Binding affinities of folic acid and related pterins with biological macromolecules under physiological conditions
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Michael Soniat
Abstract
Folic acid and pterin derivatives are important heterocyclic compounds found in a variety of biological systems and have been shown to be photochemically active. Understanding the amount of binding that various pterins have with biological macromolecules under physiological conditions is important in predicting what specific biomolecules will bind with pterins and may, therefore, result in photochemical damage from charge-transfer reactions. The relative binding of folic acid, or pteroyl-L-glutamic acid (PteGlu), 6-methylpterin (Mep), 6-hydroxymethylpterin (Hmp), 6-formylpterin (Fop), and 6-carboxypterin (Cap) with bovine serum albumin (BSA), electrically neutral lipid (ENL), polyguanylic acid (Poly G), polycytidylic acid (Poly C), polyadenylic acid (Poly A), polythymidylic acid (Poly T), Micrococcus luteus DNA (72% GC), Escherichia coli DNA (50% GC), calf thymus DNA (42% GC), and Clostridium perfrigens DNA (27% GC) in neutral phosphate buffer were studied. Our results indicate that PteGlu demonstrated strong binding to neutral lipids, while the other pterins showed minimal binding, and BSA had a significant binding to PteGlu, Cap, and especially Fop. Our results also reveal a high affinity for DNA by PteGlu, which suggests that a relatively high percentage of folic acid is bound to DNA before photochemistry occurs.
Introduction
Folic acid and pterin derivatives are important heterocyclic compounds found in a variety of biological systems. In recent years, an increasing amount of attention has focused on the photochemistry of folic acid, or pteroyl-L-glutamic acid (PteGlu), and the resulting pterin decomposition products [1–7]. Lorente and Thomas reported their experimental study on folic acid and pterins in aqueous solution assigning the absorption and emission spectra and proposed the plausible decay mechanism of excited folic acid [8, 9]. Pterins, chiefly 6-carboxy pterin (Cap), have been shown to undergo photochemistry with dGMP and to photosensitize DNA damage by both type I (electron transfer) and type II (singlet oxygen) mechanisms [3, 4, 10–14]. The sensitization mechanism has been found to be dependent on pH, the availability of oxygen, and the immediate availability of appropriate electron donors [6, 10, 11, 15, 16]. If PteGlu or pterins resulting from irradiation bind strongly to various biological macromolecules, the probability of photooxidative damage at these locations will be increased owing to the increase in the local concentration of the photosensitizer. Petroselli and coworkers have shown that the fluorescence of various pterins is quenched by deoxynucleotides by both static and dynamic quenching mechanisms [17]. When a photosensitizer is bound to biological macromolecules, the resulting photochemistry should favor the shorter-lived singlet states, which should result in charge transfer (type-I oxidation) reactions specifically with the bound biomolecule. In comparison, the longer-lived triplet states, which result in type-II oxidation, may result in less specific damage. The fact that free pterins fluoresce strongly compared to the pterin portion in folic acid has been attributed to the intramolecular electron transfer reaction after photoexcitation [18]. The resulting folic acid charge-transfer species may lead to an additional electron transfer with DNA bases such as guanosine and result in oxidative DNA damage such as 8-oxo-guanosine [15]. Conversely, Vorobey and coworkers reported in 2006 that human serum albumin (HSA) was found to have the effect of actually decreasing the rate of folic acid photodegradation into Cap, thus, slowing the production of a potential charge-transfer acceptor [19].
