- Email: [email protected]
The oxidative cleavage of folates
ANALYTICAL
BIOCHEMISTRY
84,
277-295
The Oxidative
(1978)
Cleavage
A Critical TADASHI MARUYAMA,TETSUO
of Folates
Study
SHIOTA, AND CARLOS L. KRUMDIECK
Departments of Nutrition and Microbiology University of Alabama in Birmingham,
and The Institute of Dental Research, Birmingham, Alabama 35294
Received July 6, 1977; accepted August 16, 1977 Alkaline permanganate oxidation has been used to determine the chain length of naturally occurring pteroylpolyglutamates on the assumption that all forms of folates cleave at the P-N”’ bond to produce the corresponding p-aminobenzoylpolyglutamates. The chain length of the latter could be determined by cochromatography with synthetic markers. The products of alkaline (ammonium bicarbonate buffer, pH 9.0) permanganate oxidation of a number of reduced and oxidized, onecarbon-substituted and unsubstituted folic acid derivatives have been identified, and their yields and stability to the oxidative treatment have been determined. Unsubstituted, oxidized and reduced folic acid and Ns-formyl-tetrahydrofolic acid are cleaved at the C9-N1” bond to produce p-aminobenzoylglutamic acid. N>, N”‘-methenyl-tetrahydrofolic acid, NS,N”‘-methylene-tetrahydrofolic acid, and N”‘-formyl-tetrahydrofolic acid are not cleaved but are oxidized to N”‘-formyl-folic acid which is completely stable to the oxidative treatment employed. N5-methyltetrahydrofolic acid is not cleaved either but is oxidized to Ns-methyl-dihydrofolic acid which upon continued oxidation decomposes slowly to unidentified products. The y-glutamyl peptide linkage is completely stable to oxidation. Using p-amino[3,5-3H]benzoylglutamic acid, it is also shown that this product, previously thought to be stable to the oxidative treatment is decomposed by it. The significance ofthese findings in terms of the errors that may have been introduced in prior estimations of the chain length and pool sizes of the naturally occurring pteroylpolyglutamates is discussed. The possibility of developing a method for the chain length determination of noncleavable pools of one-carbon-substituted folates using [2-‘4C]folic acid to label the folates in vivo is presented.
In nature, the folate coenzymes constitute a complex family of closely related compounds the majority of which are pteroyl-y-glutamyl derivatives differing from one another in the number of glutamyl residues, the presence of a one-carbon substituent at N5 and/or N’O and the state of reduction of the pyrazine ring. The separation, identification, and quantitation of these coenzymes are a difficult undertaking complicated by abnormalities in the chromatographic behavior of the intact pteroylpolyglutamates. These abnormalities preclude the estimation of chain length based on the elution position of the reduced one-carbon-substituted pteroylpolyglutamates from ion exchange (l-3) or molecular sieve columns (4-6). 277
0003-2697/78/0841-0277$02.00/O Copyright 0 1978 by Academic press. Inc. All rights of reproduction in any form reserved
278
MARUYAMA,
SHJOTA,
AND
KRUMDIECK
To overcome these obstacles, cleavage of the Cg-N’O bond of suitably in vivo labeled folates was attempted, and the chain lengths of the resulting radioactive p-aminobenzoylpoly-y-glutamates (pABG,) were determined by cochromatography with synthetic markers. Both reductive (1,7,8) and oxidative (9- 12) treatments were employed with apparent success. However, testing of the cleaving procedures has been largely limited to demonstrating cleavage of folic acid and lack of attack on the poly-y-glutamyl chain (1,13). The possibility that oxidative or reductive cleavage of W”formyl-tetrahydrofolic acid (lo-CHO-H,-PteGlu) and N5,Wo-methenyltetrahydrofolic acid (5,10=CH-H,-PteGlu) could result in the formation of N-formyl-p-aminobenzoylglutamic acid or N-methyl-p-aminobenzoylglutamic acid, respectively, has been considered and dismissed as unimportant by Reed et al. (14), Scott (15), and Baugh et al. (1). Scott (16) summarily reported that alkaline permanganate oxidation at room temperature quantitatively cleaved the Cs-Nl” bond of folic acid (PteGlu), N5-formyltetrahydrofolic acid (5CHO-H,-PteGlu), tetrahydrofolic acid (H,-PteGlu), N5-methyl-tetrahydrofolic acid (5CH,-H,-PteGlu), N5,Wo-methylenetetrahydrofolic acid (5,10-CH,-H,-PteGlu), 5,10=CH-H,-PteGlu, dihydrofolic acid (HZ-PteGlu), N5-methyl-dihydrofolic acid (5-CH,-H,-PteGlu), lo-CHO-H,-PteGlu, and W”-formylfolic acid (lo-CHO-PteGlu). He stated that in all instances except the latter two, the product is p-aminobenzoylglutamic acid. The IO-formyl derivatives gave two products: pABG and formylaminobenzoyl glutamate which could not be separated with the DEAE-cellulose columns employed (16). Preliminary experiments in our laboratory indicating slight but definite separations of the products of permanganate oxidation of liver folates from the cochromatographed syntheticpABG, markers strongly suggested the presence of products other than pABG, and prompted a careful examination of the yield, products, and stability thereof resulting from the alkaline permanganate oxidative treatment of one-carbon-substituted folates. The results reported here demonstrate that, under the conditions used, some folates do not cleave at the Cs-N’O bond on alkaline permanganate oxidation, others do so readily, and still others cleave only after subsequent acidification. Furthermore, a significant decomposition of pABG upon prolonged oxidation has also been demonstrated. MATERIALS
AND METHODS
PteGlu, 5-CH,-H,-PteGlu (Ba salt), and pABG were purchased from Sigma Chemical Co. Prior to use the commercial folic acid was purified according to Blakley (17) and the 5-CH,-H,-PteGlu was purified by DEAEcellulose column chromatography (1.5 x 22 cm) using a linear gradient of 500 ml of water in the mixing chamber and 500 ml of 1 .OM ammonium bicarbonate in the reservoir. Pure 5-CHO-H,-PteGlu was a gift of Dr. John Montgomery from Southern Research Institute, Birmingham, Alabama.
OXIDATIVE
CLEAVAGE
OF
FOLATES
279
p-Nitrobenzoylglutamic acid was purchased from California Foundation for Biochemical Research. 5,lO=CH-H,-PteGlu was prepared by the method of Rowe (18) from II,-PteGlu obtained by catalytic hydrogenation with platinum oxide as described by Hatefi et al. (19). The S,lO=CH-H,-PteGlu was twice recrystallized from hot 0.1 N HCI in 0.1 M 2-mercaptoethanol prior to use. 5,10-CH,-H,-FteGlu was prepared as described by Huennekens et al. (20) and was purified by chromatography through a DEAE-cellulose column eluted using a linear gradient of ammonium bicarbonate buffer, pH 9.5, 0.004 to 0.4 M, 500 ml of each. lo-CHO-PteGlu and the N”-formyl-pteroylpolyglutamates were prepared by formylation of PteGlu and the corresponding pteroylpolyglutamates with 98% formic acid as described by Blakley (21). The pteroylpoly-y-glutamates were synthesized by the solid phase method of Krumdieck and Baugh (22) and purified by DEAE-cellulose chromatography as described. Tritiated [3’,5’,9(n)-3H]folic acid, 47 Ci/ mol, and [2-14C]folic acid, 58.2 mCi/mmol, were purchased from Amersham/Searle. Tritium-labeled pABG (3,5-3H) was prepared from [3’,5’,9(n)-3H]folic acid as follows: 5 mg of folic acid were dissolved in 4.6 ml of 0.1 M ammonium bicarbonate buffer, pH 9.0, and 20 &i of [3’,5’,9(n)-3H]folic acid were added. The solution was oxidized with 0.5 ml of 2% KMnO, for 2 min. The reaction was terminated by the addition of 0.5 ml of 30% H,O,. Following removal of the MnO,, the supernatant was applied to a DEAE-cellulose column (1.5 x 22.0 cm) and eluted with a linear gradient of water to 1.O M NH4HC03, 500 ml of each. ThepABG-containing fractions were pooled, lyophilized, and a concentrated solution applied to a cellulose column (1.5 x 40 cm). The column was eluted with 0.2 M NH,HCO, to remove fluorescent materials, and thepABG-containing fractions were pooled and lyophilized. The purified product had a specific activity of 0.796 &i/~mol. Doubly labeled 5-CH,-Z~Z,-[2-~~C,3’,5’,9(n)-~H]PteGlu was prepared by catalytic reduction of 50 pmol of folic acid to which 5.0 PCi of [2-14C]folic acid and 25.0 &i of [3’,5’,9(n)-3H]folic acid had been added. The doubly labeled tetrahydrofolic acid obtained was converted to 5,lO = CH-H,F’teGlu and reduced with sodium borohydride to the desired 5-CH,-H,PteGlu (23), and this compound was purified through a DEAE-cellulose (Cl-) column using an elution gradient of 0.2 M NaCl in 0.01 M 2-mercaptoethanol to 0.7 M NaCl in 0.01 M 2-mercaptoethanol, 500 ml of each. After desalting, the spectrally pure product was lyophylized and stored in sealed evacuated ampoules. Doubly labeled 5-CH3-H,-PteGlu was prepared by oxidation of the doubly labeled 5-CH3-H,-PteGlu as follows: one milligram of carrier commercial 5-CH,-H,-PteGlu was added to 0.27 mg of 5-CH,H4-[2-14C,3’,5’,9(n)-3H]PteGlu in a total volume of 2.0 ml of 0.1 M NH, HCO,, pH 9.0. Fifty microliters of H,O, were added before and after the addition of 100 ~1 of 2% KMnO,. After removal of the precipitate, the prod-
280
MARUYAMA,
SHIOTA,
AND
KRUMDIECK
uct was purified through a DEAE-cellulose column (1.5 x 22.0 cm) eluted with a linear gradient of 0.0 to 0.5 M NH4HC03, 500 ml of each. The purified product was then lyophilized. The final specific activity was 0.022 &i/ ,umol for 14C and 0.154 pCi/pmol for 3H. Ultraviolet spectra were obtained with a Hitachi-Perkin Elmer 124 spectrophotometer. Reference spectra and molar extinction coefficients were obtained from the literature. The molar extinction coefficients used are presented in Table 1. The Bratton-Marshall test (24) was used to determine the concentration ofpABG in solution and to qualitatively detect it in thin-layer plates. High-pressure liquid chromatography (hplc) was carried out using a Waters hplc system with a weak anion exchanger column of Reeve Angel AL Pellionex wax. All separations were done at room temperature and 1.5 mllmin flow rate. The’pressure was approximately 1000 psi. A Waters Model 440 detector and a Varian A-25 recorder were used to monitor the optical density of the effluent at 254 nm. Linear gradients of potassium phosphate buffer, pH 7.0, from 0.001 to 0.1 or 0.3 M in 30 min were constructed by a Waters Model 660 solvent programmer. Five-drop fractions (approximately 0.35 ml) were collected when required using an LKB Ultrorat 7000 fraction collector. The volume of sample applied was usually 10 to 20 ~1 but was increased to 50 or 100 ~1 when fractions were collected for subsequent analysis. All radioactivity measurements were performed with a Packard Tri-Carb liquid scintillation spectrometer using Bray’s solution (25) as a counting fluid. Cellulose plates (Polygram Cel 300, Macherey-Nagel) were used for thin-layer chromatography. The solvents used were: (A) 3% NH4Cl and (B) I-propanol:l% NH,OH, 2:l by volume. TABLE SPECTRAL
Compound PteGlu IO-CHO-PteGlu S-CHO-H,-F’teGlu S-CH,-I&-PteGlu 5-CH,-H,-PteGlu 5,lO=CH-H,-PteGlu 5, IO-CH,-If,-PteGlu pABG
Molar extinction coefficient at A max (X 10-S) 23.4 20.9 37.2 31.7 31.2 26.5 32.0 15.4
1 DATA
A max (nm) 283 269 285 290 290 348 294 273
Conditions in in in in in in in in
0.1 pH pH pH pH 1N pH 0.1
N NaOH 7.0 7.0 7.0 7.0 HCl 7.2 N NaOH
References (26727) (27728)
(2% (30) (30)
cw (31) (32)
OXIDATIVE
CLEAVAGE
OF FOLATES
281
Oxidation procedure. The folates (0.175 to 1.21 mM; the amounts varied with the availability of the various forms) were dissolved in 0.1 M ammonium bicarbonate buffer, pH 9.0, and oxidized by adding 2% KMnO, in an amount equal to one-tenth the volume of the folate-ammonium bicarbonate solution. All operations were performed at room temperature under a red light. At the end of the oxidation period, the excess KMnO, was destroyed with 30% H,O, (volume equal to that of 2% KMnO,), and the MnO, precipitate was removed by centrifugation. Distilled water was added to the control samples in place of the KMnO, and H,O, solutions. The time of oxidation in all the experiments done for the purpose of identification of oxidation products was 20 min. In the experiments designed to follow the rate of oxidation by permanganate, the time-zero samples correspond to reactions where the KMnO, was initially destroyed by the prior addition of HzOz. A very brief period of oxidation by H,O, or nascent oxygen unavoidably took place. RESULTS
I. Identijication
of the Oxidation
Products of Folates
(a) Folic acid, tetrahydrofolic acid, and dihydrofolic acid. The results of hplc of folic acid before and after KMnO, oxidation are shown in Fig. 1 a. The peak of folic acid disappeared after oxidation and was replaced by O5r (b)
0.12 0 IO E 5 0.06 @ dOO6 d 004
5 Time
IO in mmtes
FIG. 1. Oxidation of folic acid. (a) The hplc elution profile of PteGlu before (---) and after (-) oxidation. Ten milliliters of 0.176 mM PteGlu were oxidized with 0.4 ml of 2% KMnO, for 20 min. (b) The uv spectra of PteGlu (before) and its oxidation product (7.4-min peak) (after).
