ARCHIVES
OF
BIOCHEMISTRY
Reduction
AND
ofPharmacology,
456-461
179,
of Methemoglobin DORIS
Department
BIOPHYSICS
by Tetrahydropterin
TAYLOR
University
(1977)
PAUL
AND
HOCHSTEIN
of Southern California School Angeles, California 90033 Received
June
and Glutathione
of Medicine,
2025 Zonal
Avenue,
Los
9, 1976
The reduced pteridine 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine nonenzymatically reduces methemoglobin in solution and in intact erythrocytes. The extent of the reaction in whole cells is markedly increased in the presence of glucose. The stimulating effect of glucose is absent in erythrocytes from individuals deficient in glucose 6-phosphate dehydrogenase. Glucose functions by maintaining levels of reduced glutathione which, in turn, reduce the dihydropterin to the active tetrahydro form. Although it appears unlikely that this mechanism could contribute in more than a minor way to the maintenance of ferrous hemoglobin in vivo, the results suggest that the interaction of glutathione with pterins might be of consequence in the regulation of pterin-dependent pathways in other tissues.
5,6,7,8-tetrahydropteridine (DMPH,)’ to reduce cytochrome c results in ATP formation over site 3 of mitochondria (11). The ability of cytochrome c to oxidize the tetrahydropterin leads to decreased phenylalanine hydroxylation in vitro in the presence of mitochondria (unpublished work). During these investigations on the interaction of pterins with cellular constituents, we have found that DMPH, is able to reduce methemoglobin in cell lysates and intact red cells and that this reaction is enhanced in whole cells by the addition of glucose in the medium. A preliminary report of these findings has appeared (12).
Tetrahydrobiopterin is an unconjugated 2-amino-4-hydroxypteridine (pterin) found in mammalian tissue which serves as a cofactor in the hydroxylation of phenylalanine (11, tyrosine (2,3), and tryptophan (4, 5). Analogs of tetrahydrobiopterin, such as the 6-methyl and 6,7-dimethyl tetrahydropterins, can substitute for the naturally occurring compound in these reactions (6). During oxidation, tetrahydropterms are first converted to the quininoid dihydropterins (7), which are reducible by such agents as NADPH and mercaptoethano1 (8) and by a reduced pyridine nucleotide-dependent dihydropterin reductase (7). Any quininoid dihydropterin which escapes reduction isomerizes spontaneously to the 7,8-dihydropterin. Dihydrofolate reductase is able to convert the naturally occurring 7,8-dihydrobiopterin to the tetrahydro form, but is not able to do so with the 6,7-dimethyl-7,8-dihydropterin (9). By these mechanisms, pterins are able to cycle between their tetrahydro and dihydro forms, donating two electrons with each cycle. Tetrahydropterins oxidize spontaneously in the presence of oxygen, producing hydrogen peroxide and, under certain conditions, superoxide anion (10). We have previously shown that the ability of 2-amino-4-hydroxy-6,7-dimethyl-
MATERIALS
AND
METHODS
Hemoglobin in erythrocytes from normal adult volunteers was converted to methemoglobin (MI-RI) by treating fresh, heparinized blood with NaNO, at a final concentration of 1%. After standing for 15 min on ice, the blood was diluted with 4 vol of 0.9% NaCl and centrifuged at 400g for 5 min. The diluted plasma and buffy coat were removed and the red cells were washed four times in 8 vol of 0.9% NaCl. Packed, washed cells were diluted with KrebsRinger phosphate buffer, pH 7.4, and kept on ice. * Abbreviations used: DMPH,, 2-amino-l-hydroxy - 6,7 - dimethyl - 5,6,7., 8 - tetrahydropteridine; MHb, methemoglobin; GSH, glutathione; DTE, dithioerythritoh G-8PD, glucose 6-phosphate dehydrogenase; NEM, N-ethylmaleimide. 456
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
TETRAHYDROPTERIN This buffer was used in all experiments unless otherwise indicated. Whole cell experiments were carried out by incubating the MI-&containing cells at a final hematocrit of 30% in buffer. Other additions are noted in individual experiments. The reaction was started with DMPH, and incubated at 37°C without shaking. The absorbance at 630 nm was recorded immediately after mixing an aliquot of the incubation mixture in 19 vol of 1 mM potassium phosphate buffer, pH 6.0, containing 0.1% Triton X-100. Cell lysates were prepared by freezing and thawing the washed, MHb-containing cells three times and diluting with buffer. Bovine hemoglobin (Sigma Chemical Co., 2x crystallized) was found to be greater than 90% in the ferric form by its absorbance in solution at 630 nm and was used without further oxidation. Experiments involving lysates or bovine MHb were carried out at 23°C in a recording spectrophotometer. Reaction components, excluding DMPH,, were mixed in the cuvette and allowed to equilibrate. The reaction was started by mixing in the DMPH, and was followed at 630 nm. The initial rates of reduction were linear with DMPH, concentration and the reaction was saturated with respect to MHb. The amount of MHb reduction in each experiment was calculated from the decrease in absorbance at 630 nm. The extinction coefficient for MHb was determined by oxidizing a solution of human hemoglobin (Hemoglobin Standard, Sigma Chemical Co.) with an excess of potassium ferricyanide (13) and measuring the absorbance at 630 nm. In the pH 6.0 buffer system described above, the extinction coefficient was found to be 4.2 mM-‘, and this value was used for the whole cell experiments. A value of 4.7 rnM-’ was found for MHb in Krebs-Ringer phosphate buffer, pH 7.4, and was used to calculate the amount of MHb reduced in the lysate experiments and experiments carried out with the recrystallized MHb. Superoxide dismutase was prepared from bovine erythrocytes by the method of McCord and Fridovich (14). Cells containing MHb were depleted of GSH by the method of Beutler et al. (15). Equal volumes of packed, MHb-containing cells and 2 mM N-ethylmaleimide were mixed and allowed to stand at room temperature for 20 min. The cells were washed four times with 4 vol of buffer. As a control, cells were carried through the procedure in the absence of Nethylmaleimide. Determination of GSH was carried out according to Beutler (16). The glucose 6-phosphate dehydrogenase content of red cells was assayed according to the method of Glock and McLean (17). DMPH, (hydrochloride form, Aldrich Chemical Co.) was prepared fresh each day in buffer made
