRD assessment has been primarily assessed in bone marrow (BM) using flow cytometry or next generation sequencing. Not only these measurements may lead to false-negative results due to spatial heterogeneity in the BM, hemodilution and/or extramedullary disease as indicated by Derman et al. in Blood Cancer Journal ((2021), 11:19) but the procedure for BM collection is invasive and painful for the patient. These authors present a peripheral blood (PB)-based method that potentially can overcome the barriers in assessing for residual disease in MM patients. We hereby present a non-invasive blood-test for measurement of MRD using mass spectrometry (intact mass approach) for the detection of Ig product in MM patients. We will describe the assay development, analytical characterization of assay performance, and data from the analysis of plasma/serum samples from MM patients enrolled in a clinical trial. A very low volume of patient plasma/serum sample (10 μL) is required to perform this assay. The plasma/serum sample is diluted in purification buffer, applied to Melon gel resin and incubated for 5 min, followed by centrifugation and reduction with DTT. The supernatant is then analyzed by LC-MS on a Q-Exactive mass spectrometer. Deconvolution of the intact protein m/z spectra is performed to yield intact mass of the specific protein target species of interest. Overall, the method can process up to 96 samples in parallel within 3 hrs, making this procedure highly scalable for clinical applications. Data obtained from the assay characterization indicate that the assay is sensitive with an LOD <500 ng/mL and differentiates MM patient from a healthy donor as well allows for the monitoring of MM patient before and after treatment. In conclusion, although still an exploratory analysis, the blood-based mass spectrometry method can be used to screen for MRD in patients who have undetectable disease by flow cytometry or next generation sequencing.

Quick and easy method for extraction and purification of Pfu-Sso7d, a high processivity DNA polymerase

The polymerase chain reaction is an extensively used technique with numerous applications in the field of biological sciences. In addition to naturally occurring DNA polymerases with varying processivity and fidelity, genetically engineered recombinant DNA polymerases are also used in PCR. The Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA binding protein, to the polymerase domain of the Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity. Expensive commercial variants of Pfu-Sso7d are sold under various trade names. Here, we report a quick, cost and time-efficient purification protocol and an optimized buffer system for Pfu-Sso7d. We evaluated precipitation efficiencies of varying concentrations of ethanol and acetone and compared the activities of the precipitated enzyme. Although both the solvents efficiently precipitated Pfu-Sso7d, acetone showed better precipitation efficiency. Purified Pfu-Sso7d showed excellent activity in the PCR of templates with varying lengths and GC contents. We also report a buffer system that works with Pfu-Sso7d as efficiently as commercially available buffers. This quick and efficient purification scheme and buffer system will provide researchers cost-efficient access to fusion polymerase.

Nuclei Purification Buffer

Nuclei Purification Buffer

Enhanced Stability of Detergent-Free Human Native STEAP1 Protein from Neoplastic Prostate Cancer Cells upon an Innovative Isolation Procedure.

Background: The STEAP1 is a cell-surface antigen over-expressed in prostate cancer, which contributes to tumor progression and aggressiveness. However, the molecular mechanisms underlying STEAP1 and its structural determinants remain elusive. Methods: The fraction capacity of Butyl- and Octyl-Sepharose matrices on LNCaP lysates was evaluated by manipulating the ionic strength of binding and elution phases, followed by a Co-Immunoprecipitation (Co-IP) polishing. Several potential stabilizing additives were assessed, and the melting temperature (Tm) values ranked the best/worst compounds. The secondary structure of STEAP1 was identified by circular dichroism. Results: The STEAP1 was not fully captured with 1.375 M (Butyl), in contrast with interfering heterologous proteins, which were strongly retained and mostly eluted with water. This single step demonstrated higher selectivity of Butyl-Sepharose for host impurities removal from injected crude samples. Co-IP allowed recovering a purified fraction of STEAP1 and contributed to unveil potential physiologically interacting counterparts with the target. A Tm of ~55 °C was determined, confirming STEAP1 stability in the purification buffer. A predominant α-helical structure was identified, ensuring the protein’s structural stability. Conclusions: A method for successfully isolating human STEAP1 from LNCaP cells was provided, avoiding the use of detergents to achieve stability, even outside a membrane-mimicking environment.