282
MARUYAMA,
SHIOTA,
AND KRUMDIECK
two new peaks. The first was identified as pABG by uv spectra (Fig. 1 b) and by tic (solvent A). Only one Bratton-Marshall positive spot migrating together with apABG marker (R, = 0.9) was detected. The second peak, which was fluorescent, cochromatographed with pterin-6-carboxylic acid (P-6-COOH). It is obvious that the Cg-N’O bond of folic acid is cleaved and that the major products of the reaction are pABG and P-6-COOH. H4PteGlu and H,-PteGlu are also cleaved at the Cg-N’O bond by KMnO, oxidation each producing pABG and P-6-COOH. (6) N5,N10-Methenyl-tetruhydrofolic acid. In neutral or alkaline conditions 5,10=CH-H,-PteGlu converts rapidly to lo-CHO-H,-PteGlu. Therefore, under the conditions of our experiments (15 min at pH 9.0 prior to oxidation), the oxidation of the bridge compound represents in effect oxidation of lo-CHO-H,-PteGlu. The possibility of formation of N-formylpABG instead ofpABG (15) was of considerable interest. Figure 2a shows the hplc pattern obtained after the oxidation of 5,10=CH-H,-PteGlu. A substance eluting as a major single peak after the position of elution of p ABG and which fluoresced blue under uv (365 nm) illumination was found. The uv spectra of this substance and of the starting material are shown in Fig. 2 b. The oxidation product was identified spectrally as loCHO-PteGlu (28) which was previously shown to be fluorescent (33). This product cochromatographed with authentic IO-CHO-PteGlu on thin-layer plates (R, = 0.77 with solvent A). Furthermore, the uv spectrum of the product treated with 1 .O N NaOH overnight at room temperature changed to that of folic acid, in keeping with the previously reported deformylation of lo-CHO-PteGlu in alkaline solutions (21,26).
5 IO Time in minutes
15
FIG. 2. Oxidation of NS,N’O-methenyl-tetrahydrofolic acid. (a) The hplc elution profile of the oxidation product of 5,10=CH-H,-F’teGlu. Five-tenths milliliter of 0.195 IIIM S,lO=CHH,-PteGlu was oxidized with 0.05 ml of 2% KMn04 for 20 min. (b) The uv spectra of 5, IO&HH,-PteGlu (before) and its oxidation product (after).
OXIDATIVE
CLEAVAGE
OF FOLATES
283
(c) N5,N10-Methylene-tetrahydrofolic acid. In contrast to 5,10=CH-H,PteGlu, 5,10-CH,-H,-PteGlu is stable at pH 9.5 and unstable in acidic solutions (34). The DEAE-cellulose column purified material had a uv spectrum (Fig. 3 b) identical to that reported by Blakley (31) but still contained some impurities as indicated by the hplc elution profile in Fig. 3 a. Following oxidation, a major product eluted in the position of IO-CHO-PteGlu and had its characteristic spectra (Fig. 3 b) and fluorescence. Of the smaller peaks, one cochromatographed with added pABG on both hplc and tic plates. The other minor peaks eluting with the solvent front and at 5 min were not identified. It is uncertain whether these minor products arise from oxidation of 5,10-CH,-H,-PteGlu or from impurities contaminating the starting material. (d) N5-Formyf-tetrahydrofolic acid. The hplc elution profile of 5-CHOH,-PteGlu is shown by the broken line of Fig. 4 a. It eluted as a single material slightly after the position of elution of pABG and had a uv spectrum identical to that previously reported (26). The major product after oxidation was identified as pABG spectrally and by cochromatography with standardpABG on both hplc and tic. Only one Bratton-Marshall positive spot was detected after tic with solvent A. A second faintly BrattonMarshall positive spot (Z?, = 0.19) separated frompABG (R, = 0.50) with solvent B. The two minor peaks appearing after oxidation (eluting with the solvent front and at 6.4 min.) were not identified. The absence of the
Time m minutes
3. Oxidation of N5,N1”-methylene-tetrahydrofolic acid. (a) The hplc elution profile of 5,10-CH,-H,-PteGlu before (---) and after (-) oxidation. One milliliter of 0.260 mM 5,10CH,-H.,-PteGlu was oxidized with 0.1 ml of 2% KMnO, for 20 min. (b) The uv spectra of 5,10-CH,-H,-PteGlu (before) and its main oxidation product (8.6min peak) (after). FIG.
284
MARUYAMA,
SHIOTA,
AND KRUMDIECK
(a)
0.06
t
o.o+
Time
in minutes
FIG. 4. Oxidation of@formyl-tetrahydrofolic acid. (a) The hplc elution profile of S-CHOH,-PteGlu before (---) and after (-) oxidation. Ten milliliters of 0.272 mM 5-CHO-H,PteGlu were oxidized with 0.5 ml of 2% KMnO, for 20 min. (b) The uv spectra of SCHOH,-F’teGlu (before) and its oxidation product (7.3-min peak) (after).
strongly fluorescent P-6-COOH worth noting.
peak seen after oxidation
of folic acid is
(e) N5-Methyl-tetrahydrofolic acid. The hplc elution profiles of 5-CH,H,-PteGlu before and after oxidation are shown in Fig. 5 a. The starting material had a uv spectrum (Fig. 5 b) identical to that reported by Gupta et al. (30). The product was spectrally identical to S-CH,-H,-FteGlu (Fig. 5 b) (30), which is known to elute before 5-CH,-H,-PteGlu from DEAEcellulose columns (35). Since 5-CH,-Hz-PteGlu is very acid labile and undergoes cleavage of the Cg-Nl” bond to producepABG (36), the product of oxidation was treated with 0.1 N HCl at room temperature for a few minutes and reapplied to the hplc after removal of the acid by flash evaporation. As expected pABG was obtained and identified spectrally and by tic. Furthermore, the product of oxidation could characteristically be converted back to 5-CH,-H,-PteGlu by reduction for 6 hr at room temperature with 1% mercaptoethanol(37). The uv spectrum of 5-CH,-H,-PteGlu was obtained after hplc purification of the reduction mixture. II. Efficiency and Rate of KMnO, Oxidation Products
Oxidation of Folates: Stability of the acid. The rate of cleavage was determined by measuring
(a) Folic acid and N5-formyl-tetrahydrofolic
of F’teGlu and 5-CHO-H,-PteGlu
to pABG
OXIDATIVE
CLEAVAGE
OF FOLATES
285
Time in minutes FIG. 5. Oxidation of W-methyl-tetrahydrofolic acid. (a) The hplc elution profile of 5-CH,H.,-Pte-Glu before (- - -) and after (-) oxidation. One milliliter of 1.21 mM 5-CH3-H,F’teGlu was oxidized with 0.1 ml of 2% KMnO, for 20 min. (b) The uv spectra of 5-CH,H,-PteGlu (before) and its oxidation product (after).
the amount ofpABG formed after various times of oxidation. The Bratton Marshall test was used to quantitate thepABG. Figure 6 is a semilogarithmic plot of the results obtained. The ordinate shows the pABG yield as a percentage of the amount of folate present in the control. Folic acid was rapidly cleaved to pABG (92% yield in 1 min) which in turn was more slowly converted to nondiazotizable products upon continued oxidation. Essentially the same results were obtained when 5-CHO-H,-PteGlu was oxidized. The most noticeable difference is that SCHO-H,-PteGlu is
IO 20 30 40 Time of oxidation (minutes)
50
FIG. 6. Time course of oxidation of folic acid (0) and N”-formyl-tetrahydrofolic acid (0). Two milliliter samples of 0.796 mM PteGlu and 0.498 mM 5-CHO-H,-F’teGlu were oxidized with 0.2 ml of 2% KMnO, for the various times indicated. The reactions were terminated by the addition of 0.2 ml of 30% H202. Duplicate samples were assayed at each time.
286
MARUYAMA,
SHIOTA,
AND KRUMDIECK
cleaved at a somewhat slower rate as indicated by a 72 to 77% yield of pABG after 1 min of oxidation. The rate of decomposition of the pABG formed was the same as in the experiment with folic acid. To confirm this unexpected finding, 3,S3H-IabeledpABG was synthesized and oxidized as described below. (6) p-Amino[S,5-3H]benzoylglutamic acid. The oxidation of 3,S3H-labeled pABG (sp act, 0.796 &i/pmol) was carried out as follows: 0.415 pmol of tritium-labeled pABG dissolved in 1.O ml of 0.1 M ammonium bicarbonate, pH 9.0, was oxidized for periods of 20 and 40 min with 0.1 ml of 2% KMnO,. One-tenth milliliter of 30% Hz02 was added at the end of the oxidation periods and prior to the addition of permanganate in the time -zero sample. The uv spectra and Bratton-Marshall determinations were carried out, O.l-ml aliquots were chromatographed on hplc, and fractions collected for radioactivity measurements. Figure 7 b shows the spectral changes in 0.1 N NaOH with increasing oxidation times. There is a pronounced drop in absorbancy at yrnax = 273 nm. The recovery of pABG calculated from the optical density at 273 nm after 20 and 40 min of oxidation is shown by the solid line of Fig. 7 a. The dot-dash line in the same figure shows recovery based on the Bratton-Marshall test, and the dotted line recovery of radioactivity eluting in the position of pABG after hplc chromatography of the oxidation mixtures. All three methods gave results that are in very close agreement. Figure 8 shows the radioactivity elution pattern obtained after hplc of the 0; 20-, and 40-min oxidation samples. A new radioactive species which is neither uv-absorbing nor BrattonMarshall-positive and that accounts for the total loss of label from pABG was formed during oxidation. The hplc cochromatography of p-nitro06-
(b)
omm-
E
IO
20
Time of oxidation
30
40
(minutes)
FIG. 7. Time course of oxidation ofp-amino[3,5-3H]benzoylglutamic acid. (a) Semilogarithmic plot of percentage recovery vs oxidation time. See text for details. (b) Spectral changes with increasing oxidation time.