AND
457
METHEMOGLOBIN
anaerobic KOH and tration of at 265 nm
with nitrogen. It was neutralized with kept on ice, under nitrogen. The concenDMPH, was determined by its extinction (18). RESULTS
When DMPH, was added to a solution of purified bovine MHb, a rapid decrease in absorbance at 630 nm was seen (Fig. 1, curve B), indicating the conversion of ferric hemoglobin (Ml%) to the ferrous form. About i min after the addition of DMPH4, reoxidation of the hemoglobin began to occur. Inclusion of superoxide dismutase did not prevent this reoxidation (curve 0, but catalase did (curve D), indicating, that H,O, was the species involved in the oxidation of hemoglobin, not the superoxide radical. If intact human erythrocytes in which hemoglobin had been converted to MHb were incubated with DMPHI, the MHb was similarly reduced (Fig. 2, curve B). Reoxidation did not begin as soon after addition of DMPH, as in the MHb solution, nor was it as rapid once it did begin, presumably because catalase was present in the cells. Maximal reduction was seen at about 30 min after addition of DMPH,. The amount of MHb reduced was about half that expected on the basis of 2 mol of heme reduced per mole of DMPH,. Inclu-
B
A
J
0 t -.l
C
-A01234561 MINUTES
FIG. 1. Reduction of purified bovine methemoglobin by DMPH,. The l.O-ml reaction mixture contained 3.2 mg of bovine methemoglobin and 1 pmol of DMPH, in Krebs-Ringer phosphate buffer at pH 7.4. The reaction was carried out at 37°C. The arrow indicates the addition of DMPH+ Curve A, no DMPHI; B, DMPH, only; C, 60 pg of superoxide dismutase added before DMPH,; D, 300 units of catalase added before DMPH,. Total reduction of MHb would result in a A A,,, of 0.94.
TAYLOR
“tI
0
30 IYC”~lTlO”
60 Till
PO ,.l”“III~
AND
vm
FIG. 2. Reduction of methemoglobin in intact erythrocytes. Hemoglobin was oxidized and incubations were carried out as described under Materials and Methods. The final reaction volume was 2.0 ml. Curve A, 5.5 mM glucose; B, 1 mM DMPH,; C, 1 mM DMPH, and 5.5 mM glucose; D, 5.5 mM glucose and 0.1 mM methylene blue; E, 1 mM DMPH, and 5 mM DTE. Results are the averages of three experiments. The maximum reduction found with DMPH, and DTE (curve E) represents more than a 90% reduction of MHb present.
sion of glucose in the medium resulted in a threefold stimulation in the conversion of MHb to hemoglobin by DMPH, (curve C). About 50% more MHb was reduced in 2 h as that expected on a stoichiometric basis, leading to the conclusion that glucose was causing the pterin to cycle between its oxidized and reduced forms. Reduction of MHb by methylene blue in the presence of glucose (curve D) is shown for comparison. The dye reduces MHb and is in turn reduced by NADPH (19). Dithioerythritol (DTE) can, like its isomer, dithiothreitol, reduce quininoid dihydropterin directly, and it caused a very rapid reduction of MHb by DMPH, (curve E). Curve A shows the effect of glucose alone on MHb reduction. Lack of any reduction of MHb in the presence of glucose alone may indicate that small amounts of sodium nitrite remained in the cells. DTE or methylene blue alone did not affect MHb reduction, but these curves have been omitted from the graph for clarity. Table I shows the effectiveness of various concentrations of DMPH, in reducing MHb in whole cells. Effects could be seen down to 10e6 M when either DTE or glucose was used to regenerate the DMPH,. The concentration of DMPH, effective in this
HOCHSTEIN
system is in the range of concentration of tetrahydropterins in mammalian tissues. For example, concentrations of 0.2 x 10V6 M have been found for 6-hydroxyalkyl pterins in human blood (20), and 1.5 x 1O-5 M has been found in a high-speed supernatant fraction of rat liver (21). In human liver, tetrahydrobiopterin levels are about 6 X lo-‘j M (22). In an effort to determine how glucose was exerting its enhancing effect on the reduction of MHb by DMPHI, red blood cells from two individuals deficient in glucose 6-phosphate dehydrogenase (G-8PD) were examined. The patients’ cells had less than 10% of the normal G-6-PD activity. The red cells from both patients showed almost no enhancement by glucose of the DMPH,-dependent MHb reduction (Table II). Unlike the other experiments reported in this paper, these studies were carried out with shaking during the incubation period. Results for shaking and nonshaking experiments were found to be qualitatively similar, but the amount of DMPH,-dependent MHb reduction was always less when the reaction mixtures were agitated during incubation because of the increased autoxidation of DMPH,. Because erythrocytes from the individuals deficient in G-6-PD did not show a glucose enhancement of DMPH,-dependent MHb reduction, it can be concluded TABLE OF MHb
REDUCTION
DMPH, DMPH, tration
concen-
0 10-S 10-4 10-S 10-G
I
BY DMPH,
AND
GLUCOSE
OR
DTEQ
AND
A MHb
reduction
(pmol)
(M)
Glucose
DTE
0.10 t 0.18 3.9 ” 0.3 2.4 t 0.3
0.02 ? 0.21 5.7 2 0.5 3.9 k 0.3
1.0
t
0.2
1.0 2 0.4
1.2
2 0.2
0.48 k 0.21
a Hemoglobin was oxidized and 30-min incubations were carried out using whole cells, as described under Materials and Methods. The final concentration of glucose was 5.5 mM, DTE was present at 5.0 mM, and the concentration of DMPH, was varied as indicated. Results are given as the increase in micromoles of MHb reduced in the 2-ml incubation mixture after 30 min over controls containing only cells, buffer, and DMPH,.
