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Formation of a thiamine artifact during chromatography: A single column procedure for the separation of thiamine and the thiamine mono-, di-, and triphosphate esters
ANALYTICAL
102, 145% 149 (1980)
BIOCHEMISTRY
Formation of a Thiamine Artifact During Chromatography: A Single Column Procedure for the Separation of Thiamine and the Thiamine Mono-, Di-, and Triphosphate Esters1 ANNE Department
D. GOUNARIS of Chemistry,
Vassar
AND MAUREEN College.
SCHULMAN’
Poughkeepsie.
New
York
12601
Received June 5, 1979 Chromatography of a preparation of [Wlthiamine pyrophosphate (thiamine-PP) on Dowex IX8 in the formate form produced an unexpected peak, X-l, which was eluted just prior to the thiamine-PP peak. An ammonium formate, pH 4.5, gradient was the eluant. Rechromatography of either peak X-l or thiamine-PP produced the same two peaks. The radioactive specific activity per micromole labile phosphate was the same for the two peaks. Peak X-l appears to be a thiamine compound formed in the presence of formate solutions. This procedural artifact was circumvented by the substitution of acetate for formate. By varying the pH as well as the ionic strength a single column procedure has been developed that separates thiamine and the three phosphate esters quantitatively in micromolar amounts.
In the course of nutrition, vitamin, and enzyme studies, it is often necessary to quantitatively separate thiamine and the individual thiamine phosphate esters. Although a variety of chromatographic procedures, including anionic exchange resins (l-6) and cationic exchange resins (1,6-8), have been reported for the separation of thiamine and the individual phosphate esters, two separate column procedures are required (7,8). By combining a change in pH as well as an increase in the ionic strength of the eluting buffer, a simplified single column procedure has been developed and is described in this report. In addition evidence is presented describing an artifact produced in the presence of formate buffers. This observation indicates that formate should be avoided in work with thiamine compounds. 1 This investigation was supported in part by Grant AM 12180 from the National Institutes of Health, USPHS. A prehminary report of this work was presented at the Federation Meetings of American Societies for Experimental Biology 1979, No. 2813. * Present address: University of Maryland, College Park, Md.
MATERIALS
AND METHODS
Thiamine hydrochloride was purchased from J. T. Baker, thiamine-p and thiaminePP were purchased from Sigma, and radioactive thiamine labeled with 14C in the C, position of the thiazole ring was purchased from Searle Chemical. Ammonium formate, reagent grade, was purchased from Allied Chemical. Formic acid, reagent grade, was obtained from J. T. Baker. All other chemicals were reagent grade. Dowex resins were purchased from Bio-Rad. Labile phosphate was determined by Bartlett’s (9) modification of the Fisk-Subbarow procedure (10). Acid-washed glassware was used for the phosphate determination. The sample plus water and 0.5 ml of 2.0 M H,SO, in a total volume of 2.0 ml was heated in a boiling water bath for 20 min. After cooling 0.75 ml of 1.O M H2S04, 1.O ml of 0.5% ammonium molybdate solution, and 0.2 ml of Fisk-Subbarow reagent were 3 Abbreviations used: thiamine-P, thiamine monophosphate; thiamine-PP, thiamine diphosphate: thiamine-PPP, thiamine triphosphate.
