Affinity Folate Research Articles

Sensitive detection of folate receptor-positive circulating tumor cells based on intracellular uptake of the PbS nanoparticle cluster-loaded phospholipid micelles decorated with folic acid in combination with E-DNA sensor

Developing methods for the circulating tumor cells (CTCs) detection is important for noninvasive cancer diagnosis. A sensitive method for homogeneous electrochemical detection of CTCs was developed based on the affinity of folic acid (FA) for folate receptor (FR), which is overexpressed on the CTC surface. The PbS nanoparticle cluster-loaded phospholipid micelles decorated with FA were prepared and used as a nanoprobe. The specific binding of FA to FR-positive CTCs leads to the uptake of the nanoprobes into the CTCs. Cellular imaging assay showed the excellent biocompatibility and selectivity of nanoprobes. After separation of intracellular nanoprobes and the residual free nanoprobes in the solution by centrifugation, nanoprobes in the cell were dissolved using acid. The amount of the target cells was quantified by the electrochemical detection of Pd2+ using the electrochemical DNA (E-DNA) sensor, which was fabricated based on the self-assembly of probe DNA on the flowerlike gold nanostructures modified the screen-printed carbon electrode (SPCE). Because of the large quantities of detectable Pd2+ contained in the nanoprobes, the signal amplification effect was achieved. The developed electrochemical assay allows the detection of ∼5 cells from 1 mL of sample. Studies with both healthy individuals and cancer patient blood samples demonstrate that the assay is sensitive and FR-positive CTCs specific.

Platinum-Based Nanocomposite Pt@BSA as an Efficient Electrochemical Biosensing Interface for Rapid and Ultrasensitive Determination of Folate Receptor-Positive Tumor Cells.

Developing a cytosensing strategy based on electrochemical approaches has attracted wide interest due to the low cost, rapid response, and simple instrumentation. In this work, an electrochemical cytosensor employing the Pt@BSA nanocomposite as the biosensing substrate along with the covalent cross-linking of targeting molecules folic acid (FA) was constructed for highly sensitive determination of folate receptor (FR)-positive tumor cells. The prepared Pt@BSA nanocomposite revealed excellent biocompatibility for cell adhesion and proliferation, which was confirmed by cell viability evaluation using thiazolyl blue tetrazolium bromide (MTT) colorimetric methods. Due to the satisfactory electrical conductivity originating from Pt@BSA and the high binding affinity of FA to FR on the cell surface, an ultrasensitive and specific cytosensing device was designed for rapid and quantitative determination of HeLa cells (a model system) by differential pulse voltammetry (DPV) tests. This proposed cytosensor resulted in a wide HeLa cell determination range of 2.8 × 101-2.8 × 106 cells mL-1 with a low DPV detection limit of 9 cells mL-1. The developed cytosensing approach exhibited highly specific recognition of FR-positive tumor cells, excellent inter-assay reproducibility with a relative standard deviation (RSD) of 4.7%, acceptable intra-assay precision, and favorable storage stability, expanding the application of electrochemical measurement technology in the biomedical field of early detection and diagnosis of cancers.

Correlation of folate receptor alpha expression with clinicopathological parameters and outcome in triple negative breast cancer

Folate receptor alpha (FRα) is a membrane-bound protein with a high affinity for folate, which is necessary for the biosynthesis of amino acids and nucleotide bases. It has been shown to be a potential prognostic and therapeutic target, primarily in lung and ovarian cancer, as well as in breast cancer. The aim of this study was to examine FRα expression in a cohort of patients with triple negative breast cancer (TNBC), in correlation with clinicopathological parameters and prognostic factors. By using polyclonal FRα antibody on archival paraffin blocks immunohistochemistry was performed. To evaluate the expression of FRα, H-score was used, which marks both the proportion of stained cells and the intensity of staining. Statistical analysis correlating FRα expression with clinicopathologic parameters and clinical outcome were performed. FRα was expressed in most of the patients (85%). Significant correlation of expression and histologic grade (Mann Whitney U test, P = 0,03) and type of tumor (P = 0,02), was found. It was noticed that with higher Ki-67 proliferation index values, H-score has lower values (r = −0,284, P = 0,006). Multivariant regression analysis (Cox regression, Stepwise method) showed H-score as a significant predictor for the risk of disease recurrence (OR = 1,005, P = 0,04). No correlation between FRα expression and overall survival (OS) and disease-free survival (DFS) was found.In conclusion, FRα is highly expressed in TNBC, and, given the correlation with clinicopathological parameters, subpopulation of patients could be identified that could be potential targets for new therapeutic perspectives in the treatment of this breast cancer subtype.