TETRAHYDROPTERIN TABLE
to
Micromoles
TABLE
1
PaNormal tient 1 control
METHEMOGLOBIN
IN RED CELLS 6-PHOSPHATE
of MHb min
Experiment
DMPH, DMPH, + glucase DMPH, + DTE Methylene blue + glucose
459
METHEMOGLOBIN
II
REDUCTION OF METHEMOGLOBIN DEFICIENT IN GLUCOSE DEHYDROGENASE~
Additions incubation
AND
reduced
PRESENCE
in 60
Reducing agent
REDUCTION OF VARIOUS
Concentration (mbr)
III BY DMPH, REDUCING
Increase in MHb duced (nmol) Lysate
Experiment
2
PaNormal tient 2 control
0.037 0.070
0.010 0.56
0.36 0.43
0.69 1.1
2.2 0.33
2.0 1.7
2.0 -
1.9 -
R Hemoglobin was oxidized and incubations and measurement of MHb reduction in whole cells were carried out as described under Materials and Methods. The hematocrit of the incubation mixtures was 18% and the tubes were shaken during the 60-min incubation period. DMPH, concentration was 1 mM, glucose was 5.5 mM, DTE was 5 mM, and methylene blue was 0.1 mM.
that the basis of the glucose effect resides in the pentose phosphate pathway. The most likely possibilities include (1) an enzymatically linked reduction of dihydropterm by NADPH similar to the wellknown NADPH-methylene blue reductase or (2) a nonenzymatic reduction of dihydropterin by GSH or NADPH. Intensive studies have failed to verify an enzymatic basis for the glucose effect. NADH, NADPH, GSH, and DTE were all able to increase the reduction of MHb by DMPH, in bovine MHb solutions and in human MHb-containing cell lysates. Table III shows the difference in effectiveness of these compounds in cell lysates. The concentration of total triphosphopyridine nucleotide in red cells has been shown to be 5-17 PM (23), but NADPH at 10 PM was ineffective in our system. Although not shown in Table III, the inclusion of generating systems for the reduced pyridine nucleotides did not enhance MHb reduction. It can be seen in Table III that GSH is an excellent reducing agent for dihydropterin even below levels of GSH normally found in mammalian tissues 11-5 mM, Ref. (2411. Table IV lends support to the postulate that GSH is important to the enhancing
IN THE
AGENTS~
DTE
5 2
GSH
5 2 0.5 0.2
42 31 20 2.1*
NADH
1.0 0.1 0.01
11 5.5 0
NADPH
1.0 0.1 0.01
10 4.3 0
re-
Purified MHb
8.7 8.7
31* 8.76
’ Each l.O-ml reaction mixture contained 6 mg of purified bovine MHb or the equivalent of 20 ~1 of MHb-containing packed red cells as cell lysate (see Materials and Methods). Reducing agent and lysate or MHb solution were mixed and the reaction was started with DMPH, (0.5 mM, final concentration). The reaction was carried out for 8 min at 25” in Krebs-Ringer phosphate buffer, pH 7.4. Results are given as the increase in nanomoles of MHb reduced compared to the control containing lysate or MHb and DMPH,. Results are the averages of duplicates or triplicates. b Reaction mixture contained 300 units of catalase.
effect of glucose. N-Ethylmaleimide sequesters much of the GSH in the cell, and little GSH reappears during the 30-min incubation period. Reduction of MHb by DMPH, alone or with glucose was less in GSH-depleted cells than in cells with normal GSH levels. Presumably, even the low level of GSH that is present in NEMtreated cells is continually regenerated when glucose is included as substrate, thus allowing some stimulation of MHb reduction. This explanation accounts for the minimal MHb reduction by DMPH, alone and the significant amount of activity seen in the presence of DMPH, and glucose in NEM-treated cells. The reason for the slight increase in the amount of MHb present when glucose alone was in-
460
TAYLOR
AND
HOCHSTEIN
TABLE REDUCTION
Incubation
OF METHEMOGLOBIN
CELLS
DEPLETED
GSH
content
(mM)
Additions Control
+ + +
None Glucose DMPH, Glucose
+ DMPH,
IV
IN RED
cells
2.1 2.2 0.37 1.5
NEM-treated 0.14 0.20 0.10 0.15
OF REDUCED
GLUTATHIONE~
Micromoles Control -0.27 1.4 2.8
of MHb cells
reduced
NEM-treated -0.30 0.69 1.8
o All procedures are described in detail under Materials and Methods. Hemoglobin was oxidized in intact cells and glutathione was alkylated with NEM. GSH and MHb levels in the NEM-treated and control cells were measured before and after a 30-min incubation with the designated additions. Glucose concentration was 5.5 mM, and DMPH, was 1.0 mM. Results are averages of duplicate determinations.