145
0003..2697/80/030145-05$02,00/O Copyright All right\
r 1980 by Academx Prey,. Inc of reproductmn in any form reerved
146
GOUNARIS
AND SCHULMAN
FIG. 1. Chromatography of thiamine phosphate esters. Column parameters for A through C were 30 x 2.2 cm, Dowex 1X8, 200-400 mesh, and fraction volume was 7.5 ml. In A and B the material that elutes prior to the gradient, indicated by the arrow in A, is designated as peak I. The next two adjacent peaks are designated X-l and X-2 in that order. The final eluted material in A and C is referred to as peak III. Following application of the sample, the column was eluted with the equilibration buffer. The arrow along thex-axis indicates the beginning of the linear gradient described. (A) Material chromatographed was an aliquot of the thiamine phosphate ester synthesis. The resin was in the formate form and the linear gradient was established with 400 ml of 0.005 M NHZHCOO-, pH 4.5, ia the mixing chamber and 400 ml of 0.4 M NH:HCOO-, pH 4.5, in the reservoir. (B) Material chromatographed was a mixture of Y-labeled peak X-2 with unlabeled thiamine-PP. The resin form and the gradient were identical to those of A. (C) Material chromatographed was an aliquot of the thiamine phosphate ester synthesis. The resin was in the acetate form and the linear gradient was established with 400 ml of 0.005 M NH:CH,COO-, pH 4.5, in the mixing chamber and 400 ml of 0.4 M NH:CH,COO-, pH 4.5, in the reservoir. The micromoles labile phosphate per micromole thiamine compound for the peaks in chromatogram A were : peak 1 (thiamine and thiamine-P), 0.0; peak IIa (thiamine-PP artifact, X-l), 1.08; peak IIb (thiamine-PP), 0.96; and peak III (thiamine-PPP), 1.98.
added. The solution was heated in a boiling water bath for 7.0 min. Optical measuremen , were determined at 880 nm. The curve was linear up to 0.15 pmol inorganic phosphate. All other experimental procedures are described below. RESULTS AND DISCUSSION
This study evolved in the process of separating and purifying the products from the synthesis of 14C-labeled thiamine-PP prepared by the method of Viscontini et al. (11). Since micromolar amounts of material were involved, isolation by alcohol precipitation as described in the original procedure was not feasible. A chromatographic procedure utilizing a volatile eluting buffer was selected for the isolation of products. The procedures reported (6-11) were
modified as follows: Dowex 1X8, 200-400 mesh, was converted to the formate form, a column 30 x 2.2 cm was equilibrated with 0.005 M ammonium formate, pH 4.5. For elution a linear gradient was established with 400 ml of 0.005 M ammonium formate, pH 4.5, in the mixing chamber and 400 ml of 0.4 M ammonium formate, pH 4.5, in the reservoir. The gradient was started at fraction 10. A typical chromatogram is illustrated in Fig. IA. The column details are described in the legend. Thiamine and thiamine-P do not bind to the resin under these conditions and are eluted immediately. About 15 fractions after the gradient is started, two successive peaks are obtained which are designated as X-l and X-2. Analyses of X-l and X-2 showed 1 prnol of labile phosphate per micromole of thiamine compound. Rechromatography following lyophilization of either
FORMATION
OF A THIAMINE
ARTIFACT
peak resulted in a redistribution into two peaks, X-l and X-2. Chromatography of either radioactive peak combined with nonradioactive thiamine-PP again yielded peak X-l and peak X-2; the radioactive specific activity of the two peaks after chromatography was the same. The chromatography of a mixture of r4C-labeled X-2 and cold thiamine-PP is illustrated in Fig. 1B. Under these conditions an equilibration of the thiamine compounds X-l and X-2 occurs. Following lyophilization and solution in I>,0 an NMR spectrum of material from each peak was performed. The proton peak for the rapidly exchanging H at the C, position of the thiazole ring was not observed in X-l but was observed in X-2. An aliquot of X- 1 was dissolved in 1.O M HCl for 30 min, evaporated to dryness, and dissolved in D,O. The NMR spectrum of this material was repeated. Following this treatment the NMR spectrum was identical to that of X-2 which was identical with that of a known sample of thiamine-PP. A detailed analysis of the compound X-l was not pursued; however, the data suggest that X-l is a thiamine derivative in which the C, hydrogen of the thiazole ring is substituted. Formic acid, the first member of the carboxylic acid series, is characterized as having some aldehydic character (12), suggesting the possibility of attack by the carbene anion derived from thiamine. The possibility of formaldehyde as a contaminant in the formic acid and ammonium formate was considered. Formaldehyde was not indicated as a contaminant by the manufacturer and an aldehyde contaminant was not detected by the fuchsin-aldehyde test described by Shriner et al. (13). The structure and characterization of X-l is a problem for future investigation. In the context of this report the principal issue is to report the observation of this artifact and to caution others in the use of formate with thiamine compounds. Under the conditions of the chromatographic procedure described thiamine and thiamine-P do not bind to the resin; thm analogous species for these compounds
DURING
/
I
0
10
147
CHROMATOGRAPHY
1
20
I
I
30 40 50 Fh’ACTlON NIJMFR
1
I
/
60
70
80
FIG. 2. Chromatography of a mixture of thiamine, thiamine-P, and thiamine-PP. Column parameters were 30 x 0.9cm, Dowex 1X8,200-400 mesh, in the acetate form, and the fraction volume was 3.0 ml. The column was equilibrated with 0.005 M NH:CH,COO-, pH 6.7. The elution was initiated with 0.005 M NH:CH,COO-, pH 6.7; at fraction 5, indicated by the arrow, the linear gradient, established with 250 ml of 0.005 M NH:CH,COO-, pH 4.5, in the mixing chamber and 250 ml of 0.25 M NH$CH&OO-, pH 4.5, in the reservoir, was started.