Impaired folate binding of serine hydroxymethyltransferase 8 from soybean underlies resistance to the soybean cyst nematode

Management of the agricultural pathogen soybean cyst nematode (SCN) relies on the use of SCN-resistant soybean cultivars, a strategy that has been failing in recent years. An underutilized source of resistance in the soybean genotype Peking is linked to two polymorphisms in serine hydroxy-methyltransferase 8 (SHMT8). SHMT is a pyridoxal 5'-phosphate-dependent enzyme that converts l-serine and (6S)-tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. Here, we determined five crystal structures of the 1884-residue SHMT8 tetramers from the SCN-susceptible cultivar (cv.) Essex and the SCN-resistant cv. Forrest (whose resistance is derived from the SHMT8 polymorphisms in Peking); the crystal structures were determined in complex with various ligands at 1.4-2.35 Å resolutions. We find that the two Forrest-specific polymorphic substitutions (P130R and N358Y) impact the mobility of a loop near the entrance of the (6S)-tetrahydrofolate-binding site. Ligand-binding and kinetic studies indicate severely reduced affinity for folate and dramatically impaired enzyme activity in Forrest SHMT8. These findings imply widespread effects on folate metabolism in soybean cv. Forrest that have implications for combating the widespread increase in virulent SCN.

Enhancement of Binding Affinity of Folate to Its Receptor by Peptide Conjugation.

(1) Background: The folate receptor (FR) is a target for cancer treatment and detection. Expression of the FR is restricted in normal cells but overexpressed in many types of tumors. Folate was conjugated with peptides for enhancing binding affinity to the FR. (2) Materials and Methods: For conjugation, folate was coupled with propargyl or dibenzocyclooctyne, and 4-azidophenylalanine was introduced in peptides for “click” reactions. We measured binding kinetics including the rate constants of association (ka) and dissociation (kd) of folate-peptide conjugates with purified FR by biolayer interferometry. After optimization of the conditions for the click reaction, we successfully conjugated folate with designed peptides. (3) Results: The binding affinity, indicated by the equilibrium dissociation constant (KD), of folate toward the FR was enhanced by peptide conjugation. The enhanced FR binding affinity by peptide conjugation is a result of an increase in the number of interaction sites. (4) Conclusion: Such peptide-ligand conjugates will be important in the design of ligands with higher affinity. These high affinity ligands can be useful for targeted drug delivery system.

Cytotoxicity of 5-ALA-conjugated bismuth oxidenanoparticles on KB cell line

Introduction: In recent years, bismuth-based nanomaterials have been widely used in medical researches as imaging, drug delivery and x-ray radiosensitizing agents. Due to their anti-microbial effects against Helicobacter pylori (HP), bismuth colloidal compounds are used to treat various types of diseases such as chronic gastritis. Despite their advantages, bismuth-based compounds have shown many toxic effects in humans. There are few studies on cytotoxicity effects of bismuth (ΙΙΙ) oxide (Bi2O3) nanoparticles (NPs). Based on the natural high affinity of folate for the folate receptor protein (FR), which is commonly expressed on the surface of many human cancers, folate-drug conjugates bind tightly to the FR and trigger cellular uptake via endocytosis. Therefore, we aimed to investigate the cytotoxicity effects of 5-ALA-conjugated Bi2O3 NPs, 5-ALA-folate-conjugated Bi2O3 NPs and free 5-ALA on oral squamous carcinoma cell line (KB cells) for the first time. 5-ALA is used in photo dynamic detection and photodynamic surgery of cancer. Being a precursor of a photosensitizer, 5-ALA is also used as an add on agent for photodynamic therapy. Materials and Methods: Bi2O3 NPs were synthesized and conjugated with folate and 5- aminolevulinic acid (5-ALA). KB cells were cultured monolayer and incubated with 10, 20, 50, 100 and 200 μg/ml concentrations of free 5-ALA, 5-ALA-folate-conjugated Bi2O3 NPs and 5-ALA-conjugated Bi2O3 NPs for 24 hours. The survival of the cells obtained after 24 h of incubation with the materials using MTT assay. Results: Bi2O3 NPs were synthesized (~20 nm) and conjugated to folate and (5-ALA). Results indicated that the significant cellular death were noted at the concentrations more than of 50 μg/ml for all compounds that cell viability reduced to 37%, 49% and 63% in groups receiving 5-ALA-folate-conjugated NPs, 5-ALA-conjugated Bi2O3 NPs and free 5-ALA, respectively. The cytotoxicity of the materials on KB cells also increased with increasing concentrations of synthesized NPs. Conclusion:5-ALA-folate-conjugated NPs, 5-ALA-conjugated Bi2O3 NPs and free 5-ALA were easily taken up by the cells and showed cytotoxic effects at high concentrations. Folic acid conjugation facilitates selective targeting of the KB cells with the overexpression of the folate receptors and improves internalization of the drugs through receptor-mediated endocytosis