eluded in the medium (Table IV) is not clear. The levels of GSH in the depleted cells were probably high enough to account for some reduction of MHb even in the absence of glucose, as illustrated by the fact that reduction of lysate MHb by DMPH, was increased in the presence of 0.2 M GSH (Table III). It is also clear from Table III that the GSH-DMPH2 interaction is nonenzymatic, since GSH-dependent stimulation of MHb reduction was similar for human cell lysates and the recrystallized bovine MHb. The low level of GSH found in control cells or NEM-treated cells in the presence of DMPH, alone may be due -m oxidation by dihydropterin or utilization by glutathione reductase for the detoxification of hydrogen peroxide (25) produced by autoxidation of DMPH+ The lack of glucose would prevent regeneration of the GSH. These results implicate GSH in the glucose stimulation of MHb reduction by DMPHI, but do not rule out the participation of other, as yet unidentified, metabolic processes. DISCUSSION
Until recently, the only known effect of tetrahydropterins on cellular functions was that they acted as cofactors for aromatic amino acid hydroxylases. We are reporting in this paper another interaction of a tetrahydropterin with a cellular component, methemoglobin. This DMPH,-dependent reduction of MHb is increased in the presence of glucose, and the effect of glucose seems to depend on its ability to generate reduced glutathione. This con-
cept is supported by the fact that red cells from individuals deficient in G-8PD, which have a diminished capacity to generate reduced glutathione, have only a marginal response to glucose. Since GSH will reduce dihydropterin directly and DMPH, can reduce MHb without enzymatic involvement, the system described does not require any metabolic component other than per&se phosphate shunt activity. It is not clear from our studies whether or not tetrahydropterins reduce MI-lb in the intact animal. In view of the fact that there are several methemoglobin reductases, but that only one of them is of clinical significance in humans (261, it is doubtful that the system we describe could contribute in more than a very minor way to maintenance of ferrous hemoglobin levels in uiuo. In addition, levels of 6-hydroxyalkylpterins are low in uiuo, i.e., 0.2 x lo-” M in human whole blood (20). These results raise the question of the physiological significance of GSH reduction of dihydropterins. Although the tetrahydropterin used in our experiments is a synthetic analog of the naturally occurring mammalian compound,’ their biological characteristics are qualitatively similar in many respects, including susceptibility to nonenzymatic reduction by endogenous agents (6). Kaufman et al. (22) have suggested that endogenous reducing compounds such as GSH and ascorbate may affect the activity of the aromatic amino acid hydroxylase in uiao. Our experiments show that, at levels of GSH found in mammalian tissues, i.e., l-5 mM, dihydropterin reduction can be affected significantly.
TETRAHYDROPTERIN ACKNOWLEDGMENT This work was supported in part by Research Grant No. HD08159 from the National Institutes of Health. REFERENCES 1. KAUFMAN, S. (1959) J. Biol. Chem. 234, 26772682. 2. HOSADA, S., AND GLICK, D. (1966) J. Biol. Chem. 241, 192-196. 3. FRIEDMAN, P. A., KAPPELMAN, A. H., AND KAUFMAN, S. (1972) J. Biol. Chem. 247, 41654173. 4. BRENNEMAN, A. R., AND KAUFMAN, S. (1964) Biochem. Biophys. Res. Commun. 17;177-183. 5. ELLENBOGEN, L., TAYLOR, R. J., JR., AND BRUNDAGE, G. B. (1965) B&hem. Biophys. Res. Commun. 19, 708-715. 6. KAUFMAN, S. (1971) in Advances in Enzymology (Nerd, F. F., ed.), Vol. 35, pp. 245-319, WileyInterscience, New York. 7. KAUFMAN, S. (1964) J. Biol. Chem. 239,332-338. 8. KAUFMAN, S. (1962) J. Biol. Chem. 237, PC2712PC2713. 9. KAUFMAN, S. (1967) J. Biol. Chem. 242, 39343943. 10. NISHIKIMI,
M. (1975) Arch. 166, 273-279. 11. TAYLOR, D., AND HOCHSTEIN,
12.
Biophys. TAYLOR,
(1975)
Biochem.
13. ANTONINI, E., AND BRUNORI, M. (1971) in Hemoglobin and Myoglobin in Their Reactions with Ligands (Antonini, E., and Brunori, M., eds.), pp. 41-42, American Elsevier, New York. 14. MCCORD, J. M., AND FRIDOVICH, I. (1969) J. Biol. Chem.
15. BEUTLER, (1970)
244, 6049-6055. E., SRIVASTAVA, Biochem. Biophys.
S. K., AND WEST, C. Res.
Commun.
38,
341-347. 16. BEUTLER, E. (1971) Red Cell Metabolism, pp. 103-105, Grune and Stratton, New York. 17. GLOCK, G. E., AND MCLEAN, P. (1953) B&hem. J. 55, 400-408. 18. KAUFMAN, S.
(1969) Arch. Biochem. Biophys. 134, 249-252. 19. BRIN, M., AND YONEMOTO, R. H. (1958) J. Biol. Chem. 230, 307-316. 20. FRANK, O., BAKER, H., AND SOBOTKA, H. (1963) Nature (London) 197, 490-491. 21. REMBOLD, H., AND BUFF, K. (1972) Eur. J. Biothem.
28, 579-585. S., HOLTZMAN, N., MILSTEIN, BUTLER, I., AND KRUMHOLZ, A. (1975) Engl. J. Med. 293, 786-790. JOCELYN, P. C. (1960)Biochem. J. 77,363-368. JOCELYN, P. C. (1959) Biochem. Sot. Symp.
22. KAUFMAN, 23. 24.
Biophys.
P. (1975) Biochem. Res. Commun. 67, 156-162. D., DESOTO, J. A., AND HOCHSTEIN, J. Cell Biol. 67, 428.
461
AND METHEMOGLOBIN
S., N. 17,
43-65. 25. COHEN, P.
26.
G., AND HOCHSTEIN, P. (1963) Biochemistry 2, 1420-1428. SCOTT, E., DUNCAN, I., AND EKSTRAND, V. (1965) J. Biol. Chem. 240. 481-485.