would not be observed. Since only trace amounts of thiamine-PPP were present an analogous artifact could easily be undetected. Schellenberger and Hubner (6) reported a chromatographic separation of thiamine phosphate esters utilizing a similar column in 1965. In their procedure the column was equilibrated with water. Under this initial condition thiamine and thiamine-P did not bind. Thiamine-PP was then eluted with 0.01 M HCOOH followed by the elution of thiamine-PPP with 0.5 M HCOONa. The elution procedure used differs from that described in this report with respect to pH and the linear concentration gradient. This difference may have masked the presence of the thiamine artifact in the earlier work. In order to avoid this artifact, ammonium acetate was substituted for ammonium formate. A typical chromatogram with this modification is presented in Fig. IC. As predicted, peak X-l is not observed under these conditions. In order to effect the separation- of thi-
148
GOUNARIS THIAMINE
AND SCHULMAN
THIAMINE-MONO-P pH 4.5
pH4.5
THAMMHE-
051
04.
P-P
pH4.5 1
i
0.3 0.2
[ffff
0
240
260
280
303
240
260
280
300
L 240
260
280
1 MO
x, nm
FIG. 3. The spectra of the thiamine compounds at pH 4.5 and 6.7. Solvents were: 0.005 NH:CH,COO-, pH 4.5, and 0.005 M NH:CH,COO-, pH 6.7.
amine and thiamine-P, a second column was necessary. A further modification was then introduced that effectively separated thiamine and the thiamine phosphate esters. A consideration of the ionization constants4 indicates that at pH 6.5 thiamine will not bind but the thiamine phosphate esters will. By decreasing the pH to 4.5 without changing the ionic strength thiamine-P is eluted and then the thiamine-PP and thiamine-PPP can q At pH 4.5 net charge thiamine = +2, net charge thiamine-P = + 1; at pH 6.5 net charge thiamine = + 1, net charge thiamine-P = - 1.
M
be eluted with the linear gradient as previously shown. With this rationale the column was planned and executed. A 2.0 M ammonium acetate, pH 6.5, solution was used to convert the resin (Dowex 1X8, -200-400 mesh) to the acetate form. The column was equilibrated with 0.005 M ammonium acetate, pH 6.5. For a 30 x 0.9TABLE RECOVERY
2
OF MATERIALFROM OF
CHROMATOGRAPHY
FIG. 2”
Amount (pmol) TABLE EXTINCTION
1
Compound
applied
recovered
Percentage recovery
Thiamine Thiamine-P Thiamine-PP
1.28 0.83 0.70
1.26 0.93 0.64
98.4 112.0 91.4
COEFFICIENTS" E260
x 10-a
(a)
(b)
Compound
pH 4.5
pH 6.7
Thiamine Thiamine-P Thiamine-PP
9.14 10.12
6.89 6.80 7.18
a Solvents were (a) 0.005 M NH,+CH,COO-, pH 4.5, and (b) 0.005 M NH,+CH,COO-, pH 6.7. The E was calculated at A,,, indicated by the arrows in Fig. 3.
a The compounds were quantitated spectroscopically with EtBO determined as described in the text and correlated with total phosphate analysis and labile phosphate. An aliquot of thiamine-PP subjected for the same period was analyzed for labile phosphate. The analysis revealed a decrease of labile phosphate during this period, indicating 10.8% hydrolysis ofthiamine-PP. Adjustment for this change accounts for the increase in thiamine-P and decrease in thiamine-PP.