Development and evaluation of a novel DNA purification buffer and protocol for blood samples on FTA cards

DNA fingerprinting is expanding its horizons by the virtue of new innovative technologies. The use of FTA cards for the preservation of blood, saliva, and other biological samples is a preferred method, owing to the reliability and ease of sample preservation provided by this technology. Across the globe, FTA cards are routinely used in crime investigations to collect blood samples of victims, suspects and from the crime scene. Owing to lack of technical know-how, the FTA matrixes are often overloaded with blood samples as the collector tries to fill the entire circle(s) on the FTA card with blood. This makes purification of FTA punches unwieldy difficult with the standard FTA protocol. As a result, the FTA punches remain reddish in color, even after several washes. The purification process may hence take excess time, spanning to several hours. Further, FTA punches that are reddish in color have a bearing on multiplex PCR success rate as they may show no amplifications or partial amplifications. A new purification buffer reagent and a new protocol were designed to purify FTA punches rapidly. A comparative study revealed that the novel purification buffer and protocol successfully cleansed overloaded FTA cards in drastically less time as compared to the conventional buffer and protocol. Further, the success rate of multiplex PCR was high with the generation of complete STR profiles from all the samples.

Extraction and purification of NUDT9 homology domain of human transient receptor potential melastatin 2 channel

To develop methods of extraction and purification of Cterminal NUDT9 homology domain of human transient receptor potential melastatin 2 (TRPM2) channel. After sonication and centrifuge of Escherichia coli strain Rosetta (DE3) which was induced by isopropylthio-β-D-galactoside, GST-NUDT9-H was collected after the binding of supernatant with GST beads and eluted with reduced glutathione. Then the elution buffer containing fusion protein was purified by size exclusion chromatography after concentration and centrifuge. Finally, with the cleavage of thrombin and binding with the GST beads, NUDT9-H with high purity in supernatant was collected. The GST-NUDT9-H fusion protein was stabilized with lysis buffer containing 0.5% n-dodecyl -β-d-maltoside (DDM), and wash buffer containing 0.025% DDM in size-exclusion chromatography system, and finally the NUDT9-H with high purity was obtained after cleaved by thrombin (1 U/2 mg fusion protein) for 24 h. Due to the poor stability of NUDT9-H, it is necessary to add DDM in extraction and purification buffer to stabilize the conformation of NUDT9-H, so as to increase its yields and purity.

Prokaryotic expression and action mechanism of antimicrobial LsGRP1C recombinant protein containing a fusion partner of small ubiquitin-like modifier.

Antimicrobial peptides (AMPs) are peptides exhibiting broad-spectrum antimicrobial activities and considered as potential therapeutic agents. LsGRP1C, a novel AMP derived from defense-related LsGRP1 protein of Lilium, was proven to inhibit kinds of bacteria and fungi via alteration of microbial membrane permeability and induction of fungal programmed cell death-like phenomena by in vitro assays using synthetic LsGRP1C. In this study, the prokaryotic production of LsGRP1C recombinant protein containing an N-terminal fusion partner of the yeast small ubiquitin-like modifier (SUMO) was achieved by using optimized Escherichia coli host and purification buffer system, which lead to a high yield of soluble SUMO-LsGRP1C fusion protein. In vitro assay revealed that E. coli-expressed SUMO-LsGRP1C exhibited even better antifungal activity as compared to synthetic LsGRP1C. Meanwhile, the ability of SUMO-LsGRP1C in conducting fungal membrane permeabilization and programmed cell death was verified by SYTOX Green staining and 4',6-diamidino-2-phenylindole staining/terminal deoxynucleotidyl transferase dUTP nick-end labeling assays, respectively, indicating that E. coli-expressed SUMO-LsGRP1C shares identical modes of action with synthetic LsGRP1C. Herein, this E. coli expression system enables the effective and convenient production of antimicrobial LsGRP1C in a form of SUMO-fused recombinant protein.

