Elsevier

Chemical Physics

Volume 358, Issues 1–2, 30 March 2009, Pages 132-136
Chemical Physics

Thermal degradation of (6R,S)-5,10-methenyltetrahydrofolate in aqueous solution at pH 8

https://doi.org/10.1016/j.chemphys.2009.01.005Get rights and content

Abstract

The degradation of the folate (6R,S)-5,10-methenyltetrahydrofolate chloride (MTHF-Cl) in aqueous solution at pH 8 at room temperature is studied by absorption spectra measurements. Samples with and without the reducing agent β-mercaptoethanol (β-ME) both under aerobic and anaerobic conditions are investigated. MTHF-Cl hydrolyses to (6R,S)-10-formyltetrahydrofolate (10-HCO-H4folate) in all four cases. 10-HCO-H4folate oxidizes to 10-formyldihydrofolate (10-HCO-H2folate) under aerobic conditions in the absence of β-ME. The degradation dynamics is analysed theoretically and conversion rate constants of hydrolysis and oxidation are determined.

Introduction

5,10-Methenyltetrahydrofolate (MTHF, 5,10-CH+-H4folate) is an important derivative of folic acid [1]. It has been also referred to as N5,N10-anhydroformyltetrahydrofolic acid [2], [3], anhydroleucovorin [2], [3], anhydrocitrovorum [4], isoleucovorin [2], [3], 5,10-methenyltetrahydopteroylglutamic acid [5], and degradation product of folinic acid [6]. It is encountered in enzymatic systems [1], [7]. It is the light harvesting cofactor in DNA repairing photolyases [8], [9], [10], [11], [12] (energy transfer from excited MTHF to anionic form of flavin adenine dinucleotide hydroquinone (FADredH)). It is present as second cofactor besides FAD in cryptochromes [13], [14], [15], [16], [17] which are one family of blue-light photoreceptors [18], [19], [20], [21].

The synthesis of MTHF is described in [5], [22], [23], [24]. Some information on the stability of MTHF is found in [1], [24]. The pH dependent conversion of MTHF to 10-formyltetrahydrofolate by ring-opening is discussed in [5], [25], [26], [27], [28], [29], [30]. At low pH MTHF was found to be rather stable. Absorption spectroscopic data of MTHF are found in [1], [8], [22], [23], [24], [31], [32]. Information on the fluorescence behaviour of MTHF is found in [16], [17], [32].

In an absorption and emission spectroscopic characterisation of the photo-cycle dynamics of the circadian blue-light photoreceptor cryptochrome dCry from Drosophila melanogaster in aqueous Tris–HCl buffer at pH 8 [17], it was found that the FAD cofactor is present in its oxidized form (FADox) and the folate cofactor is present in the form of 10-formyldihydrofolate (10-FDHF, 10-HCO-H2folate). In the in vitro preparation of dCry MTHF hydrolysed to 10-formyltetrahydrofolate (10-FTHF, 10-HCO-H4folate) which then oxidized to 10-HCO-H2folate. Here the room temperature conversion of MTHF-Cl in aqueous Tris–HCl buffer solution at pH 8 (without dCry protein) to 10-HCO-H4folate and 10-HCO-H2folate is studied in detail by absorption spectroscopy. In order to find out whether some degradation of MTHF to detectable amounts of 10-formyl-folic acid (10-HCO-folate) [1], [24], [32], [33], [34], [35], [36], [37], 5-formyltetrahydrofolic acid (5-HCO-H4folate, folinic acid) [2], [3], [6], [21], [24], [27], [32], [33], [38], [39], [40], or 5,10-methylenetetrahydrofolic acid (5,10-CH2-H4folate) [24], [30], [41], [42] also takes place, the absorption cross-section spectra of these compounds are additionally determined. The studies have been carried out to compare the MTHF behaviour in the dCry protein with the behaviour of MTHF in free aqueous solution and to better understand the MTHF behaviour in cryptochromes [16], [17].

The absorption spectroscopic behaviour of 10-formyltetrahydrofolate (10-HCO-H4folate) at various pH values is investigated in [1], [25], [26], [33], [38]. A fluorescence spectroscopic characterisation is carried out in [32]. The preparation of 10-HCO-H4folate from MTHF at neutral pH in the presence of β-mercaptoethanol (β-ME) is described in [22], [43]. The conversion of tetrafolic acid to 10-HCO-H4folate by the enzyme formyltetrahydrofolate synthetase is handled in [44], [45], [46]. The enzymatic conversion of 5-formyltetrahydrofolate to 10-HCO-H4folate is studied in [26]. 10-HCO-H4folate at neutral pH is reported to be stable under anaerobic conditions [25]. In acidic pH 10-HCO-H4folate is converted to MTHF [24]. In air 10-HCO-H4folate is oxidized [5], [6], [26].

The absorption spectrum of 10-formyldihydrofolate (10-HCO-H2folate) is presented in [5]. Fluorescence emission of 10-HCO-H2folate in the cryptochrome dCry from Drosophila melanogaster was reported in [17]. 10-HCO-H2folate is formed either by oxidation of 10-HCO-H4folate [5], [6], [43], [47], [48], [49] in aerobic solution or by reduction of 10-formylfolic acid (10-HCO-folate) with sodium dithionite (Na2S2O4) [34], [49]. Oxidation of 10-HCO-H2folate to 10-HCO-folate under prolonged exposure to air was observed in sample preparations used in [5] and [8]. The reduction of 10-HCO-H2folate to 10-HCO-H4folate in the presence of dihydrofolate reductase is discussed in [49].