High prevalence of serum folate receptor autoantibodies in children with autism spectrum disorders

Background: Supplementation of folic acid by pregnant mothers is thought to lower the risk of autism spectrum disorders (ASDs) in the offspring. Folic acid is taken up by cells via receptors with high affinity for folate and reduced folic acid derivatives. However, this is blocked by the presence of folate receptor autoantibodies (FRAA). Cerebral FRAA have been detected with high frequency in children with ASDs, suggesting the existence of a link between folic acid uptake and disease aetiology.Methods: We investigated the frequency of FRAA in serum samples from 40 children with ASDs and 42 gender- and age-matched children with typical development (TD). Serum FRAA concentrations were measured by enzyme-linked immunosorbent assay.Results: We found a significant difference in the frequency of serum FRAA in the two study cohorts. Serum FRAA were present in 77.5% (31/40) of children with ASDs compared with 54.8% (23/42) of TD children (p = 0.03746, Fischer’s exact test). Thus, serum FRAA are more prevalent in children with ASDs than in TD children.Conclusions: Our data suggest that children with ASDs may have defects in folic acid absorption that play a role in the onset of ASDs.

Quantum dots-Ru complex assembling dyads for cancer cell detection and cellular imaging based on hybridization chain reaction

An easy and fluorescent strategy is developed for the detection of cancer cells and cellular imaging. This assay relies on the quenching of Ru complex toward quantum dots (QDs) and the amplification of hybridization chain reaction (HCR). In the presence of target cancer cells, folate-modified single-stranded DNA (F-DNA) links on the cell membranes through high affinity of folate and receptors. Hybridization reaction between pre-prepared HCR products and F-DNA immobilizes HCR products on the surfaces of target cells, resulting in large amount of Ru complex into HCR products and strong red fluorescence on the surfaces of target cells. Meanwhile, QDs-Ru complex assembling dyads in supernate are reduced, leading to the fluorescence restoration of QDs. Taking human cells lines HeLa as a model analyst, the linear response for HeLa cells in a concentration range from 100 to 105 cells mL−1 is obtained with a detection limit of 100 cells mL−1. The specificity of this assay for the detection of cancer cells is demonstrated against several cell competitors. Additionally, this assay is successfully applied to inhibitor screening for cancer cells in combination with cellular imaging, demonstrating the university of this design.

Abstract A58: Bispecific anti-CD3-folate for the treatment of ovarian cancer.