Purification effect of two typical water source vegetation buffer zones on land-sourced pollutants

Two vegetation buffer zones (tree-shrub-grass pattern and tree-grass pattern) were selected as test objects around Siming reservoir in Yuyao City of China. The effect of the storm runoff intensity (low and high intensity) and the buffer zone width (1 m, 3 m, 5 m, 7 m, 9 m, 12 m, 16 m) on pollutants (suspended solids, ammonium nitrogen and total phosphorus) was studied by the artificial simulation runoff. The results showed that with the increase of the width of buffer zone, the pollutant concentration was decreased. The purification effect of the two buffer zones on suspended solids and total phosphorus was basically stable at 52-55% and 34-37%, respectively. But the purification effect on ammonium nitrogen was the tree-shrub-grass pattern (69.7%) significantly better than that of tree-grass pattern (52.1%). The purification rate at the low runoff intensity was 1.8-2.0 times that at the high runoff intensity. The relationship between the purification rate and buffer zone width can be expressed by the natural logarithm equation, and the model adjustment coefficient was greater than 0.92.

Purification and biochemical characterization of recombinant Persicaria minor β-sesquiphellandrene synthase

BackgroundSesquiterpenes are 15-carbon terpenes synthesized by sesquiterpene synthases using farnesyl diphosphate (FPP) as a substrate. Recently, a sesquiterpene synthase gene that encodes a 65 kDa protein was isolated from the aromatic plant Persicaria minor. Here, we report the expression, purification and characterization of recombinant P. minor sesquiterpene synthase protein (PmSTS). Insights into the catalytic active site were further provided by structural analysis guided by multiple sequence alignment.MethodsThe enzyme was purified in two steps using affinity and size exclusion chromatography. Enzyme assays were performed using the malachite green assay and enzymatic product was identified using gas chromatography-mass spectrometry (GC-MS) analysis. Sequence analysis of PmSTS was performed using multiple sequence alignment (MSA) against plant sesquiterpene synthase sequences. The homology model of PmSTS was generated using I-TASSER server.ResultsOur findings suggest that the recombinant PmSTS is mainly expressed as inclusion bodies and soluble aggregate in the E. coli protein expression system. However, the addition of 15% (v/v) glycerol to the protein purification buffer and the removal of N-terminal 24 amino acids of PmSTS helped to produce homogenous recombinant protein. Enzyme assay showed that recombinant PmSTS is active and specific to the C15 substrate FPP. The optimal temperature and pH for the recombinant PmSTS are 30 °C and pH 8.0, respectively. The GC-MS analysis further showed that PmSTS produces β-sesquiphellandrene as a major product and β-farnesene as a minor product. MSA analysis revealed that PmSTS adopts a modified conserved metal binding motif (NSE/DTE motif). Structural analysis suggests that PmSTS may binds to its substrate similarly to other plant sesquiterpene synthases.DiscussionThe study has revealed that homogenous PmSTS protein can be obtained with the addition of glycerol in the protein buffer. The N-terminal truncation dramatically improved the homogeneity of PmSTS during protein purification, suggesting that the disordered N-terminal region may have caused the formation of soluble aggregate. We further show that the removal of the N-terminus disordered region of PmSTS does not affect the product specificity. The optimal temperature, optimal pH, Km and kcat values of PmSTS suggests that PmSTS shares similar enzyme characteristics with other plant sesquiterpene synthases. The discovery of an altered conserved metal binding motif in PmSTS through MSA analysis shows that the NSE/DTE motif commonly found in terpene synthases is able to accommodate certain level of plasticity to accept variant amino acids. Finally, the homology structure of PmSTS that allows good fitting of substrate analog into the catalytic active site suggests that PmSTS may adopt a sesquiterpene biosynthesis mechanism similar to other plant sesquiterpene synthases.

Stabilization of tryptophan hydroxylase 2 by l‐phenylalanine‐induced dimerization

Tryptophan hydroxylase 2 (TPH2) catalyses the initial and rate‐limiting step in the biosynthesis of serotonin, which is associated with a variety of disorders such as depression, obsessive compulsive disorder, and schizophrenia. Full‐length TPH2 is poorly characterized due to low purification quantities caused by its inherent instability. Three truncated variants of human TPH2 (rch TPH2; regulatory and catalytic domain, NΔ47‐rch TPH2; truncation of 47 residues in the N terminus of rch TPH2, and ch TPH2; catalytic domain) were expressed, purified, and examined for changes in transition temperature, inactivation rate, and oligomeric state. ch TPH2 displayed 14‐ and 11‐fold higher half‐lives compared to rch TPH2 and NΔ47‐rch TPH2, respectively. Differential scanning calorimetry experiments demonstrated that this is caused by premature unfolding of the less stable regulatory domain. By differential scanning fluorimetry, the unfolding transitions of rch TPH2 and NΔ47‐rch TPH2 are found to shift from polyphasic to apparent two‐state by the addition of l‐Trp or l‐Phe. Analytical gel filtration revealed that rch TPH2 and NΔ47‐rch TPH2 reside in a monomer–dimer equilibrium which is significantly shifted toward dimer in the presence of l‐Phe. The dimerizing effect induced by l‐Phe is accompanied by a stabilizing effect, which resulted in a threefold increase in half‐lives of rch TPH2 and NΔ47‐rch TPH2. Addition of l‐Phe to the purification buffer significantly increases the purification yields, which will facilitate characterization of hTPH2.