These findings have led researchers to investigate PteGlu and other pterins as potential photosensitizers for pathogen reduction of blood-borne pathogens (BBP) in donated blood [20, 21]. The photochemistry of endogenous folic acid and pterins is also under investigation for people with vitiligo as a possible source of DNA damage as folic acid photoproducts Cap and 6-formylpterin (Fop) have been detected in depigmented regions of the skin [3, 11, 13, 22]. Pterins exist as weak acids with the pKa values ranging between 7.3 and 8.3, depending on the substituent found at the 6-position [5, 6, 23, 24]. Therefore, under physiological conditions, both acid and base forms of pterins will exist. In an attempt to understand the chemical behavior of both the acid (neutral) and base (anionic) forms, many photochemical and photophysical experiments have been reported under either acidic (pH<6) or basic (pH>8) conditions. Although this will help to simplify the chemical systems under investigation, it does not accurately represent the conditions found under physiological conditions. Although the behavior of these pterins is greatly complicated by the presence of both the acid and base forms in solution (i.e., Fop exists as a 1:1 ratio), these are the conditions found in the biological areas of interest. Understanding the amount of binding that various pterins have with biological macromolecules under physiological conditions is important in predicting what specific biomolecules will bind with pterins and may, therefore, result in charge-transfer photochemical damage. These biological macromolecules include DNA, lipids, and various proteins, among others. Understanding the binding of pterins to these molecules, and therefore where they may aggregate, may help scientists to understand and predict possible photochemically induced damage resulting from pterin irradiation.
Therefore, we have studied the relative binding of folic acid (PteGlu), 6-methylpterin (Mep), 6-hydroxymethylpterin (Hmp), 6-formylpterin (Fop), and 6-carboxypterin (Cap) with bovine serum albumin (BSA), the electrically neutral lipid L-α-phosphatidylcholine serine (ENL), polyguanylic acid (Poly G), polycytidylic acid (Poly C), polyadenylic acid (Poly A), polythymidylic acid (Poly T), Micrococcus luteus DNA (72% GC), Escherichia coli DNA (50% GC), calf thymus DNA (42% GC), and Clostridium perfrigens DNA (27% GC) in neutral phosphate-buffered saline (PBS) (Figure 1).
Chemical structures of folic acid and pterins.
Materials and methods
Folic acid dihydrate, 6-methylpterin, 6-hydroxymethylpterin, 6-formylpterin, 6-carboxypterin, calf thymus DNA type XV, polyguanylic acid, polycytidylic acid, polyadenylic acid, polythymidylic acid, M. luteus DNA, E. coli DNA, C. perfrigens DNA, and bovine albumin fraction V powder were purchased from Sigma-Aldrich (St. Louis, MO, USA). L-α-phosphatidylcholine serine lipids were purchased from Avanti Polar Lipids (Alabaster, AL, USA). All chemicals were used as received without further purification. Millipore Amicon Ultra-4 Centrifugal Filter Devices (5000 NMWL, Darmstadt, Germany) were purchased from Fisher Scientific.
PBS was prepared by dissolving 8.5 g NaCl, 0.2 g KH2PO4, and 2.9 g Na2HPO4 in 1000 mL of distilled water. After all solids had dissolved, pH was measured with a calibrated pH meter and pH was adjusted to 7.3±0.1 using 1 M NaOH.
The albumin solution was prepared by dissolving 3.75 g of bovine serum albumin in PBS buffer to achieve a total volume of 50 mL, resulting in a 1.09×10–3 M solution [20]. The procedure used for the lipid solution has proven to produce large vesicle structures sufficiently large to encapsulate large solutes [25]. The solution was prepared by dissolving 210 mg of phosphatidycholine in 8.5 mL of chloroform. The chloroform was then evaporated under vacuum, and the thin film deposited on the inside of the round bottom flask was dissolved in 25 mL of PBS. The solution was allowed to stand for 1 h and vortexed gently for 1 min, and an additional 25 mL PBS was added.
All DNA and polynucleotide stock solutions were prepared to produce a concentration of 1.0×10–3 M in nucleotides by measuring the absorbance at 260 nm using the distinct molar extinction coefficients: Poly A 15,400 M–1 cm–1, Poly C 7400 M–1 cm–1, Poly G 11,500 M–1 cm–1, Poly T 8700 M–1 cm–1, Calf Thymus 6600 M–1 cm–1, M. luteus DNA 6900 M–1 cm–1, C. perfringens DNA 6300 M–1 cm–1, and E. coli DNA 6400 M–1 cm–1 [26].