OXIDATIVE
CLEAVAGE
287
OF FOLATES
-
Omin.
. . ..
l .
.
20rn,“,
---C--40lW” I
3,000
0.06
1s
0.05
8 2 ; 2
2,000
0.04
003
.$ _o E t 2 Y
002
1.000
Fratin
number
8. Oxidation ofp-amino[3,5-3H]benzoylglutamate. The hplc elution profiles ofradioactivity from 3,5JH-labeled pABG oxidized with KMnO, for 0, 20, and 40 min. FIG.
benzoylglutamate with the oxidation mixture ofpABG (40 min) resulted in a clear separation of the nitro compound, ruling it out as one of the possible products of pABG oxidation. Oxidation of 2.0 mM 3,S3H-labeled pABG for 60 min resulted in the formation of a third minor (8.8% of total radioactivity) product which eluted after pABG and had uv absorption maxima at 330 nm at pH 7.0, 1.0, and 13.0. (c) N5,N10-Methenyl-tetrahydrofolic acid, N5,N10-methylene-tetrahydrofolic
acid, N1o-formyl-tetrahydrofolic acid, and N’O-formyl-folic acid.
The stability of lo-CHO-PteGlu, the main product of KMnO, oxidation of 5,10=CH-H,-PteGlu and 5,10-CH,-H,-PteGlu was studied next. A sample of synthetic lo-CHO-PteGlu was subjected to oxidation for 20 min and then chromatographed in the hplc system. A single peak eluted in the same position as lo-CHO-PteGlu. After oxidation, the uv spectrum of the material was unchanged and was identical to that of lo-CHO-PteGlu and the product cochromatographed on thin-layer plates with the authentic marker. Both the product of oxidation and the synthetic marker were stable for up to 2 days at room temperature in 1.0 N NH,OH and in solutions buffered at pH values of 6.0, 7.0, 9.0, and 9.5. It was completely stable in 0.1 N HCl for 60 min at room temperature. Figure 9 shows the yields and rates of formation of lo-CHO-F’teGlu by the oxidation of 5,10=CH-H,-PteGlu and 5,10-CH,-H,-PteGlu. It also shows that lo-CHO-PteGlu is stable to KMnO, oxidation for up to 40 min. The amounts of IO-CHO-PteGlu were determined spectrophotometrically from the OD at 269 nm at pH 7.0. In the case oxidation of lo-CHO-PteGlu, the optical density at 269 nm was measured directly after centrifugation to remove the MnO, precipitate. In
288
MARUYAMA,
SHIOTA,
AND KRUMDIECK
IO 20 30 40 Time of oxidation (minutes) FIG. 9. Time course of oxidation of N5, N’“-methenyl-tetrahydrofolic acid (0), N5, NLumethylene-tetrahydrofolic acid (O), and N1o-formyl-folic acid (x). Samples of 0.5 ml of 0.195 mM 5,lO=CH-H,-PteGlu, 1.0 mlof0.313 mM 5,10-CH,-If,-PteGlu, and 2.0mlof0.838 mM loCHO-PteGlu were oxidized with one-tenth their volumes of 2% KMnO, for the various times indicated. Reactions were terminated by the addition of H,O,.
the other two cases, after stopping the oxidations at the appropriate times, the reaction mixtures were chromatographed on hplc, and the fractions corresponding to IO-CHO-PteGlu were combined and concentrated by flash evaporation prior to quantitation at 269 nm. The results indicate that IO-CHO-PteGlu is completely stable to the oxidative conditions used. The 5,10=CH-H,-PteGlu was dissolved in 0.1 M ammonium bicarbonate, pH 9.0, and kept 15 min at room temperature prior to oxidation to assure its conversion to lo-CHO-H,-PteGlu. Following the addition of permanganate, lo-CHO-H,-PteGlu was rapidly oxidized to lo-CHO-PteGlu via an intermediate believed to be the same substance as the product of air oxidation of IO-CHO-H,-PteGlu. The latter elutes as a sharp single peak on hplc, and its uv spectra are shown in Fig. 10. This substance is thought to be W”formyl-dihydrofolic acid (38). The yield of the oxidation of 5,10=CH-H,PteGlu to IO-CHO-PteGlu was slightly above 100%. The yield of lo-CHOF’teGlu obtained by oxidation of 5,10-CH,-H,-F’teGlu was only 82 to 83%. This reaction also seems to proceed through the formation of an intermediate which could be detected as a minor peak eluting slightly after pABG in the hplc chromatograms of samples oxidized for less than 1 min. (d) N5-Methyl-tetrahydrofolic acid and N5-methyl-dihydrofolic acid. The formation of 5-CH,-H,-PteGlu by the oxidation of 5-CH,-II,-PteGlu proceeds very rapidly after the addition of KMnO,. A characteristic spectrum at pH 7.0 with A,,, at 249 and 289 nm and a ratio of OD valves at 290/250 nm of 1.39 is obtained. The yield of this reaction based on spectral quantitation is 99%. The time course of oxidation of 5-CH,-H,-PteGlu by permanganate is shown in Fig. 11 (solid line). The percentage recovery of 5-CH3-H,-F’teGlu was calculated from the optical density at 290 nm of solu-
OXIDATIVE
CLEAVAGE
OF FOLATES
289
I 0.6- I !I I
0.'
I I
d
0.3 -
0
>. I’ \
I/
\ \ \
0.2 -
\ .-_ \ ’ ---.*.
0.1 1
200
--., \1, h 3 4 C)O 250 300 350 Wave Length
nm
FIG. 10. The uv spectra of the air oxidation product of N’“-formyl-tetrahydrofolic
acid.
tions adjusted to pH 7.0 after removing the MnO, precipitate. The curve shows a rapid initial loss of product that changes to a much slower rate. The uv spectra of the reaction mixtures at pH 7.0 showed a rapid drop in the 289-nm peak with little or no change in the peak at 249 nm. The 290/250-nm ratio decreased from approximately 1.4 at zero time to 1.27, 1.22, 1.20, 1.17, and 1.14 at 1, 5, 10, 20, and 40 min, respectively. An hypsochromic
$1 5 a
I IO Time
I I I 20 30 40 of axidation(minutes)
FIG. 11. Time course of oxidation of W-methyl-dihydrofolic acid (0) and W-methyl-tetrahydrofolic acid (x). Duplicate samples of 2.0 ml of 0.524 mM 5-CH,-H,-PteGlu and 1.0 ml of 0.407 mM 5-CH,-Hz-PteGlu were oxidized with one-tenth their volumes of 2% KMnO, for the various times indicated. Reactions were terminated by the addition of H,O*.
290
MARUYAMA,
SHIOTA,
AND KRUMDIECK
shift to a uv maximum at 285 nm was also observed. The time course of permanganate oxidation of 5-CH,-H,-PteGlu is shown by the dotted line in Fig. 11. The material decomposes with increasing oxidation time following first-order kinetics. Spectral changes essentially identical to those described during the oxidation of 5-CH,-H,-PteGlu were observed. The 290/ 250-nm ratio dropped from 1.39 at zero time to 1.33, 1.3 1, 1.30, 1.25, and 1.19 at 1, 5, 10, 20, and 40 min of oxidation, respectively. (e) Acid cleavage of 5-methyl-dihydrofolic acid. To study the products and yield of the acid cleavage of 5-CH,-H,-F’teGlu, doubly labeled 5-CH,Hz-[2-14C,3’,5’,9(n)-3H]PteGlu was synthesized as described under Materials and Methods. The doubly labeled compound was acid treated by dissolving 0.195 pmol of it in 0.2 ml of 0.1 N HCI. After 1 min at room temperature, the acid was removed by thrice-repeated flash evaporation, and the final residue was dissolved in 0.15 ml of 0.1 M NH4HC03 buffer, pH 9.0. One-hundred microliters were injected into the hplc apparatus. A solution of the doubly labeled 5-CH,-H,-PteGlu served as control. The results are shown in Fig. 12. Two peaks appeared after acid treatment. The first peak was doubly labeled, and the second (pABG) contained almost exclusively tritium label with trace amounts of 14C (Fig. 12 b). Thirty percent of the 3H in the doubly labeled 5-CH,-H,-PteGlu appeared in the first peak and 45% in the pABG peak. The total recovery of 14C was 96%, but only 80% of the total 3H was accounted for. Some tritiated volatile compound(s) (3H20?) must have been lost during the flash evaporation step. (J> N”‘-Formyl-pteroylheptaglutamate. The hplc elution profile of loCHO-PteGlu, after KMnO, oxidation is shown in Fig. 13. The numbered
Fraction
FIG.
ment.
Number
12. 5-Methyl-dihydro[2-‘4C,3’,5’,9(n)-3H]folic
acid before (a) and after (b) acid treat-
OXIDATIVE
CLEAVAGE
OF FOLATES
291
0.145 0.12 O.lO-
0.02-
0
5
IO
15
20
Time in minutes FIG. 13. Oxidation of WO-formyl-pteroylheptaglutamate. One milliliter CHO-PteGlu, was oxidized with 0.1 ml of 2% KMnO, for 20 min.