Influence of Ammonium Acetate Concentration on Receptor-Ligand Binding Affinities Measured by Native Nano ESI-MS: A Systematic Study.

Native electrospray ionization (ESI) mass spectrometry (MS) is a powerful technique for analyzing biomolecules in their native state. However, ESI-MS is incompatible with nonvolatile solution additives. Therefore, biomolecules have to be electrosprayed from a solution that differs from their purification or storage buffer, often aqueous ammonium acetate (AmAc). In this study, the effect of the ionic strength on the dissociation constants of six different noncovalent complexes, that cover interactions present in many biological systems, was investigated. Complexes were electrosprayed from 10 mM, 50 mM, 100 mM, 300 mM, and 500 mM aqueous AmAc. For all systems, it was shown that the binding affinity is significantly influenced by the ionic strength of the solution. The determined dissociation constant (Kd) was affected more than 50% when increasing the AmAc concentration. The results are interpreted in terms of altered ionic interactions induced by the solution. This work emphasizes the modulating effect of the ions on noncovalent interactions and the importance of carefully choosing the AmAc concentration for quantifying the receptor-ligand binding strengths.

Hypothiocyanous acid oxidation of tubulin cysteines inhibits microtubule polymerization

Thiol oxidation is a probable outcome of cellular oxidative stress and is linked to degenerative disease progression. In addition, protein thiol redox reactions are increasingly identified as a mechanism to regulate protein structure and function. We assessed the effect of hypothiocyanous acid on the cytoskeletal protein tubulin. Total cysteine oxidation by hypothiocyanous and hypochlorous acids was monitored by labeling tubulin with 5-iodoacetamidofluorescein and by detecting higher molecular weight inter-chain tubulin disulfides by Western blot under nonreducing conditions. Hypothiocyanous acid induced nearly stoichiometric oxidation of tubulin cysteines (1.9mol cysteine/mol oxidant) and no methionine oxidation was observed. Because disulfide reducing agents restored all the polymerization activity that was lost due to oxidant treatment, we conclude that cysteine oxidation of tubulin inhibits microtubule polymerization. Hypothiocyanous acid oxidation of tubulin cysteines was markedly decreased in the presence of 4% glycerol, a component of the tubulin purification buffer. Due to its instability and buffer- and pH-dependent reactivity, hypothiocyanous acid studies require careful consideration of reaction conditions.

Thermofluor-based optimization strategy for the stabilization and crystallization of Campylobacter jejuni desulforubrerythrin

Desulforubrerythrin from Campylobacter jejuni has recently been biochemical and spectroscopically characterized. It is a member of the rubrerythrin family, and it is composed of three structural domains: the N-terminal desulforedoxin domain with a non-heme iron center, followed by a four-helix bundle domain harboring a binuclear iron center and finally a C-terminal rubredoxin domain. To date, this is the first example of a protein presenting this kind of structural domain organization, and therefore the determination of its crystal structure may unveil unexpected structural features. Several attempts were made in order to obtain protein crystals, but always without success. As part of our strategy the thermofluor method was used to increase protein stability and its propensity to crystallize. This approach has been recently used to optimize protein buffer formulation, thus yielding more stable and homogenous protein samples. Thermofluor has also been used to identify cofactors/ligands or small molecules that may help stabilize native protein states. A successful thermofluor approach was used to select a pH buffer condition that allowed the crystallization of Campylobacter jejuni desulforubrerythrin, by screening both buffer pH and salt concentration. A buffer formulation was obtained which increased the protein melting temperature by 7 °C relatively to the initial purification buffer. Desulforubrerythrin was seen to be stabilized by lower pH and high salt concentration, and was dialyzed into the new selected buffer, 100 mM MES pH 6.2, 500 mM NaCl. This stability study was complemented with a second thermofluor assay in which different additives were screened. A crystallization screening was carried out and protein crystals were rapidly obtained in one condition. Protein crystal optimization was done using the same additive screening. Interestingly, a correlation between the stability studies and crystallization experiments using the additive screening could be established. The work presented here shows an elegant example where thermofluor was shown to be a key biophysical method that allowed the identification of an improved buffer formulation and the applicability of this technique to increase the propensity of a protein to crystallize is discussed.