All stock pterin solutions contained 30 μM pterin and were prepared in PBS, which required gentle heating and sonication to achieve dissolution. The concentrations of the pterin solutions were confirmed using the respective extinction coefficients determined in our laboratory by examination of linear Beer’s law plots in neutral PBS solvent. The experimental extinction coefficients at 350 nm for folic acid and the pterins in this study were PteGlu 7190 cm–1 M–1, Mep 7400 cm–1 M–1, Hmp 7380 cm–1 M–1, Fop 7510 cm–1 M–1, and Cap 6850 cm–1 M–1 [The absorbance of each 30-μM pterin solution was measured at 350 nm and the concentrations verified prior to each binding experiment (A350 nm=A1)].
Binding procedure
Millipore Amicon Ultra-4 Centrifugal Filter Devices containing an Ultracel-regenerated cellulose semi-permeable membrane with a molecular weight cutoff of 5000 were used for all binding studies. All solutions were prepared and experiments performed in the absence of light and at room temperature. Control experiments showed that no macromolecules used in this study passed through the filter membrane. Likewise, control experiments showed that all pterins used in the study passed through the filter membrane without binding to the membrane or filter device itself. Equal volumes (1000 μL each) of pterin and macromolecule stock solutions were combined, and the resulting solution was mixed on an orbit mixer for 24 h at room temperature to reach equilibrium. After 24 h, the solution was centrifuged through the filter membrane for 1 h at 3500×g. The UV-Vis absorbance of the filtrate containing unbound sensitizer was measured, and the concentration was determined at 350 nm (A350 nm=A2). Biological macromolecules with the equilibrium amount of bound pterin remained in the filter apparatus. Each pterin/macromolecule solution was performed in triplicate, and the average values and standard deviations are reported. The absorbance values obtained were used to calculate the percent bound of each sensitizer with the macromolecule by the formula:
A1=initial absorbance of sensitizer (30 μM)
A2=absorbance of filtrate after experiment (15 μM less bound sensitizer)
Solubility determination procedure
Excess pterin was added to 100 mL of PBS to achieve a turbid suspension. Mixtures were stirred and lightly heated for an hour, sonicated for 5 min, and then cooled to room temperature. The undissolved solid was separated using centrifuge filters, and absorbance measurements were obtained at 350 nm on the clear, saturated pterin solutions. UV-Vis absorption was used to determine the final concentration of pterin using the respective molar extinction coefficients at A350. All solutions were prepared and handled in the absent of light, and all experiments were performed in triplicate. The average values are reported along with the statistical standard deviations.
Results and discussion
Pterin-lipid binding
The electrically neutral lipid (ENL), L-α-phosphatidylcholine serine, had a mild binding affinity to PteGlu (15%) and a low affinity to all the other pterins (<4%) (Table 1). For the pH used in this study, the glutamic acid portion of folic acid exists predominately as a dianion. The mild binding between PteGlu and ENL may be due to an electrostatic interaction between the anionic glutamate portion of the form of PteGlu and the quaterinary ammonium portion of phosphatidylcholine. There appears to be a general trend involving the intermolecular forces possible between the group at the 6-position of the pterin ring and the binding to neutral lipid. Mep, containing only a methyl group as a substituent, cannot participate in any hydrogen bonding or dipole-dipole interactions at that position and is observed to have the weakest binding to ENL of any pterin studied.
Percent of sensitizer bound to macromolecule after 24 h equilibration.
| PteGlu | Mep | Hmp | Fop | Cap | |
|---|---|---|---|---|---|
| ENL | 15±4% | 1±2% | 4±3% | 3±3% | 4±1% |
| BSA | 16±2% | 3±1% | 2±1% | 40±5% | 17±1% |
Equilibration conditions: neutral PBS; pterin: 15 μM; phosphatidycholine: 2.73 mM; albumin: 0.454 mM.