of 0.502 mM lo-
arrows indicate the position of elution of the lo-CHO derivatives of pteroylmono-, tri-, hexa-, and heptaglutamates, respectively. A single symetrical peak eluting in the same position as before was obtained after oxidation of lo-CHO-PteGlu,. Table 2 summarizes all the results obtained. DISCUSSION
Our results contradict the assumptions (i) that alkaline permanganate oxidation cleaves the Cg-Nl” bond of all forms of folates (16) and (ii) that the pABG resulting from the cleavage of susceptible folates is stable to alkaline permanganate treatment (15). Together with the herein verified stability of the y-peptide linkages of the pteroylpolyglutamates to KMnO, oxidation, the above assumptions are central to the oxidative cleavage procedure used extensively to determine the chain length of polyglutamates and to quantitate the relative size of the various polyglutamate pools in biological materials. Our findings indicate that substantial errors may be introduced in these determinations. Since the radioactive precursor commonly used to label the folates of experimental animals prior to their extraction and oxidation is [3’,5’,9(n )-3H]folic acid, the presence of uncleaved folates (still bearing the 3H atom on Cg) in a fraction presumed to contain only 3,5-3H-labeled p ABG, of a given chain length, will result in a falsely high estimation of the size of that particular polyglutamate pool. If this were the only source of error it is calculated that it would amount to an overestimation equal to one-half the percentage concentration of uncleaved folates contaminating the fraction. For exam-
Converts to S,lO=CH-H,-PteGlu Stable
Relatively stable Very labile, cleaved to pABG Stable
Stable
5, IO-CH,-PteGlu
IO-CHO-H,-PteGlu
5-CH,-H,-PteGlu S-CH,-Hz-PteGlu
pABG
IO-CHO-PteGlu,
IO-CHO-PteGlu
Unstable
S,lO=CH-If,-PteGlu
Converts to PteGlu, (slow) Stable
Stable
Converts to IO-CHO-H,-PteGlu Relatively stable at pH 9.5 Converts slowly to H,-PteGlu Converts to PteGlu (slow)
Unstable Unstable Stable
RelativeIy stable Unstable Converts to S,lO=CH-If,-PteGlu Stable
If,-PteGlu HJ’teGlu 5-CHO-H,-PteGlu
In base, pH 13.0 Stable
In acid, pH 1.0
Stability at room temperature
PteGlu
Compound
2
100
IO-CHO-PteGlu
pABG and unknown
IO-CHO-PteGlu,
65 (pABG); 35 (unknown)
100
79 93
100
lo-CHO-PteGlu
S-CH,-H,-PteGlu 5-CH&,-PteGlu
83
106
83
92
Yield (%)
IO-CHO-PteClu
IO-CHO-PteGlu
pABG pABG pABG
pABG
Product
KMnO, oxidation at pH 9.0
SUMMARYOF RESULTS
TABLE
Slow decomposition by oxidation y-Peptide linkage is stable to oxidation Yield after 20 min of oxidation
Very stable to oxidation at pH 9.0
Yield after 5 min of oxidation
Yield after 1 min of oxidation
Remarks
OXIDATIVE
CLEAVAGE
OF
FOLATES
293
ple, the presence of 20% uncleaved folates would result in 10% overestimation of the pool size. The lability of pABG to protracted oxidation, which results in loss of tritium label at a rate following first-order kinetics (Fig. 7), introduces an error ofopposite sign in the estimation of the relative size of the polyglutamate pools. Under the conditions currently used (oxidation at pH 9.0 for 20 min at room temperature) the tt ofpABG is approximately 34 min. Very substantial underestimations of the pool sizes of polyglutamates derived from cleavable folates can therefore result. Of additional importance is the observation (Krumdieck et al., unpublished observations) that the stable lo-CHO-PteGlu, formed during oxidation elute from DEAE-cellulose columns in positions different from those of PABG, markers having the same polyglutamyl chain length. Thus, the three atoms of 3H present in an uncleaved folate of chain length “n” would erroneously add to the estimation of the pool size cochromatographing with a pABG polyglutamate marker having “n + X” glutamyl residues. Furthermore, if the very acid-labile 5-CH,-H,-FteGlu,, are maintained intact during the chromatographic resolution of the oxidation mixture, three families of polyglutamates (5-CH,-Hz-PteGlu,, IO-CHO-PteGlu,, and pABG,) could be present and result in incomplete resolutions and poorly defined radioactivity elution profiles that do not follow exactly the uv absorption of the p ABG, synthetic markers. This problem has been encountered and discussed by Browner al. (10). Evidently, this source of error will be minimized if the mixture is acidified after oxidation whereby the 5-CH3II,-PteGlu, would cleave to their corresponding pABG,. The possibility of developing a method for the chain-length determination of certain pools of one-carbon-substituted folates is evident when one considers the use of [2-14C]folic acid as the labeled precursor administered to the experimental animals. Following oxidation and acidification of the folates of a tissue, the only polyglutamates bearing the 2J4C label would be the lo-CHO-BteGlu, derived from .5,lO=CH-H,-PteGlu,, 5,10-CH,-H,PteGlu,, and lo-CHO-H,-PteGlu,. The instability of the 5,10-CH,-II,PteGlu, at slightly acidic pH makes possible its destruction (34) prior to oxidation leaving only 5,10=CH-H,-PteGlu, and IO-CHO-H4-PteGlu,, the two coenzyme forms involved in purine biosynthesis, as the sole precursors of lo-CHO-F’teGlu,. An avenue for the determination of the chain length of the folates participating in vivo primarily in purine biosynthesis would be open. Furthermore, the use of doubly labeled [2-14C,3’,5’,9(n)3H]folic acid and the chromatography of the oxidation mixture before and after acidification should allow the identification and quantitation of the acid-labile pool of 5-CH,-HZ-PteGlu, derived from 5-CH,-H,-FteGlu,. It is worth noting that there is at present no chemical method that allows the simultaneous determination of chain length and nature of the one-carbon substituent. Available procedures depend heavily on differential microbiological assays combined with ion exchange fractionation procedures
294
MARUYAMA,
SHIOTA,
AND KRUMDIECK
and enzymatic cleavage of the polyglutamyl chain with pteroylpolyglutamyl hydrolases (39-41). The instability of pABG to alkaline KMnO, oxidation has been previously reported (42). The nature of the decomposition products formed remains, however, unanswered. The loss of Bratton-Marshall-positive material at the same rate at which the uv spectrum decreases without distortion suggests ring opening to an aliphatic compound. Oxidation to pnitrobenzoylglutamate has been ruled out by separation of the cochromatographed nitro compound, and the formation ofp-hydroxybenzoylglutamic acid is eliminated by the lack of uv absorption of the new radioactive peak of Fig. 7. The biphasic time course curve of oxidation of 5-CH,-H,-PteGlu suggests the instantaneous formation of an intermediate (reaction 1 in the scheme below) which can either go to 5-CH,-H,-PteGlu (reaction 2) or decompose to unknown products in the presence of KMnO, (reaction 3). 5-CH,--H,-PteGlu
(1) KMnO [intermediate] 4 KMnO, (3) Unknowi
(2)
+c 5-CH,--Hz-PteGlu
product(s)
The intermediate is thought to be an isomer of 5-CH,-H,-PteGlu occurring in equilibrium with the predominant W-methyl-5,6-dihydro form (37). Starting with 5-CH,-H,-PteGlu, KMnO, oxidation would yield an initially high concentration of the less stable intermediate, a significant fraction of which would rapidly decompose generating the early drop in the curve of Fig. 11. In keeping with this interpretation, the KMnO, oxidation of 5-CH3H,-PteGlu, which should proceed without the transient accumulation of the labile intermediate, generates a monophasic first-order curve. The rate of loss of 5-CH,-H,-PteGlu would depend on the equilibrium position of reaction 2. ACKNOWLEDGMENTS We gratefully acknowledge the skillful technical assistance of Mrs. Barbara Hudson. These investigations were supported by grants from the National Science Foundation, BMS-74-17348, and from the National Institute of Dental Research, DE-02670.
REFERENCES 1. Baugh, C. M., Braverman, E., and Nair, M. G. (1974) Biochemistry 13, 4952-4957. 2. Kisliuk, R. L., Gaumont, Y., and Baugh, C. M. (1974)J. Biol. Chem. 249, 4100-4103. 3. Moran, R. G., Werkheiser, W. C., and Zakrzewski, S. F. (1976) J. Biof. Chem. 251, 3569-3575. 4. Gawthome, J. M., and Smith, R. M. (1973) Biochem. J. 136, 295-301. 5. Shin, Y. S., Buehring, K. U., and Stokstad, E. L. R. (1972)J. Biol. Chem. 247, 72667269.
OXIDATJVE
CLEAVAGE
OF FOLATES
295
6. Buehring, K. U., Tamura, T., and Stokstad, E. L. R. (1974)J. Biol. Chem. 249, 10811089. 7. Hintze, D. N., and Farmer, J. L. (1975) J. Bacterial. 124, 1236-1239. 8. Leslie, G. I., and Baugh, C. M. (1974)Biochemistry 13, 4957-4961. 9. Houlihan, C. M., and Scott, J. M. (1972) Biochem. Biophys. Res. Commun. 48, 16751681. 10. Brown, .I. P., Davidson, G. E., and Scott, J. M. (1974) Eiochim. Biophys. ACM 343, 78-88. 11. Brown, J. P., Dobbs, F., Davidson, G. E., and Scott, J. M. (1974)J. Gen. Microbial. 84, 163-172. 12. Hoffbrand, A. V., Trippe, E., and Lavoie, A. (1976) C/in. Sci. Mol. Med. 50, 61-68. 13. Thompson, R. W., and Krumdieck, C. L. (1977) Amer. J. Clin. Nutr., in press. 14. Reed, B., Weir, D., and Scott. J. M. (1976) Biochem. Sot. Trans. 4. 906-907. 15. Scott, J. M. (1976) Biochem. Sot. Trans. 4, 845-850. 16. Scott, J. M. (1977) in Folic Acid, Biochemistry and Physiology in Relation to the Human Nutrition Requirements, pp. 43-55, Food and Nutrition Board, N.R.C., N.A.S., Washington, D. C. 17. Blakley, R. L. (1957) Biochem. J. 65, 331-342. 18. Rowe, P. B. (1968) Anal. Biochem. 22, 166-177. 19. Hatefi, Y., Talbert, P. T., Osborn, M. J., and Huennekens, F. M. (l%O)Biochem. Prep. 7, 89-92. 20. Huennekens, F. M., Ho, P. P. K., and Scrimgeour, K. G. (1963) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 6, pp. 806-811, Academic Press, New York. 21. Blakley, R. L. (1959) Biochem. J. 72, 707-715. 22. Krumdieck, C. L., and Baugh, C. M. (1969) Biochemistry 8, 1568-1572. 23. Chanarin, I., and Perry, .I. (1967) Biochem. J. 105, 633-634. 24. Flaks, J. G., and Lukens, L. N. (1963) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 6, pp. 52-99, Academic Press, New York. 25. Bray, G. A. (196O)Anal. Biochem. 1, 279-285. 26. Pohland, A., Flynn, E. H., Jones, R. G., and Shive, W. (195l)J. Amer. Chem. Sot. 73, 3247-3252. 27. Blakley, R. L. (1969) The Biochemistry of Folic Acid and Related Pteridines, p. 569, North-Holland, Amsterdam. 28. Rabinowitz, J. C. (1960) in The Enzymes (Boyer, P. D., Lardy, H., and MyrbBck, eds.), Vol. 2, pp. 185-252, Academic Press, New York. 29. Uyeda, K., and Rabinowitz, J. C. (1965)J. Biol. Chem. 240, 1701-1710. 30. Gupta, V. S., and Huennekens, F. M. (1967) Arch. Eiochem. Biophys. 120, 712-718. 31. Blakley, R. L. (1960)Eiochem. J. 74, 71-82. 32. Kallen, R. G., and Jencks, W. P. (1966) J. Biol. Chem. 241, 5845-5850. 33. Uyeda, K., and Rabinowitz, J. C. (1963) Anal. Biochem. 6, 100-108. 34. Osbom, M. J., Talbert, P. T., and Huennekens, F. M. (196O)J. Amer. Chem. Sot. 82, 4921-4927. 35. Scrimgeour, K. G., and Vitols, K. S. (1966) Biochemistry 5, 1438-1443. 36. Deits, T. L., Russell, A., Fujii, K., and Whitely, J. M. (1975) in Chemistry and Biology of Pteridins (Pfteiderer, W., ed.), pp. 525-534, Walter deGruyter, New York. 37. Donaldson, K. O., and Keresztesy, J. C. (1962) J. Biol. Chem. 237, 3815-3819. 38. May, M., Bardos, T. J., Barger, F. L., Landsford, M., Ravel, J. M., Sutherland, G. L., and Shive, W. (1951) J. Amer. Chem. Sot. 73, 3067-3075. 39. Osborne-White, W. S., and Smith, R. M. (1973) Eiochem. J. 136, 265-278. 40. Corrocher, R., Bhuyan, B. K., and Hoffbrand, A. V. (1972) Clin. Sci. 43, 799-813. 41. Brody, T., Shin, Y. S., and Stokstad, E. L. R. (1976) J. Neurochem. 27, 409-413. 42. Gapski, G. R., Whiteley, J. M., and Huennekens, F. M. (1971) Biochemistry 10, 29302934.