Biochemical Characterization of the Transcriptional Regulator BzdR from Azoarcus sp. CIB

The BzdR transcriptional regulator that controls the P(N) promoter responsible for the anaerobic catabolism of benzoate in Azoarcus sp. CIB constitutes the prototype of a new subfamily of transcriptional regulators. Here, we provide some insights about the functional-structural relationships of the BzdR protein. Analytical ultracentrifugation studies revealed that BzdR is homodimeric in solution. An electron microscopy three-dimensional reconstruction of the BzdR dimer has been obtained, and the predicted structures of the respective N- and C-terminal domains of each BzdR monomer could be fitted into such a reconstruction. Gel retardation and ultracentrifugation experiments have shown that the binding of BzdR to its cognate promoter is cooperative. Different biochemical approaches revealed that the effector molecule benzoyl-CoA induces conformational changes in BzdR without affecting its oligomeric state. The BzdR-dependent inhibition of the P(N) promoter and its activation in the presence of benzoyl-CoA have been established by in vitro transcription assays. The monomeric BzdR4 and BzdR5 mutant regulators revealed that dimerization of BzdR is essential for DNA binding. Remarkably, a BzdRΔL protein lacking the linker region connecting the N- and C-terminal domains of BzdR is also dimeric and behaves as a super-repressor of the P(N) promoter. These data suggest that the linker region of BzdR is not essential for protein dimerization, but rather it is required to transfer the conformational changes induced by the benzoyl-CoA to the DNA binding domain leading to the release of the repressor. A model of action of the BzdR regulator has been proposed.

Deciphering the Catalytic Domain of Colicin M, a Peptidoglycan Lipid II-degrading Enzyme

Colicin M inhibits Escherichia coli peptidoglycan synthesis through cleavage of its lipid-linked precursors. It has a compact structure, whereas other related toxins are organized in three independent domains, each devoted to a particular function: translocation through the outer membrane, receptor binding, and toxicity, from the N to the C termini, respectively. To establish whether colicin M displays such an organization despite its structural characteristics, protein dissection experiments were performed, which allowed us to delineate an independent toxicity domain encompassing exactly the C-terminal region conserved among colicin M-like proteins and covering about half of colicin M (residues 124-271). Surprisingly, the in vitro activity of the isolated domain was 45-fold higher than that of the full-length protein, suggesting a mechanism by which the toxicity of this domain is revealed following primary protein maturation. In vivo, the isolated toxicity domain appeared as toxic as the full-length protein under conditions where the reception and translocation steps were by-passed. Contrary to the full-length colicin M, the isolated domain did not require the presence of the periplasmic FkpA protein to be toxic under these conditions, demonstrating that FkpA is involved in the maturation process. Mutational analysis further identified five residues that are essential for cytotoxicity as well as in vitro lipid II-degrading activity: Asp-229, His-235, Asp-226, Tyr-228, and Arg-236. Most of these residues are surface-exposed and located relatively close to each other, hence suggesting they belong to the colicin M active site.