FORMATION
OF A THIAMINE
ARTIFACT
cm column the flow rate was 24 ml/h and 3.0-ml fractions were collected. A 2.0-ml aliquot containing thiamine, thiamine-P, and thiamine-PP was applied to the 30 x 0.9-cm column. The column was eluted with 0.005 M ammonium acetate, pH 6.5, until the first five fractions were collected and then the linear gradient was established with 250 ml of 0.005 M ammonium acetate, pH 4.5, in the mixing chamber and 250 ml of 0.25 M ammonium acetate, pH 4.5, in the reservoir. All column operations were performed at 4°C. This separation is illustrated in Fig. 2. The column was monitored at 260 nm for the thiamine moiety and for phosphate content using Bartlett’s procedure (9). Total phosphate analyses (9) at these concentrations were not reproducible; however, qualitatively they demonstrated the absence of phosphate in peak I and the presence of phosphate in peaks II and III. The absence of phosphate in peak I in conjunction with the uv spectrum identifies peak I as thiamine. The labile phosphate determination of peak II was negative but for peak III 1 pmol labile phosphate per micromole thiamine compound was found. Hence peak II is identified as thiamine-P and peak III as thiamine-PP. In order to quantitate the thiamine compounds, the extinction coefficients at the conditions of elution were determined and utilized for calculating recovery. The spectra at pH 4.5 and 6.7 are illustrated in Fig. 3 and the extinction coefficients calculated are summarized in Table 1. With this procedure the three thiamine compounds are completely resolved and quantitatively recovered (see Table 2). A review of these data demonstrates a loss of thiamine diphosphate and an increase of thiamine-P during the procedure. Hydrolysis of the labile phosphate of thiamine-PP could account for this exchange. An aliquot of the thiamine-PP solution used to prepare the sample was left standing during the time span of the chromatographic procedure.
149
DURING CHROMATOGRAPHY
When analyzed, it was found that the labile phosphate content had decreased by 10.8%. With this adjustment the material balance for each thiamine compound is between 98 and 102%. CONCLUSION
In this report a simplified single column procedure for the separation of thiamine and the thiamine phosphate esters, which requires routine laboratory facilities and standard techniques, has been described. In addition evidence has been presented demonstrating the production of a thiamine artifact formed when a Dowex-1 formate column and formate buffers were used to separate the thiamine compounds, thus indicating that formate should be avoided when working with these compounds. REFERENCES 1. Rindi, G., and Giuseppe, L. (1961) Biochem. J. 78, 602-606. 2. Matsukama, T., Hirano, H., and Yurugi, S. (1970) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.), Vol. 18A, p. 147, Academic Press, New York. 3. Koike, H., and Yusa, T. (1970) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.), Vol. lSA, pp. 105-108, Academic Press, New York. 4. Suzuoki, Z., Yoneda, M., and Hori, M. (1957) J. Biochem.
44, 783-786.
Koike, H., Wada, T., and Minakami, J. Biochem.
H. (1967)
62, 492-494.
Schellenberger,
A., and Hiibner, G. (1965) Z. 343, 189-192. Siliprandi, D., and Siliprandi, N. (1954) Biochim. Biophys. Acta 14, 52-61. Rossi-Fanelli, A., Ipata, P., and Fasella, P. (1961) Physiol.
Biochem.
Chem.
Biophys.
Res. Commun.
4, 23-27.
9. Bartlett, G. (1959) J. Biol. Chem. 234, 466-468. 10. Fiske, C. H., and Subbarow, Y. (1925) J. Biol. Chem.
66, 375-400.