Abstract Folic acid receptor 1 (FOLR1, FRα) is highly expressed in 80% of ovarian tumors and has a low nanomolar affinity for folic acid. Although ovarian tumors are intrinsically immunogenic, immune reactions to them are inhibited by a suppressive environment. We are developing a bispecific anti-CD3-folate to redirect T cells to engage with and kill tumor cells. Folic acid was site-specifically conjugated to a FAb fragment of an anti-CD3 antibody through an engineered linkable non-natural amino acid. Anti-CD3-folate bispecific FAb is able to engage T cells and exert cytotoxicity in a variety of tumor cell lines including ovarian; OV-90, OVCAR3, and breast cancer; SKOV-3. Cytotoxicity assays generated EC50 in the femtomolar range. This cytotoxicity could be modulated by varying folic acid concentrations in the assay medium. T-cell activation was confirmed by appearance of activation markers on the cell surface and secretion of pro-inflammatory cytokines after treatment of tumor cells in combination with human PBMC's. T cell activation and subsequent cytokine release is dependent on simultaneous binding of tumor and T cell to anti-CD3-Folate. In a xenograft mouse model of ovarian cancer, anti-CD3-folate significantly inhibited tumor growth, and increased life span (ILS)of 30% and 50% for SKOV-3 and OV-90, respectively when compared to control treated animals. These data suggest that anti-CD3-folate bispecifc FAb- mediated T cell engagement could be a powerful tool for immune-ablation of ovarian cancer. Citation Format: Jinming Xia, Kristine deDios, Evelyn Rodrigo, Robin Humphreys, Alan Wahl, Marco Gymnopoulos. Bispecific anti-CD3-folate for the treatment of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A58.

Comparative Evaluation of Two Different Novel Formulations of Quercetin Against Non Melanoma Skin Cancer in Human Subjects

Nonmelanoma skin cancer (NMSC) is the steadily increasing cancer in the India. Various studies have put forward strong evidences of NMSC incidence due to UV radiation exposure. Keratinocyte macrophage cells express macrophage which represents both type of receptor viz. folate binding receptor having affinity for folic acid and mannose-binding receptor which are having affinity towards mannose. This study was designed to identify the correlation parameter in evaluation of in vivo doses and epidermal changes following UVB irradiation using noninvasive techniques. We in our study have developed two different formulations embedded with endogenous ligands conjugated nanoparticles of same drug (quercetin) and have analyzed its effect on human volunteers against the release of MIF and MED analysis for which prior permission from Human Ethical Committee has been obtained accordingly. The result of the study showed that skin biopsies sections of human subjects showed that there was a dose dependent effect on human skin in response to the UV radiations. The pattern was nearly same for NL-4C and NL-5C for MED analysis. While for MIF release study, for first 12 hours the MIF level ranged from 12.34 ± 9.90 ng/ml to 79.4 ± 23.65 ng/ml which was the result of acute exposure of UV radiations. For next 24-48 hours, the MIF levels was found to be increased from 22.43 ± 12.56 ng/ml to 102.34 ± 20.4 ng/ml which shows that the prepared formulations were significant in inhibiting the release of MIF.

Folate-decorated poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) nanoparticles for targeting delivery: optimization and in vivo antitumor activity

Context: Doxorubicin (DOX)-loaded folate-targeted poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HB-HO)] nanoparticles [DOX/FA-PEG-P(HB-HO) NPs] have potential application in clinical treatments for cervical cancer due to specific affinity of folate and folate receptor in HeLa cells.Objective: The aim of this study was to develop an optimized formulation for DOX/FA-PEG-P(HB-HO) NPs, and investigate the targeting and efficacies of the nanoparticles.Materials and methods: DOX/FA-PEG-P(HB-HO) NPs were prepared by W1/O/W2 solvent extraction/evaporation method, and an orthogonal experimental design [L9 (34)] was applied to establish the optimum conditions. The physico–chemical characteristics, microscopic observation and in vivo antitumor study of the nanoparticles were evaluated.Results: The optimum formulation was obtained with DOX 10% (w/v), FA-PEG-P(HB-HO) 6.5% (w/v), PVA 3%(w/v) and oil phase/internal water phase volume ratio of 3/1. The size distribution, drug loading and encapsulation efficiency of the optimized nanoparticles were 150–350 nm, 29.6 ± 2.9% and 83.5 ± 5.7%, respectively. In vitro release study demonstrated that 80% of the drug could release from the nanoparticles within 11 days. Furthermore, in vitro microscopic observation and in vivo antitumor study showed that DOX/FA-PEG-P(HB-HO) NPs could inhibit HeLa cells effectively, and the tumor inhibition rate (TIR) in vivo was 76.91%.Discussion and conclusions: DOX/FA-PEG-P(HB-HO) NPs have been successfully developed and optimized. In vitro drug release study suggested a sustained release profile. Moreover, DOX/FA-PEG-P(HB-HO) NPs could effectively inhibit HeLa cells with satisfying targeting, and reduce side effects and toxicity to normal tissues. DOX/FA-PEG-P(HB-HO) NPs were superior in terms of inhibiting HeLa tumor over non-targeted formulations therapy.