OF
BIOCHEMISTRY
Reduction
AND
ofPharmacology,
456-461
179,
of Methemoglobin DORIS
Department
BIOPHYSICS
by Tetrahydropterin
TAYLOR
University
(1977)
PAUL
AND
HOCHSTEIN
of Southern California School Angeles, California 90033 Received
June
and Glutathione
of Medicine,
2025 Zonal
Avenue,
Los
9, 1976
The reduced pteridine 2-amino-4-hydroxy-6,7-dimethyl-5,6,7,8-tetrahydropteridine nonenzymatically reduces methemoglobin in solution and in intact erythrocytes. The extent of the reaction in whole cells is markedly increased in the presence of glucose. The stimulating effect of glucose is absent in erythrocytes from individuals deficient in glucose 6-phosphate dehydrogenase. Glucose functions by maintaining levels of reduced glutathione which, in turn, reduce the dihydropterin to the active tetrahydro form. Although it appears unlikely that this mechanism could contribute in more than a minor way to the maintenance of ferrous hemoglobin in vivo, the results suggest that the interaction of glutathione with pterins might be of consequence in the regulation of pterin-dependent pathways in other tissues.
5,6,7,8-tetrahydropteridine (DMPH,)’ to reduce cytochrome c results in ATP formation over site 3 of mitochondria (11). The ability of cytochrome c to oxidize the tetrahydropterin leads to decreased phenylalanine hydroxylation in vitro in the presence of mitochondria (unpublished work). During these investigations on the interaction of pterins with cellular constituents, we have found that DMPH, is able to reduce methemoglobin in cell lysates and intact red cells and that this reaction is enhanced in whole cells by the addition of glucose in the medium. A preliminary report of these findings has appeared (12).
Tetrahydrobiopterin is an unconjugated 2-amino-4-hydroxypteridine (pterin) found in mammalian tissue which serves as a cofactor in the hydroxylation of phenylalanine (11, tyrosine (2,3), and tryptophan (4, 5). Analogs of tetrahydrobiopterin, such as the 6-methyl and 6,7-dimethyl tetrahydropterins, can substitute for the naturally occurring compound in these reactions (6). During oxidation, tetrahydropterms are first converted to the quininoid dihydropterins (7), which are reducible by such agents as NADPH and mercaptoethano1 (8) and by a reduced pyridine nucleotide-dependent dihydropterin reductase (7). Any quininoid dihydropterin which escapes reduction isomerizes spontaneously to the 7,8-dihydropterin. Dihydrofolate reductase is able to convert the naturally occurring 7,8-dihydrobiopterin to the tetrahydro form, but is not able to do so with the 6,7-dimethyl-7,8-dihydropterin (9). By these mechanisms, pterins are able to cycle between their tetrahydro and dihydro forms, donating two electrons with each cycle. Tetrahydropterins oxidize spontaneously in the presence of oxygen, producing hydrogen peroxide and, under certain conditions, superoxide anion (10). We have previously shown that the ability of 2-amino-4-hydroxy-6,7-dimethyl-
MATERIALS
AND
METHODS
Hemoglobin in erythrocytes from normal adult volunteers was converted to methemoglobin (MI-RI) by treating fresh, heparinized blood with NaNO, at a final concentration of 1%. After standing for 15 min on ice, the blood was diluted with 4 vol of 0.9% NaCl and centrifuged at 400g for 5 min. The diluted plasma and buffy coat were removed and the red cells were washed four times in 8 vol of 0.9% NaCl. Packed, washed cells were diluted with KrebsRinger phosphate buffer, pH 7.4, and kept on ice. * Abbreviations used: DMPH,, 2-amino-l-hydroxy - 6,7 - dimethyl - 5,6,7., 8 - tetrahydropteridine; MHb, methemoglobin; GSH, glutathione; DTE, dithioerythritoh G-8PD, glucose 6-phosphate dehydrogenase; NEM, N-ethylmaleimide. 456
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN
0003-9861
TETRAHYDROPTERIN This buffer was used in all experiments unless otherwise indicated. Whole cell experiments were carried out by incubating the MI-&containing cells at a final hematocrit of 30% in buffer. Other additions are noted in individual experiments. The reaction was started with DMPH, and incubated at 37°C without shaking. The absorbance at 630 nm was recorded immediately after mixing an aliquot of the incubation mixture in 19 vol of 1 mM potassium phosphate buffer, pH 6.0, containing 0.1% Triton X-100. Cell lysates were prepared by freezing and thawing the washed, MHb-containing cells three times and diluting with buffer. Bovine hemoglobin (Sigma Chemical Co., 2x crystallized) was found to be greater than 90% in the ferric form by its absorbance in solution at 630 nm and was used without further oxidation. Experiments involving lysates or bovine MHb were carried out at 23°C in a recording spectrophotometer. Reaction components, excluding DMPH,, were mixed in the cuvette and allowed to equilibrate. The reaction was started by mixing in the DMPH, and was followed at 630 nm. The initial rates of reduction were linear with DMPH, concentration and the reaction was saturated with respect to MHb. The amount of MHb reduction in each experiment was calculated from the decrease in absorbance at 630 nm. The extinction coefficient for MHb was determined by oxidizing a solution of human hemoglobin (Hemoglobin Standard, Sigma Chemical Co.) with an excess of potassium ferricyanide (13) and measuring the absorbance at 630 nm. In the pH 6.0 buffer system described above, the extinction coefficient was found to be 4.2 mM-‘, and this value was used for the whole cell experiments. A value of 4.7 rnM-’ was found for MHb in Krebs-Ringer phosphate buffer, pH 7.4, and was used to calculate the amount of MHb reduced in the lysate experiments and experiments carried out with the recrystallized MHb. Superoxide dismutase was prepared from bovine erythrocytes by the method of McCord and Fridovich (14). Cells containing MHb were depleted of GSH by the method of Beutler et al. (15). Equal volumes of packed, MHb-containing cells and 2 mM N-ethylmaleimide were mixed and allowed to stand at room temperature for 20 min. The cells were washed four times with 4 vol of buffer. As a control, cells were carried through the procedure in the absence of Nethylmaleimide. Determination of GSH was carried out according to Beutler (16). The glucose 6-phosphate dehydrogenase content of red cells was assayed according to the method of Glock and McLean (17). DMPH, (hydrochloride form, Aldrich Chemical Co.) was prepared fresh each day in buffer made