11. Viscontini, M., Bonnetti, G., and Karrer, P. (1949) Helv. Chim. Acta 32, 1478-1485. 12. Fieser, L., and Fieser, M. (1950) Organic Chemistry, 2nd ed., p. 169, Heath, Boston. 13. Shriner, R., Fuson, R., and Curtin, D. (1956) The systematic Identification of Organic Compounds, 5th ed., p. 129, Wiley, New York.
102, 145% 149 (1980)
BIOCHEMISTRY
Formation of a Thiamine Artifact During Chromatography: A Single Column Procedure for the Separation of Thiamine and the Thiamine Mono-, Di-, and Triphosphate Esters1 ANNE Department
D. GOUNARIS of Chemistry,
Vassar
AND MAUREEN College.
SCHULMAN’
Poughkeepsie.
New
York
12601
Received June 5, 1979 Chromatography of a preparation of [Wlthiamine pyrophosphate (thiamine-PP) on Dowex IX8 in the formate form produced an unexpected peak, X-l, which was eluted just prior to the thiamine-PP peak. An ammonium formate, pH 4.5, gradient was the eluant. Rechromatography of either peak X-l or thiamine-PP produced the same two peaks. The radioactive specific activity per micromole labile phosphate was the same for the two peaks. Peak X-l appears to be a thiamine compound formed in the presence of formate solutions. This procedural artifact was circumvented by the substitution of acetate for formate. By varying the pH as well as the ionic strength a single column procedure has been developed that separates thiamine and the three phosphate esters quantitatively in micromolar amounts.
In the course of nutrition, vitamin, and enzyme studies, it is often necessary to quantitatively separate thiamine and the individual thiamine phosphate esters. Although a variety of chromatographic procedures, including anionic exchange resins (l-6) and cationic exchange resins (1,6-8), have been reported for the separation of thiamine and the individual phosphate esters, two separate column procedures are required (7,8). By combining a change in pH as well as an increase in the ionic strength of the eluting buffer, a simplified single column procedure has been developed and is described in this report. In addition evidence is presented describing an artifact produced in the presence of formate buffers. This observation indicates that formate should be avoided in work with thiamine compounds. 1 This investigation was supported in part by Grant AM 12180 from the National Institutes of Health, USPHS. A prehminary report of this work was presented at the Federation Meetings of American Societies for Experimental Biology 1979, No. 2813. * Present address: University of Maryland, College Park, Md.
MATERIALS
AND METHODS
Thiamine hydrochloride was purchased from J. T. Baker, thiamine-p and thiaminePP were purchased from Sigma, and radioactive thiamine labeled with 14C in the C, position of the thiazole ring was purchased from Searle Chemical. Ammonium formate, reagent grade, was purchased from Allied Chemical. Formic acid, reagent grade, was obtained from J. T. Baker. All other chemicals were reagent grade. Dowex resins were purchased from Bio-Rad. Labile phosphate was determined by Bartlett’s (9) modification of the Fisk-Subbarow procedure (10). Acid-washed glassware was used for the phosphate determination. The sample plus water and 0.5 ml of 2.0 M H,SO, in a total volume of 2.0 ml was heated in a boiling water bath for 20 min. After cooling 0.75 ml of 1.O M H2S04, 1.O ml of 0.5% ammonium molybdate solution, and 0.2 ml of Fisk-Subbarow reagent were 3 Abbreviations used: thiamine-P, thiamine monophosphate; thiamine-PP, thiamine diphosphate: thiamine-PPP, thiamine triphosphate.