BIOCHEMISTRY
84,
277-295
The Oxidative
(1978)
Cleavage
A Critical TADASHI MARUYAMA,TETSUO
of Folates
Study
SHIOTA, AND CARLOS L. KRUMDIECK
Departments of Nutrition and Microbiology University of Alabama in Birmingham,
and The Institute of Dental Research, Birmingham, Alabama 35294
Received July 6, 1977; accepted August 16, 1977 Alkaline permanganate oxidation has been used to determine the chain length of naturally occurring pteroylpolyglutamates on the assumption that all forms of folates cleave at the P-N”’ bond to produce the corresponding p-aminobenzoylpolyglutamates. The chain length of the latter could be determined by cochromatography with synthetic markers. The products of alkaline (ammonium bicarbonate buffer, pH 9.0) permanganate oxidation of a number of reduced and oxidized, onecarbon-substituted and unsubstituted folic acid derivatives have been identified, and their yields and stability to the oxidative treatment have been determined. Unsubstituted, oxidized and reduced folic acid and Ns-formyl-tetrahydrofolic acid are cleaved at the C9-N1” bond to produce p-aminobenzoylglutamic acid. N>, N”‘-methenyl-tetrahydrofolic acid, NS,N”‘-methylene-tetrahydrofolic acid, and N”‘-formyl-tetrahydrofolic acid are not cleaved but are oxidized to N”‘-formyl-folic acid which is completely stable to the oxidative treatment employed. N5-methyltetrahydrofolic acid is not cleaved either but is oxidized to Ns-methyl-dihydrofolic acid which upon continued oxidation decomposes slowly to unidentified products. The y-glutamyl peptide linkage is completely stable to oxidation. Using p-amino[3,5-3H]benzoylglutamic acid, it is also shown that this product, previously thought to be stable to the oxidative treatment is decomposed by it. The significance ofthese findings in terms of the errors that may have been introduced in prior estimations of the chain length and pool sizes of the naturally occurring pteroylpolyglutamates is discussed. The possibility of developing a method for the chain length determination of noncleavable pools of one-carbon-substituted folates using [2-‘4C]folic acid to label the folates in vivo is presented.
In nature, the folate coenzymes constitute a complex family of closely related compounds the majority of which are pteroyl-y-glutamyl derivatives differing from one another in the number of glutamyl residues, the presence of a one-carbon substituent at N5 and/or N’O and the state of reduction of the pyrazine ring. The separation, identification, and quantitation of these coenzymes are a difficult undertaking complicated by abnormalities in the chromatographic behavior of the intact pteroylpolyglutamates. These abnormalities preclude the estimation of chain length based on the elution position of the reduced one-carbon-substituted pteroylpolyglutamates from ion exchange (l-3) or molecular sieve columns (4-6). 277
0003-2697/78/0841-0277$02.00/O Copyright 0 1978 by Academic press. Inc. All rights of reproduction in any form reserved
278
MARUYAMA,
SHJOTA,
AND
KRUMDIECK
To overcome these obstacles, cleavage of the Cg-N’O bond of suitably in vivo labeled folates was attempted, and the chain lengths of the resulting radioactive p-aminobenzoylpoly-y-glutamates (pABG,) were determined by cochromatography with synthetic markers. Both reductive (1,7,8) and oxidative (9- 12) treatments were employed with apparent success. However, testing of the cleaving procedures has been largely limited to demonstrating cleavage of folic acid and lack of attack on the poly-y-glutamyl chain (1,13). The possibility that oxidative or reductive cleavage of W”formyl-tetrahydrofolic acid (lo-CHO-H,-PteGlu) and N5,Wo-methenyltetrahydrofolic acid (5,10=CH-H,-PteGlu) could result in the formation of N-formyl-p-aminobenzoylglutamic acid or N-methyl-p-aminobenzoylglutamic acid, respectively, has been considered and dismissed as unimportant by Reed et al. (14), Scott (15), and Baugh et al. (1). Scott (16) summarily reported that alkaline permanganate oxidation at room temperature quantitatively cleaved the Cs-Nl” bond of folic acid (PteGlu), N5-formyltetrahydrofolic acid (5CHO-H,-PteGlu), tetrahydrofolic acid (H,-PteGlu), N5-methyl-tetrahydrofolic acid (5CH,-H,-PteGlu), N5,Wo-methylenetetrahydrofolic acid (5,10-CH,-H,-PteGlu), 5,10=CH-H,-PteGlu, dihydrofolic acid (HZ-PteGlu), N5-methyl-dihydrofolic acid (5-CH,-H,-PteGlu), lo-CHO-H,-PteGlu, and W”-formylfolic acid (lo-CHO-PteGlu). He stated that in all instances except the latter two, the product is p-aminobenzoylglutamic acid. The IO-formyl derivatives gave two products: pABG and formylaminobenzoyl glutamate which could not be separated with the DEAE-cellulose columns employed (16). Preliminary experiments in our laboratory indicating slight but definite separations of the products of permanganate oxidation of liver folates from the cochromatographed syntheticpABG, markers strongly suggested the presence of products other than pABG, and prompted a careful examination of the yield, products, and stability thereof resulting from the alkaline permanganate oxidative treatment of one-carbon-substituted folates. The results reported here demonstrate that, under the conditions used, some folates do not cleave at the Cs-N’O bond on alkaline permanganate oxidation, others do so readily, and still others cleave only after subsequent acidification. Furthermore, a significant decomposition of pABG upon prolonged oxidation has also been demonstrated. MATERIALS
AND METHODS
PteGlu, 5-CH,-H,-PteGlu (Ba salt), and pABG were purchased from Sigma Chemical Co. Prior to use the commercial folic acid was purified according to Blakley (17) and the 5-CH,-H,-PteGlu was purified by DEAEcellulose column chromatography (1.5 x 22 cm) using a linear gradient of 500 ml of water in the mixing chamber and 500 ml of 1 .OM ammonium bicarbonate in the reservoir. Pure 5-CHO-H,-PteGlu was a gift of Dr. John Montgomery from Southern Research Institute, Birmingham, Alabama.
OXIDATIVE
CLEAVAGE
OF
FOLATES
279
p-Nitrobenzoylglutamic acid was purchased from California Foundation for Biochemical Research. 5,lO=CH-H,-PteGlu was prepared by the method of Rowe (18) from II,-PteGlu obtained by catalytic hydrogenation with platinum oxide as described by Hatefi et al. (19). The S,lO=CH-H,-PteGlu was twice recrystallized from hot 0.1 N HCI in 0.1 M 2-mercaptoethanol prior to use. 5,10-CH,-H,-FteGlu was prepared as described by Huennekens et al. (20) and was purified by chromatography through a DEAE-cellulose column eluted using a linear gradient of ammonium bicarbonate buffer, pH 9.5, 0.004 to 0.4 M, 500 ml of each. lo-CHO-PteGlu and the N”-formyl-pteroylpolyglutamates were prepared by formylation of PteGlu and the corresponding pteroylpolyglutamates with 98% formic acid as described by Blakley (21). The pteroylpoly-y-glutamates were synthesized by the solid phase method of Krumdieck and Baugh (22) and purified by DEAE-cellulose chromatography as described. Tritiated [3’,5’,9(n)-3H]folic acid, 47 Ci/ mol, and [2-14C]folic acid, 58.2 mCi/mmol, were purchased from Amersham/Searle. Tritium-labeled pABG (3,5-3H) was prepared from [3’,5’,9(n)-3H]folic acid as follows: 5 mg of folic acid were dissolved in 4.6 ml of 0.1 M ammonium bicarbonate buffer, pH 9.0, and 20 &i of [3’,5’,9(n)-3H]folic acid were added. The solution was oxidized with 0.5 ml of 2% KMnO, for 2 min. The reaction was terminated by the addition of 0.5 ml of 30% H,O,. Following removal of the MnO,, the supernatant was applied to a DEAE-cellulose column (1.5 x 22.0 cm) and eluted with a linear gradient of water to 1.O M NH4HC03, 500 ml of each. ThepABG-containing fractions were pooled, lyophilized, and a concentrated solution applied to a cellulose column (1.5 x 40 cm). The column was eluted with 0.2 M NH,HCO, to remove fluorescent materials, and thepABG-containing fractions were pooled and lyophilized. The purified product had a specific activity of 0.796 &i/~mol. Doubly labeled 5-CH,-Z~Z,-[2-~~C,3’,5’,9(n)-~H]PteGlu was prepared by catalytic reduction of 50 pmol of folic acid to which 5.0 PCi of [2-14C]folic acid and 25.0 &i of [3’,5’,9(n)-3H]folic acid had been added. The doubly labeled tetrahydrofolic acid obtained was converted to 5,lO = CH-H,F’teGlu and reduced with sodium borohydride to the desired 5-CH,-H,PteGlu (23), and this compound was purified through a DEAE-cellulose (Cl-) column using an elution gradient of 0.2 M NaCl in 0.01 M 2-mercaptoethanol to 0.7 M NaCl in 0.01 M 2-mercaptoethanol, 500 ml of each. After desalting, the spectrally pure product was lyophylized and stored in sealed evacuated ampoules. Doubly labeled 5-CH3-H,-PteGlu was prepared by oxidation of the doubly labeled 5-CH3-H,-PteGlu as follows: one milligram of carrier commercial 5-CH,-H,-PteGlu was added to 0.27 mg of 5-CH,H4-[2-14C,3’,5’,9(n)-3H]PteGlu in a total volume of 2.0 ml of 0.1 M NH, HCO,, pH 9.0. Fifty microliters of H,O, were added before and after the addition of 100 ~1 of 2% KMnO,. After removal of the precipitate, the prod-
280
MARUYAMA,
SHIOTA,
AND
KRUMDIECK
uct was purified through a DEAE-cellulose column (1.5 x 22.0 cm) eluted with a linear gradient of 0.0 to 0.5 M NH4HC03, 500 ml of each. The purified product was then lyophilized. The final specific activity was 0.022 &i/ ,umol for 14C and 0.154 pCi/pmol for 3H. Ultraviolet spectra were obtained with a Hitachi-Perkin Elmer 124 spectrophotometer. Reference spectra and molar extinction coefficients were obtained from the literature. The molar extinction coefficients used are presented in Table 1. The Bratton-Marshall test (24) was used to determine the concentration ofpABG in solution and to qualitatively detect it in thin-layer plates. High-pressure liquid chromatography (hplc) was carried out using a Waters hplc system with a weak anion exchanger column of Reeve Angel AL Pellionex wax. All separations were done at room temperature and 1.5 mllmin flow rate. The’pressure was approximately 1000 psi. A Waters Model 440 detector and a Varian A-25 recorder were used to monitor the optical density of the effluent at 254 nm. Linear gradients of potassium phosphate buffer, pH 7.0, from 0.001 to 0.1 or 0.3 M in 30 min were constructed by a Waters Model 660 solvent programmer. Five-drop fractions (approximately 0.35 ml) were collected when required using an LKB Ultrorat 7000 fraction collector. The volume of sample applied was usually 10 to 20 ~1 but was increased to 50 or 100 ~1 when fractions were collected for subsequent analysis. All radioactivity measurements were performed with a Packard Tri-Carb liquid scintillation spectrometer using Bray’s solution (25) as a counting fluid. Cellulose plates (Polygram Cel 300, Macherey-Nagel) were used for thin-layer chromatography. The solvents used were: (A) 3% NH4Cl and (B) I-propanol:l% NH,OH, 2:l by volume. TABLE SPECTRAL
Compound PteGlu IO-CHO-PteGlu S-CHO-H,-F’teGlu S-CH,-I&-PteGlu 5-CH,-H,-PteGlu 5,lO=CH-H,-PteGlu 5, IO-CH,-If,-PteGlu pABG
Molar extinction coefficient at A max (X 10-S) 23.4 20.9 37.2 31.7 31.2 26.5 32.0 15.4
1 DATA
A max (nm) 283 269 285 290 290 348 294 273
Conditions in in in in in in in in
0.1 pH pH pH pH 1N pH 0.1
N NaOH 7.0 7.0 7.0 7.0 HCl 7.2 N NaOH
References (26727) (27728)
(2% (30) (30)
cw (31) (32)
OXIDATIVE
CLEAVAGE
OF FOLATES
281
Oxidation procedure. The folates (0.175 to 1.21 mM; the amounts varied with the availability of the various forms) were dissolved in 0.1 M ammonium bicarbonate buffer, pH 9.0, and oxidized by adding 2% KMnO, in an amount equal to one-tenth the volume of the folate-ammonium bicarbonate solution. All operations were performed at room temperature under a red light. At the end of the oxidation period, the excess KMnO, was destroyed with 30% H,O, (volume equal to that of 2% KMnO,), and the MnO, precipitate was removed by centrifugation. Distilled water was added to the control samples in place of the KMnO, and H,O, solutions. The time of oxidation in all the experiments done for the purpose of identification of oxidation products was 20 min. In the experiments designed to follow the rate of oxidation by permanganate, the time-zero samples correspond to reactions where the KMnO, was initially destroyed by the prior addition of HzOz. A very brief period of oxidation by H,O, or nascent oxygen unavoidably took place. RESULTS
I. Identijication
of the Oxidation
Products of Folates
(a) Folic acid, tetrahydrofolic acid, and dihydrofolic acid. The results of hplc of folic acid before and after KMnO, oxidation are shown in Fig. 1 a. The peak of folic acid disappeared after oxidation and was replaced by O5r (b)
0.12 0 IO E 5 0.06 @ dOO6 d 004
5 Time
IO in mmtes
FIG. 1. Oxidation of folic acid. (a) The hplc elution profile of PteGlu before (---) and after (-) oxidation. Ten milliliters of 0.176 mM PteGlu were oxidized with 0.4 ml of 2% KMnO, for 20 min. (b) The uv spectra of PteGlu (before) and its oxidation product (7.4-min peak) (after).