Pterin-BSA binding
BSA had a relatively high binding affinity for Fop (40%), a mild affinity for Cap (17%) and PteGlu (16%), and almost no affinity for Mep and Hmp. Our results showing a weak affinity between BSA and PteGlu is consistent with the work reported by Zhang and Jia [27]. The major acting force binding PteGlu to BSA has been determined by Zhang and Jia is hydrogen bonding where PteGlu is the acceptor, and BSA is the donor. In 1976, Soliman and coworkers reported that folic acid bound was 50% bound and 50% free in solution to human serum albumin at approximately the same concentrations employed in this study [28]. They found that maximal binding to albumin occurred at pH=6; slightly more acidic than in the studies performed here, which accounts for the slight difference. Although the aldehyde shows a very strong binding to BSA, the concentration of Fop under physiological conditions in the skin should be very low as it is readily oxidized to Cap under aerobic conditions. One possible reason for the high binding of the aldehyde to BSA may be imine formation with the free amines of the proteins, although this has not yet been proven.
Pterin-polynucleotide binding
The relative binding between 15 μM PteGlu, Mep, Hmp, Fop, and Cap with 500 μM nucleotide of Poly G, Poly C, Poly A, Poly T, M. luteus DNA (72% GC), E. coli DNA (50% GC), calf thymus DNA (42% GC), and C. perfrigens DNA (27% GC) in neutral PBS were determined experimentally (Table 2).
Percent of sensitizer bound to polynucleotides after 24 h equilibrium.
| PteGlu | Mep | Hmp | Fop | Cap | |
|---|---|---|---|---|---|
| Poly G | 18±1% | 5±4% | 16±1% | 12±2% | 45±4% |
| Poly C | 17±1% | 7±1% | 41±2% | 10±4% | 14±3% |
| Poly A | 64±1% | 19±2% | 27±4% | 6±4% | 19±1% |
| Poly T | 61±5% | 0±1% | 57±5% | 35±3% | 15±1% |
Equilibration conditions: neutral PBS; pterin concentration: 15 μM; polynucleotide concentration: 500 μM.
Poly G had a high affinity to Cap (45%), a moderate affinity for PteGlu, Hmp, and Fop (12%–18%), and a low affinity (5%) with Mep. Poly C had a relatively high affinity to Hmp (41%), moderate binding with PteGlu, Cap, and Fop (10%–17%), and a relatively low affinity (7%) to Mep. Poly A had a very high affinity to PteGlu (64%), strong binding to Hmp (27%), moderate binding with Mep, and Cap (19% each), and a relatively low affinity for Fop (6%). Poly T was observed to have a very high affinity for both PteGlu (61%) and Hmp (57%) and Fop (35%), while Cap only bound moderately (15%), while Mep showed no detectable binding. Specifically, Mep demonstrated the lowest affinity for three of the four polynucleotides studied. Since the only difference between Mep and folic acid is the presence of the para-aminobenzoic glutamate portion, it is clear that the substituent at the 6-position affects the affinity of pterins with polynucleotides greatly. This effect is not predominately due to a shift in the acid/base equilibrium of the pterin portion as the pKa for PteGlu and Hmp is 8.1, while Mep=8.3. The subtle change in acidity does not appear to account for the strong difference in binding observed.
The difference in binding between PteGlu and pterins with DNA and polynucleotides shows that the binding is based on the 6-position of pterin and also that the binding is sequence specific and not just affinity for phosphates. If the pterins were primarily interacting with the phosphate backbone, then there would be little or no difference between the binding of a specific pterin and the different polynucleotides. It is interesting to note that the average polynucleotide binding to each pterin is generally related to the extent of additional hydrogen bonding sites available due to the different substituents at the 6-position. More specifically, PteGlu is observed to have the highest overall binding and contains many additional hydrogen bonding sites followed by Hmp and Cap, respectively, each containing a donor and an acceptor. Fop contains only an acceptor, and Mep contains no additional hydrogen bonding sites at the 6-position.