AND
457
METHEMOGLOBIN
anaerobic KOH and tration of at 265 nm
with nitrogen. It was neutralized with kept on ice, under nitrogen. The concenDMPH, was determined by its extinction (18). RESULTS
When DMPH, was added to a solution of purified bovine MHb, a rapid decrease in absorbance at 630 nm was seen (Fig. 1, curve B), indicating the conversion of ferric hemoglobin (Ml%) to the ferrous form. About i min after the addition of DMPH4, reoxidation of the hemoglobin began to occur. Inclusion of superoxide dismutase did not prevent this reoxidation (curve 0, but catalase did (curve D), indicating, that H,O, was the species involved in the oxidation of hemoglobin, not the superoxide radical. If intact human erythrocytes in which hemoglobin had been converted to MHb were incubated with DMPHI, the MHb was similarly reduced (Fig. 2, curve B). Reoxidation did not begin as soon after addition of DMPH, as in the MHb solution, nor was it as rapid once it did begin, presumably because catalase was present in the cells. Maximal reduction was seen at about 30 min after addition of DMPH,. The amount of MHb reduced was about half that expected on the basis of 2 mol of heme reduced per mole of DMPH,. Inclu-
B
A
J
0 t -.l
C
-A01234561 MINUTES
FIG. 1. Reduction of purified bovine methemoglobin by DMPH,. The l.O-ml reaction mixture contained 3.2 mg of bovine methemoglobin and 1 pmol of DMPH, in Krebs-Ringer phosphate buffer at pH 7.4. The reaction was carried out at 37°C. The arrow indicates the addition of DMPH+ Curve A, no DMPHI; B, DMPH, only; C, 60 pg of superoxide dismutase added before DMPH,; D, 300 units of catalase added before DMPH,. Total reduction of MHb would result in a A A,,, of 0.94.
TAYLOR
“tI
0
30 IYC”~lTlO”
60 Till
PO ,.l”“III~
AND
vm
FIG. 2. Reduction of methemoglobin in intact erythrocytes. Hemoglobin was oxidized and incubations were carried out as described under Materials and Methods. The final reaction volume was 2.0 ml. Curve A, 5.5 mM glucose; B, 1 mM DMPH,; C, 1 mM DMPH, and 5.5 mM glucose; D, 5.5 mM glucose and 0.1 mM methylene blue; E, 1 mM DMPH, and 5 mM DTE. Results are the averages of three experiments. The maximum reduction found with DMPH, and DTE (curve E) represents more than a 90% reduction of MHb present.
sion of glucose in the medium resulted in a threefold stimulation in the conversion of MHb to hemoglobin by DMPH, (curve C). About 50% more MHb was reduced in 2 h as that expected on a stoichiometric basis, leading to the conclusion that glucose was causing the pterin to cycle between its oxidized and reduced forms. Reduction of MHb by methylene blue in the presence of glucose (curve D) is shown for comparison. The dye reduces MHb and is in turn reduced by NADPH (19). Dithioerythritol (DTE) can, like its isomer, dithiothreitol, reduce quininoid dihydropterin directly, and it caused a very rapid reduction of MHb by DMPH, (curve E). Curve A shows the effect of glucose alone on MHb reduction. Lack of any reduction of MHb in the presence of glucose alone may indicate that small amounts of sodium nitrite remained in the cells. DTE or methylene blue alone did not affect MHb reduction, but these curves have been omitted from the graph for clarity. Table I shows the effectiveness of various concentrations of DMPH, in reducing MHb in whole cells. Effects could be seen down to 10e6 M when either DTE or glucose was used to regenerate the DMPH,. The concentration of DMPH, effective in this
HOCHSTEIN
system is in the range of concentration of tetrahydropterins in mammalian tissues. For example, concentrations of 0.2 x 10V6 M have been found for 6-hydroxyalkyl pterins in human blood (20), and 1.5 x 1O-5 M has been found in a high-speed supernatant fraction of rat liver (21). In human liver, tetrahydrobiopterin levels are about 6 X lo-‘j M (22). In an effort to determine how glucose was exerting its enhancing effect on the reduction of MHb by DMPHI, red blood cells from two individuals deficient in glucose 6-phosphate dehydrogenase (G-8PD) were examined. The patients’ cells had less than 10% of the normal G-6-PD activity. The red cells from both patients showed almost no enhancement by glucose of the DMPH,-dependent MHb reduction (Table II). Unlike the other experiments reported in this paper, these studies were carried out with shaking during the incubation period. Results for shaking and nonshaking experiments were found to be qualitatively similar, but the amount of DMPH,-dependent MHb reduction was always less when the reaction mixtures were agitated during incubation because of the increased autoxidation of DMPH,. Because erythrocytes from the individuals deficient in G-6-PD did not show a glucose enhancement of DMPH,-dependent MHb reduction, it can be concluded TABLE OF MHb
REDUCTION
DMPH, DMPH, tration
concen-
0 10-S 10-4 10-S 10-G
I
BY DMPH,
AND
GLUCOSE
OR
DTEQ
AND
A MHb
reduction
(pmol)
(M)
Glucose
DTE
0.10 t 0.18 3.9 ” 0.3 2.4 t 0.3
0.02 ? 0.21 5.7 2 0.5 3.9 k 0.3
1.0
t
0.2
1.0 2 0.4
1.2
2 0.2
0.48 k 0.21
a Hemoglobin was oxidized and 30-min incubations were carried out using whole cells, as described under Materials and Methods. The final concentration of glucose was 5.5 mM, DTE was present at 5.0 mM, and the concentration of DMPH, was varied as indicated. Results are given as the increase in micromoles of MHb reduced in the 2-ml incubation mixture after 30 min over controls containing only cells, buffer, and DMPH,.