145
0003..2697/80/030145-05$02,00/O Copyright All right\
r 1980 by Academx Prey,. Inc of reproductmn in any form reerved
146
GOUNARIS
AND SCHULMAN
FIG. 1. Chromatography of thiamine phosphate esters. Column parameters for A through C were 30 x 2.2 cm, Dowex 1X8, 200-400 mesh, and fraction volume was 7.5 ml. In A and B the material that elutes prior to the gradient, indicated by the arrow in A, is designated as peak I. The next two adjacent peaks are designated X-l and X-2 in that order. The final eluted material in A and C is referred to as peak III. Following application of the sample, the column was eluted with the equilibration buffer. The arrow along thex-axis indicates the beginning of the linear gradient described. (A) Material chromatographed was an aliquot of the thiamine phosphate ester synthesis. The resin was in the formate form and the linear gradient was established with 400 ml of 0.005 M NHZHCOO-, pH 4.5, ia the mixing chamber and 400 ml of 0.4 M NH:HCOO-, pH 4.5, in the reservoir. (B) Material chromatographed was a mixture of Y-labeled peak X-2 with unlabeled thiamine-PP. The resin form and the gradient were identical to those of A. (C) Material chromatographed was an aliquot of the thiamine phosphate ester synthesis. The resin was in the acetate form and the linear gradient was established with 400 ml of 0.005 M NH:CH,COO-, pH 4.5, in the mixing chamber and 400 ml of 0.4 M NH:CH,COO-, pH 4.5, in the reservoir. The micromoles labile phosphate per micromole thiamine compound for the peaks in chromatogram A were : peak 1 (thiamine and thiamine-P), 0.0; peak IIa (thiamine-PP artifact, X-l), 1.08; peak IIb (thiamine-PP), 0.96; and peak III (thiamine-PPP), 1.98.
added. The solution was heated in a boiling water bath for 7.0 min. Optical measuremen , were determined at 880 nm. The curve was linear up to 0.15 pmol inorganic phosphate. All other experimental procedures are described below. RESULTS AND DISCUSSION
This study evolved in the process of separating and purifying the products from the synthesis of 14C-labeled thiamine-PP prepared by the method of Viscontini et al. (11). Since micromolar amounts of material were involved, isolation by alcohol precipitation as described in the original procedure was not feasible. A chromatographic procedure utilizing a volatile eluting buffer was selected for the isolation of products. The procedures reported (6-11) were
modified as follows: Dowex 1X8, 200-400 mesh, was converted to the formate form, a column 30 x 2.2 cm was equilibrated with 0.005 M ammonium formate, pH 4.5. For elution a linear gradient was established with 400 ml of 0.005 M ammonium formate, pH 4.5, in the mixing chamber and 400 ml of 0.4 M ammonium formate, pH 4.5, in the reservoir. The gradient was started at fraction 10. A typical chromatogram is illustrated in Fig. IA. The column details are described in the legend. Thiamine and thiamine-P do not bind to the resin under these conditions and are eluted immediately. About 15 fractions after the gradient is started, two successive peaks are obtained which are designated as X-l and X-2. Analyses of X-l and X-2 showed 1 prnol of labile phosphate per micromole of thiamine compound. Rechromatography following lyophilization of either
FORMATION
OF A THIAMINE
ARTIFACT
peak resulted in a redistribution into two peaks, X-l and X-2. Chromatography of either radioactive peak combined with nonradioactive thiamine-PP again yielded peak X-l and peak X-2; the radioactive specific activity of the two peaks after chromatography was the same. The chromatography of a mixture of r4C-labeled X-2 and cold thiamine-PP is illustrated in Fig. 1B. Under these conditions an equilibration of the thiamine compounds X-l and X-2 occurs. Following lyophilization and solution in I>,0 an NMR spectrum of material from each peak was performed. The proton peak for the rapidly exchanging H at the C, position of the thiazole ring was not observed in X-l but was observed in X-2. An aliquot of X- 1 was dissolved in 1.O M HCl for 30 min, evaporated to dryness, and dissolved in D,O. The NMR spectrum of this material was repeated. Following this treatment