Self-Assembly of Folate onto Polyethyleneimine-Coated CdS/ZnS Quantum Dots for Targeted Turn-On Fluorescence Imaging of Folate Receptor Overexpressed Cancer Cells

Folate receptor (FR) can be overexpressed by a number of epithelial-derived tumors, but minimally expressed in normal tissues. As folic acid (FA) is a high-affinity ligand to FR, and not produced endogenously, development of FA-conjugated probes for targeted imaging FR overexpressed cancer cells is of significance for assessing cancer therapeutics and for better understanding the expression profile of FR in cancer. Here we report a novel turn-on fluorescence probe for imaging FR overexpressed cancer cells. The probe was easily fabricated via electrostatic self-assembly of FA and polyethyleneimine-coated CdS/ZnS quantum dots (PEI-CdS/ZnS QDs). The primary fluorescence of PEI-CdS/ZnS QDs turned off first upon the electrostatic adsorption of FA onto PEI-CdS/ZnS QDs based on electron transfer to produce negligible fluorescence background. The presence of FR expressed on the surface of cancer cells then made FA desorb from PEI-CdS/ZnS QDs due to specific and high affinity of FA to FR. As a result, the primary fluorescence of PEI-CdS/ZnS QDs adhering to the cells turned on due to the inhibition of electron transfer. The most important merits of the developed probe are its simplicity and the effective avoidance of the false positive results due to the simple electrostatic self-assembly of FA onto the surface of PEI-CdS/ZnS QDs and the involved fluorescence "off-on" mechanism. The probe was demonstrated to be sensitive and selective for targeted imaging of FR overexpressed cancer cells in turn-on mode.

Highly sensitive and selective detection of cancer cell with a label-free electrochemical cytosensor

Electrochemical methods have attracted considerable attention for developing cytosensing system since they can decrease the cost and time requirement for cell detection with simple instrumentation. Herein, a label-free electrochemical cytosensor with surface-confined ferrocene as signal indicator was developed for highly sensitive and selective detection of cancer cell. With layer-by-layer (LBL) self-assembly technique, positively charged poly(ethylene imine) functionalized with ferrocene (Fc-PEI) and negatively charged single-wall carbon nanotubes (SWNTs) were alternately assembled on 3-mercaptopropionic acid (MPA) modified gold substrate. Folic acid (FA) was covalently bonded onto SWNTs surface to specifically recognize cancer cells according to the high affinity of FA for folate receptor (FR) on cellular surface. The developed cytosensor presented high sensitivity and selectivity for the detection of human cervical carcinoma (HeLa) cell. By using fast-response differential pulse voltammetry (DPV) method, a wide detection range from 10 to 106cells/mL with a detection limit as low as 10cells/mL was reached even in the presence of a large amount of non-cancerous cells.

Abstract IA6-1: The evolution of folate analogs: Drugs targeted to folate transporters, drugs targeted to novel enzyme sites, effects on signaling pathways

Abstract IA6-1: The evolution of folate analogs: Drugs targeted to folate transporters, drugs targeted to novel enzyme sites, effects on signaling pathways

Folic Acid-Anchored PEGgylated Phospholipid Bioconjugate and Its Application in a Liposomal Immunodiagnostic Assay for Folic Acid