TETRAHYDROPTERIN TABLE
to
Micromoles
TABLE
1
PaNormal tient 1 control
METHEMOGLOBIN
IN RED CELLS 6-PHOSPHATE
of MHb min
Experiment
DMPH, DMPH, + glucase DMPH, + DTE Methylene blue + glucose
459
METHEMOGLOBIN
II
REDUCTION OF METHEMOGLOBIN DEFICIENT IN GLUCOSE DEHYDROGENASE~
Additions incubation
AND
reduced
PRESENCE
in 60
Reducing agent
REDUCTION OF VARIOUS
Concentration (mbr)
III BY DMPH, REDUCING
Increase in MHb duced (nmol) Lysate
Experiment
2
PaNormal tient 2 control
0.037 0.070
0.010 0.56
0.36 0.43
0.69 1.1
2.2 0.33
2.0 1.7
2.0 -
1.9 -
R Hemoglobin was oxidized and incubations and measurement of MHb reduction in whole cells were carried out as described under Materials and Methods. The hematocrit of the incubation mixtures was 18% and the tubes were shaken during the 60-min incubation period. DMPH, concentration was 1 mM, glucose was 5.5 mM, DTE was 5 mM, and methylene blue was 0.1 mM.
that the basis of the glucose effect resides in the pentose phosphate pathway. The most likely possibilities include (1) an enzymatically linked reduction of dihydropterm by NADPH similar to the wellknown NADPH-methylene blue reductase or (2) a nonenzymatic reduction of dihydropterin by GSH or NADPH. Intensive studies have failed to verify an enzymatic basis for the glucose effect. NADH, NADPH, GSH, and DTE were all able to increase the reduction of MHb by DMPH, in bovine MHb solutions and in human MHb-containing cell lysates. Table III shows the difference in effectiveness of these compounds in cell lysates. The concentration of total triphosphopyridine nucleotide in red cells has been shown to be 5-17 PM (23), but NADPH at 10 PM was ineffective in our system. Although not shown in Table III, the inclusion of generating systems for the reduced pyridine nucleotides did not enhance MHb reduction. It can be seen in Table III that GSH is an excellent reducing agent for dihydropterin even below levels of GSH normally found in mammalian tissues 11-5 mM, Ref. (2411. Table IV lends support to the postulate that GSH is important to the enhancing
IN THE
AGENTS~
DTE
5 2
GSH
5 2 0.5 0.2
42 31 20 2.1*
NADH
1.0 0.1 0.01
11 5.5 0
NADPH
1.0 0.1 0.01
10 4.3 0
re-
Purified MHb
8.7 8.7
31* 8.76
’ Each l.O-ml reaction mixture contained 6 mg of purified bovine MHb or the equivalent of 20 ~1 of MHb-containing packed red cells as cell lysate (see Materials and Methods). Reducing agent and lysate or MHb solution were mixed and the reaction was started with DMPH, (0.5 mM, final concentration). The reaction was carried out for 8 min at 25” in Krebs-Ringer phosphate buffer, pH 7.4. Results are given as the increase in nanomoles of MHb reduced compared to the control containing lysate or MHb and DMPH,. Results are the averages of duplicates or triplicates. b Reaction mixture contained 300 units of catalase.
effect of glucose. N-Ethylmaleimide sequesters much of the GSH in the cell, and little GSH reappears during the 30-min incubation period. Reduction of MHb by DMPH, alone or with glucose was less in GSH-depleted cells than in cells with normal GSH levels. Presumably, even the low level of GSH that is present in NEMtreated cells is continually regenerated when glucose is included as substrate, thus allowing some stimulation of MHb reduction. This explanation accounts for the minimal MHb reduction by DMPH, alone and the significant amount of activity seen in the presence of DMPH, and glucose in NEM-treated cells. The reason for the slight increase in the amount of MHb present when glucose alone was in-
460
TAYLOR
AND
HOCHSTEIN
TABLE REDUCTION
Incubation
OF METHEMOGLOBIN
CELLS
DEPLETED
GSH
content
(mM)
Additions Control
+ + +
None Glucose DMPH, Glucose
+ DMPH,
IV
IN RED
cells
2.1 2.2 0.37 1.5
NEM-treated 0.14 0.20 0.10 0.15
OF REDUCED
GLUTATHIONE~
Micromoles Control -0.27 1.4 2.8
of MHb cells
reduced
NEM-treated -0.30 0.69 1.8
o All procedures are described in detail under Materials and Methods. Hemoglobin was oxidized in intact cells and glutathione was alkylated with NEM. GSH and MHb levels in the NEM-treated and control cells were measured before and after a 30-min incubation with the designated additions. Glucose concentration was 5.5 mM, and DMPH, was 1.0 mM. Results are averages of duplicate determinations.