Pterin-DNA binding
Cap (45%) and Hmp (43%) had a relatively high affinity for M. luteus (72% GC). PteGlu (35%) and Fop (27%) had a relatively medium affinity, and Mep had no affinity for M. luteus DNA. Hmp (60%) and PteGlu (54%) had a relatively high affinity for E. coli (50% GC). Fop (23%) had a relatively medium affinity, and Cap and Mep had almost no affinity for E. coli DNA. PteGlu (23%) had a relatively medium affinity for calf thymus (42% GC), and the rest of the pterins had almost no affinity (<10%) for calf thymus. PteGlu (49%), Hmp (48%), and Cap (39%) had a relatively high affinity for C. perfringens (27% GC). Fop and Mep had almost no affinity (<6%) for C. perfringens DNA (Table 3).
Percent of sensitizer bound to DNA after 24 h equilibrium.
| PteGlu | Mep | Hmp | Fop | Cap | |
|---|---|---|---|---|---|
| M. luteus (72% GC) | 35±6% | 0±1% | 43±2% | 27±2% | 45±4% |
| E. coli (50% GC) | 54±1% | 0±1% | 60±6% | 23±6% | 1±1% |
| Calf thymus (42% GC) | 23±3% | 0±1% | 8±2% | 3±1% | 0±1% |
| C. perfringens (27% GC) | 49±1% | 0±1% | 48±2% | 5±1% | 39±1% |
Equilibration conditions: Neutral PBS; pterin concentration: 15 μM; DNA nucleotide concentration: 500 μM.
The difference in binding between PteGlu and pterins to DNA is difficult to rationalize with the data presented here. If the binding is purely through interchelation between the DNA bases, then there should be a clear pattern of binding as the percentage of CG bases in the DNA increases, which is not observed. Another possible explanation involves the binding affinity based on the different DNA tertiary structures. The effect of GC content on DNA structure may change the area where binding occurs, for example, the availability of major and minor grooves. PteGlu appears to have the greatest overall binding to DNA. As PteGlu contains the larger and more solubilizing glutamic acid chain, this would likely inhibit base interchelation while promoting the ribose/phosphate hydrogen binding in the DNA backbone. Double-stranded DNA participates in hydrogen bonding between nucleotides of opposing strands, while the polynucleotides do not. Mep demonstrated no affinity for any DNAs, Fop favored higher GC content DNA, and Cap binds most strongly with both extremes of GC content. Therefore, it is reasonable to conclude that Mep does not interact with the DNA backbone the way that PteGlu does. Any between Mep and DNA/polynucleptides is likely due to hydrogen bonding directly with bases in a Watson-Crick way. Further investigations into the possible perturbation of the tertiary structure of the various DNA samples used along with potential pterin-binding sites is needed.
Solubility
One common motif for DNA interchelation is to employ hydrophobic moieties, which will stack between bases in the DNA helix. The solubilities of PteGlu, Mep, Hmp, Fop, and Cap were determined in PBS at room temperature to investigate if the relative binding between these pterins and the biological macromolecules is related to the solubility and subsequent hydrophobicity. Although PteGlu is not extremely water soluble, it is approximately 100 times more soluble in aqueous media than the other pterins due to the glutamic acid portion. Hmp and Cap have a higher solubility in neutral PBS, 130 μM and 125 μM, respectively, and are, therefore, less hydrophobic than Mep and Fop, 61 μM and 55 μM, respectively (Table 4). If the pterins are binding to DNA or polynucleotides via a hydrophobic interchelation mechanism, then the more hydrophobic pterins, Mep and Fop should show stronger binding than the more hydrophilic pterins, Cap and Hmp.