282
MARUYAMA,
SHIOTA,
AND KRUMDIECK
two new peaks. The first was identified as pABG by uv spectra (Fig. 1 b) and by tic (solvent A). Only one Bratton-Marshall positive spot migrating together with apABG marker (R, = 0.9) was detected. The second peak, which was fluorescent, cochromatographed with pterin-6-carboxylic acid (P-6-COOH). It is obvious that the Cg-N’O bond of folic acid is cleaved and that the major products of the reaction are pABG and P-6-COOH. H4PteGlu and H,-PteGlu are also cleaved at the Cg-N’O bond by KMnO, oxidation each producing pABG and P-6-COOH. (6) N5,N10-Methenyl-tetruhydrofolic acid. In neutral or alkaline conditions 5,10=CH-H,-PteGlu converts rapidly to lo-CHO-H,-PteGlu. Therefore, under the conditions of our experiments (15 min at pH 9.0 prior to oxidation), the oxidation of the bridge compound represents in effect oxidation of lo-CHO-H,-PteGlu. The possibility of formation of N-formylpABG instead ofpABG (15) was of considerable interest. Figure 2a shows the hplc pattern obtained after the oxidation of 5,10=CH-H,-PteGlu. A substance eluting as a major single peak after the position of elution of p ABG and which fluoresced blue under uv (365 nm) illumination was found. The uv spectra of this substance and of the starting material are shown in Fig. 2 b. The oxidation product was identified spectrally as loCHO-PteGlu (28) which was previously shown to be fluorescent (33). This product cochromatographed with authentic IO-CHO-PteGlu on thin-layer plates (R, = 0.77 with solvent A). Furthermore, the uv spectrum of the product treated with 1 .O N NaOH overnight at room temperature changed to that of folic acid, in keeping with the previously reported deformylation of lo-CHO-PteGlu in alkaline solutions (21,26).
5 IO Time in minutes
15
FIG. 2. Oxidation of NS,N’O-methenyl-tetrahydrofolic acid. (a) The hplc elution profile of the oxidation product of 5,10=CH-H,-F’teGlu. Five-tenths milliliter of 0.195 IIIM S,lO=CHH,-PteGlu was oxidized with 0.05 ml of 2% KMn04 for 20 min. (b) The uv spectra of 5, IO&HH,-PteGlu (before) and its oxidation product (after).
OXIDATIVE
CLEAVAGE
OF FOLATES
283
(c) N5,N10-Methylene-tetrahydrofolic acid. In contrast to 5,10=CH-H,PteGlu, 5,10-CH,-H,-PteGlu is stable at pH 9.5 and unstable in acidic solutions (34). The DEAE-cellulose column purified material had a uv spectrum (Fig. 3 b) identical to that reported by Blakley (31) but still contained some impurities as indicated by the hplc elution profile in Fig. 3 a. Following oxidation, a major product eluted in the position of IO-CHO-PteGlu and had its characteristic spectra (Fig. 3 b) and fluorescence. Of the smaller peaks, one cochromatographed with added pABG on both hplc and tic plates. The other minor peaks eluting with the solvent front and at 5 min were not identified. It is uncertain whether these minor products arise from oxidation of 5,10-CH,-H,-PteGlu or from impurities contaminating the starting material. (d) N5-Formyf-tetrahydrofolic acid. The hplc elution profile of 5-CHOH,-PteGlu is shown by the broken line of Fig. 4 a. It eluted as a single material slightly after the position of elution of pABG and had a uv spectrum identical to that previously reported (26). The major product after oxidation was identified as pABG spectrally and by cochromatography with standardpABG on both hplc and tic. Only one Bratton-Marshall positive spot was detected after tic with solvent A. A second faintly BrattonMarshall positive spot (Z?, = 0.19) separated frompABG (R, = 0.50) with solvent B. The two minor peaks appearing after oxidation (eluting with the solvent front and at 6.4 min.) were not identified. The absence of the
Time m minutes
3. Oxidation of N5,N1”-methylene-tetrahydrofolic acid. (a) The hplc elution profile of 5,10-CH,-H,-PteGlu before (---) and after (-) oxidation. One milliliter of 0.260 mM 5,10CH,-H.,-PteGlu was oxidized with 0.1 ml of 2% KMnO, for 20 min. (b) The uv spectra of 5,10-CH,-H,-PteGlu (before) and its main oxidation product (8.6min peak) (after). FIG.
284
MARUYAMA,
SHIOTA,
AND KRUMDIECK
(a)
0.06
t
o.o+
Time
in minutes
FIG. 4. Oxidation of@formyl-tetrahydrofolic acid. (a) The hplc elution profile of S-CHOH,-PteGlu before (---) and after (-) oxidation. Ten milliliters of 0.272 mM 5-CHO-H,PteGlu were oxidized with 0.5 ml of 2% KMnO, for 20 min. (b) The uv spectra of SCHOH,-F’teGlu (before) and its oxidation product (7.3-min peak) (after).
strongly fluorescent P-6-COOH worth noting.
peak seen after oxidation
of folic acid is
(e) N5-Methyl-tetrahydrofolic acid. The hplc elution profiles of 5-CH,H,-PteGlu before and after oxidation are shown in Fig. 5 a. The starting material had a uv spectrum (Fig. 5 b) identical to that reported by Gupta et al. (30). The product was spectrally identical to S-CH,-H,-FteGlu (Fig. 5 b) (30), which is known to elute before 5-CH,-H,-PteGlu from DEAEcellulose columns (35). Since 5-CH,-Hz-PteGlu is very acid labile and undergoes cleavage of the Cg-Nl” bond to producepABG (36), the product of oxidation was treated with 0.1 N HCl at room temperature for a few minutes and reapplied to the hplc after removal of the acid by flash evaporation. As expected pABG was obtained and identified spectrally and by tic. Furthermore, the product of oxidation could characteristically be converted back to 5-CH,-H,-PteGlu by reduction for 6 hr at room temperature with 1% mercaptoethanol(37). The uv spectrum of 5-CH,-H,-PteGlu was obtained after hplc purification of the reduction mixture. II. Efficiency and Rate of KMnO, Oxidation Products
Oxidation of Folates: Stability of the acid. The rate of cleavage was determined by measuring
(a) Folic acid and N5-formyl-tetrahydrofolic
of F’teGlu and 5-CHO-H,-PteGlu
to pABG
OXIDATIVE
CLEAVAGE
OF FOLATES
285
Time in minutes FIG. 5. Oxidation of W-methyl-tetrahydrofolic acid. (a) The hplc elution profile of 5-CH,H.,-Pte-Glu before (- - -) and after (-) oxidation. One milliliter of 1.21 mM 5-CH3-H,F’teGlu was oxidized with 0.1 ml of 2% KMnO, for 20 min. (b) The uv spectra of 5-CH,H,-PteGlu (before) and its oxidation product (after).
the amount ofpABG formed after various times of oxidation. The Bratton Marshall test was used to quantitate thepABG. Figure 6 is a semilogarithmic plot of the results obtained. The ordinate shows the pABG yield as a percentage of the amount of folate present in the control. Folic acid was rapidly cleaved to pABG (92% yield in 1 min) which in turn was more slowly converted to nondiazotizable products upon continued oxidation. Essentially the same results were obtained when 5-CHO-H,-PteGlu was oxidized. The most noticeable difference is that SCHO-H,-PteGlu is
IO 20 30 40 Time of oxidation (minutes)
50
FIG. 6. Time course of oxidation of folic acid (0) and N”-formyl-tetrahydrofolic acid (0). Two milliliter samples of 0.796 mM PteGlu and 0.498 mM 5-CHO-H,-F’teGlu were oxidized with 0.2 ml of 2% KMnO, for the various times indicated. The reactions were terminated by the addition of 0.2 ml of 30% H202. Duplicate samples were assayed at each time.
286
MARUYAMA,
SHIOTA,
AND KRUMDIECK
cleaved at a somewhat slower rate as indicated by a 72 to 77% yield of pABG after 1 min of oxidation. The rate of decomposition of the pABG formed was the same as in the experiment with folic acid. To confirm this unexpected finding, 3,S3H-IabeledpABG was synthesized and oxidized as described below. (6) p-Amino[S,5-3H]benzoylglutamic acid. The oxidation of 3,S3H-labeled pABG (sp act, 0.796 &i/pmol) was carried out as follows: 0.415 pmol of tritium-labeled pABG dissolved in 1.O ml of 0.1 M ammonium bicarbonate, pH 9.0, was oxidized for periods of 20 and 40 min with 0.1 ml of 2% KMnO,. One-tenth milliliter of 30% Hz02 was added at the end of the oxidation periods and prior to the addition of permanganate in the time -zero sample. The uv spectra and Bratton-Marshall determinations were carried out, O.l-ml aliquots were chromatographed on hplc, and fractions collected for radioactivity measurements. Figure 7 b shows the spectral changes in 0.1 N NaOH with increasing oxidation times. There is a pronounced drop in absorbancy at yrnax = 273 nm. The recovery of pABG calculated from the optical density at 273 nm after 20 and 40 min of oxidation is shown by the solid line of Fig. 7 a. The dot-dash line in the same figure shows recovery based on the Bratton-Marshall test, and the dotted line recovery of radioactivity eluting in the position of pABG after hplc chromatography of the oxidation mixtures. All three methods gave results that are in very close agreement. Figure 8 shows the radioactivity elution pattern obtained after hplc of the 0; 20-, and 40-min oxidation samples. A new radioactive species which is neither uv-absorbing nor BrattonMarshall-positive and that accounts for the total loss of label from pABG was formed during oxidation. The hplc cochromatography of p-nitro06-
(b)
omm-
E
IO
20
Time of oxidation
30
40
(minutes)
FIG. 7. Time course of oxidation ofp-amino[3,5-3H]benzoylglutamic acid. (a) Semilogarithmic plot of percentage recovery vs oxidation time. See text for details. (b) Spectral changes with increasing oxidation time.