A folic acid-anchored, poly(ethylene glycol)-linked (PEGgylated) phospholipid and an immunoaffinity chromatographic column were prepared and employed to develop a liposomal immunodiagnostic assay for the direct determination of folic acid (FA) in this study. Distearoylphosphatidylethanolamine-poly(ethylene glycol)2000-folic acid (DSPE-PEG2000-FA) was synthesized through carbodiimide-mediated coupling of FA and DSPE-PEG2000-amine and characterized using thin layer chromatography, 1H nuclear magnetic resonance spectroscopy, and electrospray ionization-mass spectrometry. Liposomal biolabels were constructed using the synthesized DSPE-PEG2000-FA in conjunction with other phospholipids. A stationary phase having affinity for FA was prepared by covalently linking purified anti-FA monoclonal antibodies onto N-hydroxysuccinimide-activated Sepharose beads, which were subsequently packed into a 1.9 cm diameter polypropylene column. The calibration curve for FA had a linear range from 10(-8) to 10(-4) M. The limit of detection was 6.8 ng (equivalent to 500 microL of 3.1 x 10(-8) M FA). The elution buffer (35% methanol in Tris buffered saline containing 0.1% Tween 20) also served as the regeneration buffer, which allowed the same column to be used for up to 50 times without any observable loss of reactivity. The immunoaffinity chromatographic column was reusable and capable of concentrating analytes from sample solution; in conjunction with folic acid-sensitized liposomal biolabels, however, they hold great potential as sensitive immunoaffinity assays for the determination for FA. To confirm the feasibility of using this system in the analysis of real samples, the folic acid contents of three over-the-counter vitamin supplements were tested. The recoveries of folic acid of 90-112% for these three samples were obtained, suggesting contents that were consistent with the information obtained from their nutritional facts panels.

Molecular and Functional Characteristics of Proton-Coupled Folate Transporter

Proton-coupled folate transporter (PCFT) has recently been identified as the molecular entity of the carrier-mediated intestinal folate transport system. PCFT has been demonstrated to be most abundantly expressed in the upper small intestine, localizing at the brush border membrane of epithelial cells, transport folate and its analogs more efficiently at lower (acidic) pH by a H+-coupled cotransport mechanism, and have a high affinity for folate with a Michaelis constant (Km) of a few μM at pH5.5 and somewhat lower affinities for reduced folates and methotrexate (MTX). A loss of PCFT function due to a homozygous mutation in its gene has been indicated to be responsible for hereditary folate malabsorption. Thus, PCFT has all the characteristics of the brush border H+-coupled cotransporter for folate and analogs, which has long been suggested to be present in the intestine. Furthermore, sulfasalazine was found to be a potent inhibitor of PCFT, suggesting that it is a risk factor that would cause malabsorption of folate and also MTX, when coadministered in the treatment of rheumatoid arthritis. Understanding the molecular and functional characteristics of PCFT should be important and helpful in exploring therapeutic strategies for folate malabsorption and in optimizing therapies using antifolate drugs.

5-Formyltetrahydrofolate Is an Inhibitory but Well Tolerated Metabolite in Arabidopsis Leaves

5-Formyltetrahydrofolate (5-CHO-THF) is formed via a second catalytic activity of serine hydroxymethyltransferase (SHMT) and strongly inhibits SHMT and other folate-dependent enzymes in vitro. The only enzyme known to metabolize 5-CHO-THF is 5-CHO-THF cycloligase (5-FCL), which catalyzes its conversion to 5,10-methenyltetrahydrofolate. Because 5-FCL is mitochondrial in plants and mitochondrial SHMT is central to photorespiration, we examined the impact of an insertional mutation in the Arabidopsis 5-FCL gene (At5g13050) under photorespiratory (30 and 370 micromol of CO2 mol(-1)) and non-photorespiratory (3200 micromol of CO2 mol(-1)) conditions. The mutation had only mild visible effects at 370 micromol of CO2 mol(-1), reducing growth rate by approximately 20% and delaying flowering by 1 week. However, the mutation doubled leaf 5-CHO-THF level under all conditions and, under photorespiratory conditions, quadrupled the pool of 10-formyl-/5,10-methenyltetrahydrofolates (which could not be distinguished analytically). At 370 micromol of CO2 mol(-1), the mitochondrial 5-CHO-THF pool was 8-fold larger in the mutant and contained most of the 5-CHO-THF in the leaf. In contrast, the buildup of 10-formyl-/5,10-methenyltetrahydrofolates was extramitochondrial. In photorespiratory conditions, leaf glycine levels were up to 46-fold higher in the mutant than in the wild type. Furthermore, when leaves were supplied with 5-CHO-THF, glycine accumulated in both wild type and mutant. These data establish that 5-CHO-THF can inhibit SHMT in vivo and thereby influence glycine pool size. However, the near-normal growth of the mutant shows that even exceptionally high 5-CHO-THF levels do not much affect fluxes through SHMT or any other folate-dependent reaction, i.e. that 5-CHO-THF is well tolerated in plants.