eluded in the medium (Table IV) is not clear. The levels of GSH in the depleted cells were probably high enough to account for some reduction of MHb even in the absence of glucose, as illustrated by the fact that reduction of lysate MHb by DMPH, was increased in the presence of 0.2 M GSH (Table III). It is also clear from Table III that the GSH-DMPH2 interaction is nonenzymatic, since GSH-dependent stimulation of MHb reduction was similar for human cell lysates and the recrystallized bovine MHb. The low level of GSH found in control cells or NEM-treated cells in the presence of DMPH, alone may be due -m oxidation by dihydropterin or utilization by glutathione reductase for the detoxification of hydrogen peroxide (25) produced by autoxidation of DMPH+ The lack of glucose would prevent regeneration of the GSH. These results implicate GSH in the glucose stimulation of MHb reduction by DMPHI, but do not rule out the participation of other, as yet unidentified, metabolic processes. DISCUSSION
Until recently, the only known effect of tetrahydropterins on cellular functions was that they acted as cofactors for aromatic amino acid hydroxylases. We are reporting in this paper another interaction of a tetrahydropterin with a cellular component, methemoglobin. This DMPH,-dependent reduction of MHb is increased in the presence of glucose, and the effect of glucose seems to depend on its ability to generate reduced glutathione. This con-
cept is supported by the fact that red cells from individuals deficient in G-8PD, which have a diminished capacity to generate reduced glutathione, have only a marginal response to glucose. Since GSH will reduce dihydropterin directly and DMPH, can reduce MHb without enzymatic involvement, the system described does not require any metabolic component other than per&se phosphate shunt activity. It is not clear from our studies whether or not tetrahydropterins reduce MI-lb in the intact animal. In view of the fact that there are several methemoglobin reductases, but that only one of them is of clinical significance in humans (261, it is doubtful that the system we describe could contribute in more than a very minor way to maintenance of ferrous hemoglobin levels in uiuo. In addition, levels of 6-hydroxyalkylpterins are low in uiuo, i.e., 0.2 x lo-” M in human whole blood (20). These results raise the question of the physiological significance of GSH reduction of dihydropterins. Although the tetrahydropterin used in our experiments is a synthetic analog of the naturally occurring mammalian compound,’ their biological characteristics are qualitatively similar in many respects, including susceptibility to nonenzymatic reduction by endogenous agents (6). Kaufman et al. (22) have suggested that endogenous reducing compounds such as GSH and ascorbate may affect the activity of the aromatic amino acid hydroxylase in uiao. Our experiments show that, at levels of GSH found in mammalian tissues, i.e., l-5 mM, dihydropterin reduction can be affected significantly.
TETRAHYDROPTERIN ACKNOWLEDGMENT This work was supported in part by Research Grant No. HD08159 from the National Institutes of Health. REFERENCES 1. KAUFMAN, S. (1959) J. Biol. Chem. 234, 26772682. 2. HOSADA, S., AND GLICK, D. (1966) J. Biol. Chem. 241, 192-196. 3. FRIEDMAN, P. A., KAPPELMAN, A. H., AND KAUFMAN, S. (1972) J. Biol. Chem. 247, 41654173. 4. BRENNEMAN, A. R., AND KAUFMAN, S. (1964) Biochem. Biophys. Res. Commun. 17;177-183. 5. ELLENBOGEN, L., TAYLOR, R. J., JR., AND BRUNDAGE, G. B. (1965) B&hem. Biophys. Res. Commun. 19, 708-715. 6. KAUFMAN, S. (1971) in Advances in Enzymology (Nerd, F. F., ed.), Vol. 35, pp. 245-319, WileyInterscience, New York. 7. KAUFMAN, S. (1964) J. Biol. Chem. 239,332-338. 8. KAUFMAN, S. (1962) J. Biol. Chem. 237, PC2712PC2713. 9. KAUFMAN, S. (1967) J. Biol. Chem. 242, 39343943. 10. NISHIKIMI,
M. (1975) Arch. 166, 273-279. 11. TAYLOR, D., AND HOCHSTEIN,
12.
Biophys. TAYLOR,
(1975)
Biochem.
13. ANTONINI, E., AND BRUNORI, M. (1971) in Hemoglobin and Myoglobin in Their Reactions with Ligands (Antonini, E., and Brunori, M., eds.), pp. 41-42, American Elsevier, New York. 14. MCCORD, J. M., AND FRIDOVICH, I. (1969) J. Biol. Chem.
15. BEUTLER, (1970)
244, 6049-6055. E., SRIVASTAVA, Biochem. Biophys.
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Commun.
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341-347. 16. BEUTLER, E. (1971) Red Cell Metabolism, pp. 103-105, Grune and Stratton, New York. 17. GLOCK, G. E., AND MCLEAN, P. (1953) B&hem. J. 55, 400-408. 18. KAUFMAN, S.
(1969) Arch. Biochem. Biophys. 134, 249-252. 19. BRIN, M., AND YONEMOTO, R. H. (1958) J. Biol. Chem. 230, 307-316. 20. FRANK, O., BAKER, H., AND SOBOTKA, H. (1963) Nature (London) 197, 490-491. 21. REMBOLD, H., AND BUFF, K. (1972) Eur. J. Biothem.
28, 579-585. S., HOLTZMAN, N., MILSTEIN, BUTLER, I., AND KRUMHOLZ, A. (1975) Engl. J. Med. 293, 786-790. JOCELYN, P. C. (1960)Biochem. J. 77,363-368. JOCELYN, P. C. (1959) Biochem. Sot. Symp.
22. KAUFMAN, 23. 24.
Biophys.
P. (1975) Biochem. Res. Commun. 67, 156-162. D., DESOTO, J. A., AND HOCHSTEIN, J. Cell Biol. 67, 428.
461
AND METHEMOGLOBIN
S., N. 17,
43-65. 25. COHEN, P.
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