Structure of a Eukaryotic Nonribosomal Peptide Synthetase Adenylation Domain That Activates a Large Hydroxamate Amino Acid in Siderophore Biosynthesis

Nonribosomal peptide synthetases (NRPSs) are large, multidomain proteins that are involved in the biosynthesis of an array of secondary metabolites. We report the structure of the third adenylation domain from the siderophore-synthesizing NRPS, SidN, from the endophytic fungus Neotyphodium lolii. This is the first structure of a eukaryotic NRPS domain, and it reveals a large binding pocket required to accommodate the unusual amino acid substrate, N(delta)-cis-anhydromevalonyl-N(delta)-hydroxy-L-ornithine (cis-AMHO). The specific activation of cis-AMHO was confirmed biochemically, and an AMHO moiety was unambiguously identified as a component of the fungal siderophore using mass spectroscopy. The protein structure shows that the substrate binding pocket is defined by 17 amino acid residues, in contrast to both prokaryotic adenylation domains and to previous predictions based on modeling. Existing substrate prediction methods for NRPS adenylation domains fail for domains from eukaryotes due to the divergence of their signature sequences from those of prokaryotes. Thus, this new structure will provide a basis for improving prediction methods for eukaryotic NRPS enzymes that play important and diverse roles in the biology of fungi.

Extraction and Purification Method of Rice DNA from Rice Powder Containing Konjak Flour

Rice powder containing Konjak flour made with tuberous roots of Amorphophallus konjac is imported as a rice-processed product from China to Japan. An improved DNA purification method for the polymerase chain reaction (PCR) analysis of rice in such products is necessary, since Konjak flour constituents absorb the DNA purification buffer to form a gel, and cause problems in the subsequent purification steps. Here, we present a simple preparative system for isolation of the rice and a purification method of the rice DNA from the product. The purified DNA was confirmed to be a good template for both PCR and real-time PCR.

Mitochondrial DNA Damage Initiates a Cell Cycle Arrest by a Chk2-associated Mechanism in Mammalian Cells

Previous work from our laboratory has focused on mitochondrial DNA (mtDNA) repair and cellular viability. However, other events occur prior to the initiation of apoptosis in cells. Because of the importance of mtDNA in ATP production and of ATP in fuel cell cycle progression, we asked whether mtDNA damage was an upstream signal leading to cell cycle arrest. Using quantitative alkaline Southern blot technology, we found that exposure to menadione produced detectable mtDNA damage in HeLa cells that correlated with an S phase cell cycle arrest. To determine whether mtDNA damage was causatively linked to the observed cell cycle arrest, experiments were performed utilizing a MTS-hOGG1-Tat fusion protein to target the hOGG1 repair enzyme to mitochondria and enhance mtDNA repair. The results revealed that the transduction of MTS-hOGG1-Tat into HeLa cells alleviated the cell cycle block following an oxidative insult. Furthermore, mechanistic studies showed that Chk2 phosphorylation was enhanced following menadione exposure. Treatment of the HeLa cells with the hOGG1 fusion protein prior to menadione exposure resulted in an increase in the rate of Chk2 dephosphorylation. These results strongly support a direct link between mtDNA damage and cell cycle arrest.

Spectroscopic characterization of a (6-4) photolyase from the green alga Ostreococcus tauri

The cofactor content of OtCPF1, a (6-4) photolyase isolated from the green marine alga Ostreococcus tauri, was characterized by steady-state absorption and fluorescence spectroscopy. The heterologously expressed, GST-fused, purified protein (MW: 89 kDa) is non-covalently bound to flavin adenine dinucleotide (FAD), with a flavin to apoprotein molecular ratio of 64%. No light-harvesting chromophore was found in this protein. In freshly purified OtCPF1, FAD is present in three different redox states: the fully oxidized form (FAD ox, 82%), the neutral semiquinone (FADH , 14%) and the fully reduced anion (FADH −, 4%). Keeping the sample in the dark, at 5 °C, yields oxidation of FADH and FADH −, partial release of FAD to the solution and slow degradation of the protein. Upon steady-state blue-light irradiation of OtCPF1 at 450 nm, photoreduction processes leading to an accumulation of stable FADH and FADH − species are observed. We demonstrate that this accumulation is due to the presence of an external electron donor agent in the purification buffer. Composition changes observed under steady-state photoexcitation are interpreted in terms of photoinduced reductions of FAD ox and FADH states and competitive back reactions. Specific irradiation by red light at 620 nm shows both photoreduction of FADH to FADH − and irreversible oxidation of FADH to FAD ox. The photoinduced oxidation reaction is believed to be indirectly caused by the external donor agent present in the buffer. Photoexcitation is also shown to stabilize the binding of FAD to the protein. We suggest this effect to be due to slight changes in the protein conformation, possibly strengthening the hydrogen-bonding network surrounding FAD.