Quantitative description of the interaction between folate and the folate-binding protein from cow's milk.

A detailed study has been carried out on the dependence of folate binding on the concentration of FBP (folate-binding protein) at pH 5.0, conditions selected to prevent complications arising from the pre-existing self-association of the acceptor. In contrast with the mandatory requirement that reversible interaction of ligand with a single acceptor site should exhibit a unique, rectangular hyperbolic binding curve, results obtained by ultrafiltration for the FBP-folate system required description in terms of (i) a sigmoidal relationship between concentrations of bound and free folate and (ii) an inverse dependence of affinity on FBP concentration. These findings have been attributed to the difficulties in determining the free ligand concentration in the FBP-folate mixtures for which reaction is essentially stoichiometric. This explanation also accounts for the similar published behaviour of the FBP-folate system at neutral pH, which had been attributed erroneously to acceptor self-association, a phenomenon incompatible with the experimental findings because of its prediction of a greater affinity for folate with increasing FBP concentration.

Binding of radiolabeled folate and 5-methyltetrahydrofolate to cow's milk folate binding protein at pH 7.4 and 5.0. Relationship to concentration and polymerization equilibrium of the purified protein.

Binding of folate (pteroylglutamate) and 5-methyltetrahydrofolate, the major endogenous form of folate, to folate binding protein purified from cow's milk was studied at 7 degrees C to avoid degradation of 5-methyltetrahydrofolate. Both folates dissociate rapidly from the protein at pH 3.5, but extremely slowly at pH 7.4, most likely due to drastic changes in protein conformation occurring after folate binding. Dissociation of 5-methyltetrahydrofolate showed no increase at 37 degrees C suggesting that protein-bound-5-methyltetrahydrofolate is protected against degradation. Binding displayed two characteristics, positive cooperativity and a binding affinity that increased with decreasing concentrations of the protein. The binding affinity of folate was somewhat greater than that of 5-methyl tetrahydrofolate, in particular at pH 5.0. Ligand-bound protein exhibited concentration-dependent polymerization (8-mers formed at 13 microM) at pH 7.4. At pH 5.0, only folate-bound forms showed noticeable polymerization. The fact that folate at pH 5.0 surpasses 5-methyltetrahydrofolate both with regard to binding affinity and ability to induce polymerization suggests that ligand binding is associated with conformational changes of the protein which favor polymerization.

Target analogue imprinted polymers with affinity for folic acid and related compounds.

Two approaches to synthesize molecularly imprinted polymers with affinity for folic acid and other substituted pteridines have been compared. In the first approach, the folic acid analogue methotrexate was used as template and functional monomers capable of generating selective binding sites were searched in a miniaturized screening system based on binding assessment in the batch mode. Highest selectivity was seen using 2-vinylpyridine as functional monomer, which was confirmed in the chromatographic mode for a batch synthesized on a gram scale. However, the retentivity and selectivity of this phase were insufficient for anticipated applications. In a second approach, using methacrylic acid as the functional monomer, organic soluble inhibitors for the enzyme dihydrofolate reductase were used to develop sites complementary toward the pteridine substructure. This resulted in materials showing enhanced selectivity for substituted pteridines when evaluated by HPLC. Thus, methotrexate and leucovorine were selectively retained in mobile phases of either low or high aqueous content, thus showing the typical bimodal retention behavior of previously reported MIPs. In organic mobile-phase systems, the inhibitor used as template had an influence on the retentivity and selectivity of the MIP. The polymer imprinted with trimethoprim retained all folic acid analogues strongly and showed the highest selectivity among the MIPs in an organic mobile-phase system. This was supported by Scatchard analysis resulting in biphasic plots and a quantitative yield of high-energy binding sites. All templates were shown to associate strongly with MAA in CDCl(3), the strength of association correlating roughly with the template basicity and the selectivity observed in chromatography. Nonparallel complexation-induced shifts indicated formation of 1:2 template monomer complexes at concentrations corresponding to those of the prepolymerization solutions.