OXIDATIVE
CLEAVAGE
287
OF FOLATES
-
Omin.
. . ..
l .
.
20rn,“,
---C--40lW” I
3,000
0.06
1s
0.05
8 2 ; 2
2,000
0.04
003
.$ _o E t 2 Y
002
1.000
Fratin
number
8. Oxidation ofp-amino[3,5-3H]benzoylglutamate. The hplc elution profiles ofradioactivity from 3,5JH-labeled pABG oxidized with KMnO, for 0, 20, and 40 min. FIG.
benzoylglutamate with the oxidation mixture ofpABG (40 min) resulted in a clear separation of the nitro compound, ruling it out as one of the possible products of pABG oxidation. Oxidation of 2.0 mM 3,S3H-labeled pABG for 60 min resulted in the formation of a third minor (8.8% of total radioactivity) product which eluted after pABG and had uv absorption maxima at 330 nm at pH 7.0, 1.0, and 13.0. (c) N5,N10-Methenyl-tetrahydrofolic acid, N5,N10-methylene-tetrahydrofolic
acid, N1o-formyl-tetrahydrofolic acid, and N’O-formyl-folic acid.
The stability of lo-CHO-PteGlu, the main product of KMnO, oxidation of 5,10=CH-H,-PteGlu and 5,10-CH,-H,-PteGlu was studied next. A sample of synthetic lo-CHO-PteGlu was subjected to oxidation for 20 min and then chromatographed in the hplc system. A single peak eluted in the same position as lo-CHO-PteGlu. After oxidation, the uv spectrum of the material was unchanged and was identical to that of lo-CHO-PteGlu and the product cochromatographed on thin-layer plates with the authentic marker. Both the product of oxidation and the synthetic marker were stable for up to 2 days at room temperature in 1.0 N NH,OH and in solutions buffered at pH values of 6.0, 7.0, 9.0, and 9.5. It was completely stable in 0.1 N HCl for 60 min at room temperature. Figure 9 shows the yields and rates of formation of lo-CHO-F’teGlu by the oxidation of 5,10=CH-H,-PteGlu and 5,10-CH,-H,-PteGlu. It also shows that lo-CHO-PteGlu is stable to KMnO, oxidation for up to 40 min. The amounts of IO-CHO-PteGlu were determined spectrophotometrically from the OD at 269 nm at pH 7.0. In the case oxidation of lo-CHO-PteGlu, the optical density at 269 nm was measured directly after centrifugation to remove the MnO, precipitate. In
288
MARUYAMA,
SHIOTA,
AND KRUMDIECK
IO 20 30 40 Time of oxidation (minutes) FIG. 9. Time course of oxidation of N5, N’“-methenyl-tetrahydrofolic acid (0), N5, NLumethylene-tetrahydrofolic acid (O), and N1o-formyl-folic acid (x). Samples of 0.5 ml of 0.195 mM 5,lO=CH-H,-PteGlu, 1.0 mlof0.313 mM 5,10-CH,-If,-PteGlu, and 2.0mlof0.838 mM loCHO-PteGlu were oxidized with one-tenth their volumes of 2% KMnO, for the various times indicated. Reactions were terminated by the addition of H,O,.
the other two cases, after stopping the oxidations at the appropriate times, the reaction mixtures were chromatographed on hplc, and the fractions corresponding to IO-CHO-PteGlu were combined and concentrated by flash evaporation prior to quantitation at 269 nm. The results indicate that IO-CHO-PteGlu is completely stable to the oxidative conditions used. The 5,10=CH-H,-PteGlu was dissolved in 0.1 M ammonium bicarbonate, pH 9.0, and kept 15 min at room temperature prior to oxidation to assure its conversion to lo-CHO-H,-PteGlu. Following the addition of permanganate, lo-CHO-H,-PteGlu was rapidly oxidized to lo-CHO-PteGlu via an intermediate believed to be the same substance as the product of air oxidation of IO-CHO-H,-PteGlu. The latter elutes as a sharp single peak on hplc, and its uv spectra are shown in Fig. 10. This substance is thought to be W”formyl-dihydrofolic acid (38). The yield of the oxidation of 5,10=CH-H,PteGlu to IO-CHO-PteGlu was slightly above 100%. The yield of lo-CHOF’teGlu obtained by oxidation of 5,10-CH,-H,-F’teGlu was only 82 to 83%. This reaction also seems to proceed through the formation of an intermediate which could be detected as a minor peak eluting slightly after pABG in the hplc chromatograms of samples oxidized for less than 1 min. (d) N5-Methyl-tetrahydrofolic acid and N5-methyl-dihydrofolic acid. The formation of 5-CH,-H,-PteGlu by the oxidation of 5-CH,-II,-PteGlu proceeds very rapidly after the addition of KMnO,. A characteristic spectrum at pH 7.0 with A,,, at 249 and 289 nm and a ratio of OD valves at 290/250 nm of 1.39 is obtained. The yield of this reaction based on spectral quantitation is 99%. The time course of oxidation of 5-CH,-H,-PteGlu by permanganate is shown in Fig. 11 (solid line). The percentage recovery of 5-CH3-H,-F’teGlu was calculated from the optical density at 290 nm of solu-
OXIDATIVE
CLEAVAGE
OF FOLATES
289
I 0.6- I !I I
0.'
I I
d
0.3 -
0
>. I’ \
I/
\ \ \
0.2 -
\ .-_ \ ’ ---.*.
0.1 1
200
--., \1, h 3 4 C)O 250 300 350 Wave Length
nm
FIG. 10. The uv spectra of the air oxidation product of N’“-formyl-tetrahydrofolic
acid.
tions adjusted to pH 7.0 after removing the MnO, precipitate. The curve shows a rapid initial loss of product that changes to a much slower rate. The uv spectra of the reaction mixtures at pH 7.0 showed a rapid drop in the 289-nm peak with little or no change in the peak at 249 nm. The 290/250-nm ratio decreased from approximately 1.4 at zero time to 1.27, 1.22, 1.20, 1.17, and 1.14 at 1, 5, 10, 20, and 40 min, respectively. An hypsochromic
$1 5 a
I IO Time
I I I 20 30 40 of axidation(minutes)
FIG. 11. Time course of oxidation of W-methyl-dihydrofolic acid (0) and W-methyl-tetrahydrofolic acid (x). Duplicate samples of 2.0 ml of 0.524 mM 5-CH,-H,-PteGlu and 1.0 ml of 0.407 mM 5-CH,-Hz-PteGlu were oxidized with one-tenth their volumes of 2% KMnO, for the various times indicated. Reactions were terminated by the addition of H,O*.
290
MARUYAMA,
SHIOTA,
AND KRUMDIECK
shift to a uv maximum at 285 nm was also observed. The time course of permanganate oxidation of 5-CH,-H,-PteGlu is shown by the dotted line in Fig. 11. The material decomposes with increasing oxidation time following first-order kinetics. Spectral changes essentially identical to those described during the oxidation of 5-CH,-H,-PteGlu were observed. The 290/ 250-nm ratio dropped from 1.39 at zero time to 1.33, 1.3 1, 1.30, 1.25, and 1.19 at 1, 5, 10, 20, and 40 min of oxidation, respectively. (e) Acid cleavage of 5-methyl-dihydrofolic acid. To study the products and yield of the acid cleavage of 5-CH,-H,-F’teGlu, doubly labeled 5-CH,Hz-[2-14C,3’,5’,9(n)-3H]PteGlu was synthesized as described under Materials and Methods. The doubly labeled compound was acid treated by dissolving 0.195 pmol of it in 0.2 ml of 0.1 N HCI. After 1 min at room temperature, the acid was removed by thrice-repeated flash evaporation, and the final residue was dissolved in 0.15 ml of 0.1 M NH4HC03 buffer, pH 9.0. One-hundred microliters were injected into the hplc apparatus. A solution of the doubly labeled 5-CH,-H,-PteGlu served as control. The results are shown in Fig. 12. Two peaks appeared after acid treatment. The first peak was doubly labeled, and the second (pABG) contained almost exclusively tritium label with trace amounts of 14C (Fig. 12 b). Thirty percent of the 3H in the doubly labeled 5-CH,-H,-PteGlu appeared in the first peak and 45% in the pABG peak. The total recovery of 14C was 96%, but only 80% of the total 3H was accounted for. Some tritiated volatile compound(s) (3H20?) must have been lost during the flash evaporation step. (J> N”‘-Formyl-pteroylheptaglutamate. The hplc elution profile of loCHO-PteGlu, after KMnO, oxidation is shown in Fig. 13. The numbered
Fraction
FIG.
ment.
Number
12. 5-Methyl-dihydro[2-‘4C,3’,5’,9(n)-3H]folic
acid before (a) and after (b) acid treat-
OXIDATIVE
CLEAVAGE
OF FOLATES
291
0.145 0.12 O.lO-
0.02-
0
5
IO
15
20
Time in minutes FIG. 13. Oxidation of WO-formyl-pteroylheptaglutamate. One milliliter CHO-PteGlu, was oxidized with 0.1 ml of 2% KMnO, for 20 min.