Comparison of PteGlu and pterin solubility, pKa, and relative binding with polynucleotides and DNAs (references for pKa values listed).
| Pterins | Saturation concentration in PBS | Ranges of percent pterin bound to polynucleotides | Ranges of percent pterin bound to various DNAs | pKa | Reference no. |
|---|---|---|---|---|---|
| PteGlu | 6 mM | 17%–64% | 23%–54% | 2.4 | [29] |
| Mep | 61 μM | 0%–19% | 0% | 8.3 | [5] |
| Hmp | 130 μM | 16%–57% | 8%–60% | 8.1 | [23] |
| Fop | 55 μM | 6%–35% | 3%–27% | 7.3 | [6] |
| Cap | 125 μM | 14%–45% | 0%–45% | 7.9 | [24] |
Despite the obvious differences in chemical structures, slight differences in pKa values, and other significant differences, the observed pterin binding to polynucleotides, observed pterin binding to DNA, and pterin solubility appear to all follow the same trend (Table 4). The least soluble pterins also have the weakest binding to polynucleotides and DNA. We hypothesize that the low aqueous pterin solubility, i.e., hydrophobicity, is due to strong intramolecular hydrogen bonding between pterin molecules. To the best of our knowledge, no crystal structures of the simpler pterins used in these studies have been reported in the chemical literature. Complete aqueous solvation of the individual molecules would require that these hydrogen bonds be broken resulting in discreet solvated pterin molecules or smaller pterin aggregates. Structures with low solubility have stronger intramolecular forces between solute molecules than with the solvent and probably exist in solution as larger aggregates compared to the more soluble pterins. These larger aggregates are less likely to bind to nucleotides or DNA and, therefore, will result in a low observed binding, as the experimental data shows. The clear solutions collected after being passed through the centrifuge filter may still contain small aggregates that were small enough the pass through the pores. Considering Cap as an example with a molecular weight of 207.15 g/mol, an aggregate as large as a 24-mer would still pass through a filter with a molecular weight cutoff of 5000 and may contain aggregates that are not visually detectable.
Conclusions
Little information of the possible binding between folic acid and its photodecomposition product’s binding to different biological macromolecules is known. Therefore, we have studied the relative binding of PteGlu, Mep, Hmp, Fop, and Cap (15 μM) with BSA, ENL, Poly G, Poly C, Poly A, Poly T, M. luteus DNA (72% GC), E. coli DNA (50% GC), calf thymus DNA (42% GC), and C. perfrigens DNA (27% GC) in neutral PBS. Folic acid demonstrated binding to neutral lipids, while the other pterins showed minimal binding. Bovine serum albumin had a significant binding to PteGlu, Cap, and especially Fop. The strong interaction between Fop and proteins was quite noteworthy as Fop showed little binding to any other macromolecule in the study. The binding between the PteGlu or pterin and the polynucleotides and DNAs with different GC content are based on the 6-position of pterin. These results indicate both nucleotide-dependent binding and in addition to affinity for the sugar phosphate backbone. As the solubility of each pterin follows the same trend of the observed DNA binding, it is proposed that pterin aggregates in aqueous media may contribute to the observed binding results. It has been previously established that binding to DNA by photosensitive molecules may lead to DNA damage when exposed to irradiation [30]. The high binding affinity of PteGlu and related photodecomposition products means that the photochemistry may take place in the short-lived singlet state in addition to the long-lived triplet state. Various reports show that PteGlu and pterins participate in type I sensitization and type II sensitization. We believe that these differences may be explained by considering the different binding affinities reported here. With the singular exception of Fop with BSA, PteGlu binds to all macromolecules studied as strongly or stronger than all the pterins under investigation. The high affinity for DNA by folic acid means that a high percentage of PteGlu is bound to genomic material before photochemistry occurs, which means that 6-carboxypterin may not be the only product destroying DNA when UV-A light is applied.
Funding: Welch Foundation (Grant/Award Number: ‘V-0004’).
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- Theoretical study on the relative energies of cationic pterin tautomers
- Binding affinities of folic acid and related pterins with biological macromolecules under physiological conditions
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Articles in the same Issue
- Frontmatter
- Review
- Folate receptor: a potential target in ovarian cancer
- Original articles
- Theoretical study on the relative energies of cationic pterin tautomers
- Binding affinities of folic acid and related pterins with biological macromolecules under physiological conditions
- Serum tryptophan, kynurenine, phenylalanine, tyrosine and neopterin concentrations in 100 healthy blood donors
- Correlation of trisomy 13 with atelencephalic aprosencephaly