The structural formulae of MTHF-Cl, 10-HCO-H4folate, 10-HCO-H2folate, 10-HCO-folate, 5-HCO-H4folate-Ca, and 5,10-CH2-H4folate-Ca are shown in Fig. 1. Their absorption cross-section spectra are shown in Fig. 2. General spectroscopic information on folic acid and folic acid derivatives is found in [1], [24], [37], [50].

Section snippets

Experimental

The folates MTHF-Cl, 10-HCO-folate, 5-HCO-H4folate-Ca, and 5,10-CH2-H4folate were purchased from Schircks Laboratories, CH-8645 Jona, Switzerland. The other chemicals like Tris (tris(hydroxymethyl)aminomethane, (HOCH2)3CNH2), HCl, β-mercaptoethanol (HSCH2CH2OH) were bought from Sigma–Aldrich. The samples were used without further purification. The experiments were carried out at room temperature.

MTHF-Cl was dissolved in aqueous Tris–HCl pH 8 buffer solution (25 mM Tris + 150 mM NaCl + 5% glycerol, pH

Results

The change of the absorption spectra of original MTHF-Cl in Tris–HCl pH 8 buffer solution with time after preparation is shown in Fig. 3. The sample absorption versus time after preparation at the selected wavelengths of λ = 360 nm (absorption peak of original MTHF-Cl), λ = 259 nm (absorption peak of 10-HCO-H4folate), and 332 nm (absorption peak of 10-HCO-H2folate) are shown in Fig. 4.

In Fig. 3a an air-saturated solution of MTHF-Cl without β-ME is investigated. Within 80 min (see Fig. 4a) the

Discussion

The absorption coefficient development of the samples is generally given byα(λ,t)=iNi(t)σa,i(λ),where i indicates the different species, Ni is the number density of species i, and σa,i is the absorption cross-section of species i.

The room temperature degradation of MTHF-Cl in aqueous solution at pH 8 is described by Scheme 1.

k1=τ1-1 is the rate constant of 10-HCO-H4folate formation (τ1 is time constant of MTHF-Cl degradation). k2=τ2-1 is the rate constant of 10-HCO-H2folate formation (τ2 is

Conclusions

The degradation of MTHF-Cl at room temperature has been studied at pH 8 under aerobic and anaerobic conditions in the presence of β-ME and without β-ME. In all cases the hydrolysis of MTHF-Cl to 10-HCO-H4folate occurred with approximately the same time constant of τ1  14 min. 10-HCO-H4folate oxidizes to 10-HCO-H2folate under aerobic conditions in the absence of any reducing agent. The reducing agent β-mercaptoethanol acts as an oxygen scavenger and hinders 10-HCO-H4folate oxidation.

The performed

Acknowledgements

The authors thank Dr. J. Shirdel for preliminary measurements on MTHF degradation at pH 8. They thank the Deutsche Forschungsgemeinschaft for financial support in the Graduate College, GK-640/3, “Sensory Photoreceptors in Natural and Artificial Systems” and in the Research Group, FOR 526, “Sensory Blue-light Receptors”. E.W. is supported by a Grant of the Deutsche Forschungsgemeinschaft (WO-695/4). A.P. thanks Prof. F.J. Gießibl for his kind hospitality.

References (51)

  • M. Silverman et al.

    J. Biol. Chem.

    (1956)
  • S.-H. Song et al.

    J. Photochem. Photobiol.

    (2006)
  • J. Shirdel et al.

    Chem. Phys.

    (2008)
  • J.C. Rabinowitz

    Methods in Enzymol.

    (1963)
  • F.M. Huennekens et al.

    Methods in Enzymol.

    (1963)
  • L.D. Kay et al.

    J. Biol. Chem.

    (1960)
  • P. Stover et al.

    Trends in Biochem. Sci.

    (1993)
  • K. Uyeda et al.

    Anal. Biochem.

    (1963)
  • C.K. Mathews et al.

    J. Biol. Chem.

    (1963)
  • J.A. Blair et al.

    Anal. Biochem.

    (1970)
  • J.M. Scott

    Meth. Enzym.

    (1980)
  • J.C. Rabinowitz et al.

    J. Biol. Chem.

    (1962)
  • R.H. Himes et al.

    J. Biol. Chem.

    (1962)
  • R.H. Himes et al.

    J. Biol. Chem.

    (1962)
  • I. Eto et al.

    Anal. Biochem.

    (1980)
  • J. Baram et al.

    J. Biol. Chem.

    (1988)
  • A. Penzkofer et al.

    Chem. Phys.

    (2007)
  • J.C. Rabinowitz
  • D.B. Cosulich et al.

    J. Am. Chem. Soc.

    (1951)
  • D.B. Cosulich et al.

    J. Am. Chem. Soc.

    (1952)
  • J.E. Baggott et al.

    Biochem. J.

    (1995)
  • M. May et al.

    J. Am. Chem. Soc.

    (1951)
  • F.M. Huennekens et al.

    Adv. Enzym.

    (1959)
  • J.L. Johnson et al.

    PNAS

    (1988)
  • S.-T. Kim et al.

    Biochem.

    (1991)
  • Cited by (0)

    View full text