of 0.502 mM lo-
arrows indicate the position of elution of the lo-CHO derivatives of pteroylmono-, tri-, hexa-, and heptaglutamates, respectively. A single symetrical peak eluting in the same position as before was obtained after oxidation of lo-CHO-PteGlu,. Table 2 summarizes all the results obtained. DISCUSSION
Our results contradict the assumptions (i) that alkaline permanganate oxidation cleaves the Cg-Nl” bond of all forms of folates (16) and (ii) that the pABG resulting from the cleavage of susceptible folates is stable to alkaline permanganate treatment (15). Together with the herein verified stability of the y-peptide linkages of the pteroylpolyglutamates to KMnO, oxidation, the above assumptions are central to the oxidative cleavage procedure used extensively to determine the chain length of polyglutamates and to quantitate the relative size of the various polyglutamate pools in biological materials. Our findings indicate that substantial errors may be introduced in these determinations. Since the radioactive precursor commonly used to label the folates of experimental animals prior to their extraction and oxidation is [3’,5’,9(n )-3H]folic acid, the presence of uncleaved folates (still bearing the 3H atom on Cg) in a fraction presumed to contain only 3,5-3H-labeled p ABG, of a given chain length, will result in a falsely high estimation of the size of that particular polyglutamate pool. If this were the only source of error it is calculated that it would amount to an overestimation equal to one-half the percentage concentration of uncleaved folates contaminating the fraction. For exam-
Converts to S,lO=CH-H,-PteGlu Stable
Relatively stable Very labile, cleaved to pABG Stable
Stable
5, IO-CH,-PteGlu
IO-CHO-H,-PteGlu
5-CH,-H,-PteGlu S-CH,-Hz-PteGlu
pABG
IO-CHO-PteGlu,
IO-CHO-PteGlu
Unstable
S,lO=CH-If,-PteGlu
Converts to PteGlu, (slow) Stable
Stable
Converts to IO-CHO-H,-PteGlu Relatively stable at pH 9.5 Converts slowly to H,-PteGlu Converts to PteGlu (slow)
Unstable Unstable Stable
RelativeIy stable Unstable Converts to S,lO=CH-If,-PteGlu Stable
If,-PteGlu HJ’teGlu 5-CHO-H,-PteGlu
In base, pH 13.0 Stable
In acid, pH 1.0
Stability at room temperature
PteGlu
Compound
2
100
IO-CHO-PteGlu
pABG and unknown
IO-CHO-PteGlu,
65 (pABG); 35 (unknown)
100
79 93
100
lo-CHO-PteGlu
S-CH,-H,-PteGlu 5-CH&,-PteGlu
83
106
83
92
Yield (%)
IO-CHO-PteClu
IO-CHO-PteGlu
pABG pABG pABG
pABG
Product
KMnO, oxidation at pH 9.0
SUMMARYOF RESULTS
TABLE
Slow decomposition by oxidation y-Peptide linkage is stable to oxidation Yield after 20 min of oxidation
Very stable to oxidation at pH 9.0
Yield after 5 min of oxidation
Yield after 1 min of oxidation
Remarks
OXIDATIVE
CLEAVAGE
OF
FOLATES
293
ple, the presence of 20% uncleaved folates would result in 10% overestimation of the pool size. The lability of pABG to protracted oxidation, which results in loss of tritium label at a rate following first-order kinetics (Fig. 7), introduces an error ofopposite sign in the estimation of the relative size of the polyglutamate pools. Under the conditions currently used (oxidation at pH 9.0 for 20 min at room temperature) the tt ofpABG is approximately 34 min. Very substantial underestimations of the pool sizes of polyglutamates derived from cleavable folates can therefore result. Of additional importance is the observation (Krumdieck et al., unpublished observations) that the stable lo-CHO-PteGlu, formed during oxidation elute from DEAE-cellulose columns in positions different from those of PABG, markers having the same polyglutamyl chain length. Thus, the three atoms of 3H present in an uncleaved folate of chain length “n” would erroneously add to the estimation of the pool size cochromatographing with a pABG polyglutamate marker having “n + X” glutamyl residues. Furthermore, if the very acid-labile 5-CH,-H,-FteGlu,, are maintained intact during the chromatographic resolution of the oxidation mixture, three families of polyglutamates (5-CH,-Hz-PteGlu,, IO-CHO-PteGlu,, and pABG,) could be present and result in incomplete resolutions and poorly defined radioactivity elution profiles that do not follow exactly the uv absorption of the p ABG, synthetic markers. This problem has been encountered and discussed by Browner al. (10). Evidently, this source of error will be minimized if the mixture is acidified after oxidation whereby the 5-CH3II,-PteGlu, would cleave to their corresponding pABG,. The possibility of developing a method for the chain-length determination of certain pools of one-carbon-substituted folates is evident when one considers the use of [2-14C]folic acid as the labeled precursor administered to the experimental animals. Following oxidation and acidification of the folates of a tissue, the only polyglutamates bearing the 2J4C label would be the lo-CHO-BteGlu, derived from .5,lO=CH-H,-PteGlu,, 5,10-CH,-H,PteGlu,, and lo-CHO-H,-PteGlu,. The instability of the 5,10-CH,-II,PteGlu, at slightly acidic pH makes possible its destruction (34) prior to oxidation leaving only 5,10=CH-H,-PteGlu, and IO-CHO-H4-PteGlu,, the two coenzyme forms involved in purine biosynthesis, as the sole precursors of lo-CHO-F’teGlu,. An avenue for the determination of the chain length of the folates participating in vivo primarily in purine biosynthesis would be open. Furthermore, the use of doubly labeled [2-14C,3’,5’,9(n)3H]folic acid and the chromatography of the oxidation mixture before and after acidification should allow the identification and quantitation of the acid-labile pool of 5-CH,-HZ-PteGlu, derived from 5-CH,-H,-FteGlu,. It is worth noting that there is at present no chemical method that allows the simultaneous determination of chain length and nature of the one-carbon substituent. Available procedures depend heavily on differential microbiological assays combined with ion exchange fractionation procedures
294
MARUYAMA,
SHIOTA,
AND KRUMDIECK
and enzymatic cleavage of the polyglutamyl chain with pteroylpolyglutamyl hydrolases (39-41). The instability of pABG to alkaline KMnO, oxidation has been previously reported (42). The nature of the decomposition products formed remains, however, unanswered. The loss of Bratton-Marshall-positive material at the same rate at which the uv spectrum decreases without distortion suggests ring opening to an aliphatic compound. Oxidation to pnitrobenzoylglutamate has been ruled out by separation of the cochromatographed nitro compound, and the formation ofp-hydroxybenzoylglutamic acid is eliminated by the lack of uv absorption of the new radioactive peak of Fig. 7. The biphasic time course curve of oxidation of 5-CH,-H,-PteGlu suggests the instantaneous formation of an intermediate (reaction 1 in the scheme below) which can either go to 5-CH,-H,-PteGlu (reaction 2) or decompose to unknown products in the presence of KMnO, (reaction 3). 5-CH,--H,-PteGlu
(1) KMnO [intermediate] 4 KMnO, (3) Unknowi
(2)
+c 5-CH,--Hz-PteGlu
product(s)
The intermediate is thought to be an isomer of 5-CH,-H,-PteGlu occurring in equilibrium with the predominant W-methyl-5,6-dihydro form (37). Starting with 5-CH,-H,-PteGlu, KMnO, oxidation would yield an initially high concentration of the less stable intermediate, a significant fraction of which would rapidly decompose generating the early drop in the curve of Fig. 11. In keeping with this interpretation, the KMnO, oxidation of 5-CH3H,-PteGlu, which should proceed without the transient accumulation of the labile intermediate, generates a monophasic first-order curve. The rate of loss of 5-CH,-H,-PteGlu would depend on the equilibrium position of reaction 2. ACKNOWLEDGMENTS We gratefully acknowledge the skillful technical assistance of Mrs. Barbara Hudson. These investigations were supported by grants from the National Science Foundation, BMS-74-17348, and from the National Institute of Dental Research, DE-02670.
REFERENCES 1. Baugh, C. M., Braverman, E., and Nair, M. G. (1974) Biochemistry 13, 4952-4957. 2. Kisliuk, R. L., Gaumont, Y., and Baugh, C. M. (1974)J. Biol. Chem. 249, 4100-4103. 3. Moran, R. G., Werkheiser, W. C., and Zakrzewski, S. F. (1976) J. Biof. Chem. 251, 3569-3575. 4. Gawthome, J. M., and Smith, R. M. (1973) Biochem. J. 136, 295-301. 5. Shin, Y. S., Buehring, K. U., and Stokstad, E. L. R. (1972)J. Biol. Chem. 247, 72667269.
OXIDATJVE
CLEAVAGE
OF FOLATES
295
6. Buehring, K. U., Tamura, T., and Stokstad, E. L. R. (1974)J. Biol. Chem. 249, 10811089. 7. Hintze, D. N., and Farmer, J. L. (1975) J. Bacterial. 124, 1236-1239. 8. Leslie, G. I., and Baugh, C. M. (1974)Biochemistry 13, 4957-4961. 9. Houlihan, C. M., and Scott, J. M. (1972) Biochem. Biophys. Res. Commun. 48, 16751681. 10. Brown, .I. P., Davidson, G. E., and Scott, J. M. (1974) Eiochim. Biophys. ACM 343, 78-88. 11. Brown, J. P., Dobbs, F., Davidson, G. E., and Scott, J. M. (1974)J. Gen. Microbial. 84, 163-172. 12. Hoffbrand, A. V., Trippe, E., and Lavoie, A. (1976) C/in. Sci. Mol. Med. 50, 61-68. 13. Thompson, R. W., and Krumdieck, C. L. (1977) Amer. J. Clin. Nutr., in press. 14. Reed, B., Weir, D., and Scott. J. M. (1976) Biochem. Sot. Trans. 4. 906-907. 15. Scott, J. M. (1976) Biochem. Sot. Trans. 4, 845-850. 16. Scott, J. M. (1977) in Folic Acid, Biochemistry and Physiology in Relation to the Human Nutrition Requirements, pp. 43-55, Food and Nutrition Board, N.R.C., N.A.S., Washington, D. C. 17. Blakley, R. L. (1957) Biochem. J. 65, 331-342. 18. Rowe, P. B. (1968) Anal. Biochem. 22, 166-177. 19. Hatefi, Y., Talbert, P. T., Osborn, M. J., and Huennekens, F. M. (l%O)Biochem. Prep. 7, 89-92. 20. Huennekens, F. M., Ho, P. P. K., and Scrimgeour, K. G. (1963) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 6, pp. 806-811, Academic Press, New York. 21. Blakley, R. L. (1959) Biochem. J. 72, 707-715. 22. Krumdieck, C. L., and Baugh, C. M. (1969) Biochemistry 8, 1568-1572. 23. Chanarin, I., and Perry, .I. (1967) Biochem. J. 105, 633-634. 24. Flaks, J. G., and Lukens, L. N. (1963) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 6, pp. 52-99, Academic Press, New York. 25. Bray, G. A. (196O)Anal. Biochem. 1, 279-285. 26. Pohland, A., Flynn, E. H., Jones, R. G., and Shive, W. (195l)J. Amer. Chem. Sot. 73, 3247-3252. 27. Blakley, R. L. (1969) The Biochemistry of Folic Acid and Related Pteridines, p. 569, North-Holland, Amsterdam. 28. Rabinowitz, J. C. (1960) in The Enzymes (Boyer, P. D., Lardy, H., and MyrbBck, eds.), Vol. 2, pp. 185-252, Academic Press, New York. 29. Uyeda, K., and Rabinowitz, J. C. (1965)J. Biol. Chem. 240, 1701-1710. 30. Gupta, V. S., and Huennekens, F. M. (1967) Arch. Eiochem. Biophys. 120, 712-718. 31. Blakley, R. L. (1960)Eiochem. J. 74, 71-82. 32. Kallen, R. G., and Jencks, W. P. (1966) J. Biol. Chem. 241, 5845-5850. 33. Uyeda, K., and Rabinowitz, J. C. (1963) Anal. Biochem. 6, 100-108. 34. Osbom, M. J., Talbert, P. T., and Huennekens, F. M. (196O)J. Amer. Chem. Sot. 82, 4921-4927. 35. Scrimgeour, K. G., and Vitols, K. S. (1966) Biochemistry 5, 1438-1443. 36. Deits, T. L., Russell, A., Fujii, K., and Whitely, J. M. (1975) in Chemistry and Biology of Pteridins (Pfteiderer, W., ed.), pp. 525-534, Walter deGruyter, New York. 37. Donaldson, K. O., and Keresztesy, J. C. (1962) J. Biol. Chem. 237, 3815-3819. 38. May, M., Bardos, T. J., Barger, F. L., Landsford, M., Ravel, J. M., Sutherland, G. L., and Shive, W. (1951) J. Amer. Chem. Sot. 73, 3067-3075. 39. Osborne-White, W. S., and Smith, R. M. (1973) Eiochem. J. 136, 265-278. 40. Corrocher, R., Bhuyan, B. K., and Hoffbrand, A. V. (1972) Clin. Sci. 43, 799-813. 41. Brody, T., Shin, Y. S., and Stokstad, E. L. R. (1976) J. Neurochem. 27, 409-413. 42. Gapski, G. R., Whiteley, J. M., and Huennekens, F. M. (1971) Biochemistry 10, 29302934.