the NMR spectrum was identical to that of X-2 which was identical with that of a known sample of thiamine-PP. A detailed analysis of the compound X-l was not pursued; however, the data suggest that X-l is a thiamine derivative in which the C, hydrogen of the thiazole ring is substituted. Formic acid, the first member of the carboxylic acid series, is characterized as having some aldehydic character (12), suggesting the possibility of attack by the carbene anion derived from thiamine. The possibility of formaldehyde as a contaminant in the formic acid and ammonium formate was considered. Formaldehyde was not indicated as a contaminant by the manufacturer and an aldehyde contaminant was not detected by the fuchsin-aldehyde test described by Shriner et al. (13). The structure and characterization of X-l is a problem for future investigation. In the context of this report the principal issue is to report the observation of this artifact and to caution others in the use of formate with thiamine compounds. Under the conditions of the chromatographic procedure described thiamine and thiamine-P do not bind to the resin; thm analogous species for these compounds
DURING
/
I
0
10
147
CHROMATOGRAPHY
1
20
I
I
30 40 50 Fh’ACTlON NIJMFR
1
I
/
60
70
80
FIG. 2. Chromatography of a mixture of thiamine, thiamine-P, and thiamine-PP. Column parameters were 30 x 0.9cm, Dowex 1X8,200-400 mesh, in the acetate form, and the fraction volume was 3.0 ml. The column was equilibrated with 0.005 M NH:CH,COO-, pH 6.7. The elution was initiated with 0.005 M NH:CH,COO-, pH 6.7; at fraction 5, indicated by the arrow, the linear gradient, established with 250 ml of 0.005 M NH:CH,COO-, pH 4.5, in the mixing chamber and 250 ml of 0.25 M NH$CH&OO-, pH 4.5, in the reservoir, was started.
would not be observed. Since only trace amounts of thiamine-PPP were present an analogous artifact could easily be undetected. Schellenberger and Hubner (6) reported a chromatographic separation of thiamine phosphate esters utilizing a similar column in 1965. In their procedure the column was equilibrated with water. Under this initial condition thiamine and thiamine-P did not bind. Thiamine-PP was then eluted with 0.01 M HCOOH followed by the elution of thiamine-PPP with 0.5 M HCOONa. The elution procedure used differs from that described in this report with respect to pH and the linear concentration gradient. This difference may have masked the presence of the thiamine artifact in the earlier work. In order to avoid this artifact, ammonium acetate was substituted for ammonium formate. A typical chromatogram with this modification is presented in Fig. IC. As predicted, peak X-l is not observed under these conditions. In order to effect the separation- of thi-
148
GOUNARIS THIAMINE
AND SCHULMAN
THIAMINE-MONO-P pH 4.5
pH4.5
THAMMHE-
051
04.
P-P
pH4.5 1
i
0.3 0.2
[ffff
0
240
260
280
303
240
260
280
300
L 240
260
280
1 MO
x, nm
FIG. 3. The spectra of the thiamine compounds at pH 4.5 and 6.7. Solvents were: 0.005 NH:CH,COO-, pH 4.5, and 0.005 M NH:CH,COO-, pH 6.7.
amine and thiamine-P, a second column was necessary. A further modification was then introduced that effectively separated thiamine and the thiamine phosphate esters. A consideration of the ionization constants4 indicates that at pH 6.5 thiamine will not bind but the thiamine phosphate esters will. By decreasing the pH to 4.5 without changing the ionic strength thiamine-P is eluted and then the thiamine-PP and thiamine-PPP can q At pH 4.5 net charge thiamine = +2, net charge thiamine-P = + 1; at pH 6.5 net charge thiamine = + 1, net charge thiamine-P = - 1.
M
be eluted with the linear gradient as previously shown. With this rationale the column was planned and executed. A 2.0 M ammonium acetate, pH 6.5, solution was used to convert the resin (Dowex 1X8, -200-400 mesh) to the acetate form. The column was equilibrated with 0.005 M ammonium acetate, pH 6.5. For a 30 x 0.9TABLE RECOVERY
2
OF MATERIALFROM OF
CHROMATOGRAPHY
FIG. 2”
Amount (pmol) TABLE EXTINCTION
1
Compound
applied
recovered
Percentage recovery
Thiamine Thiamine-P Thiamine-PP
1.28 0.83 0.70
1.26 0.93 0.64
98.4 112.0 91.4
COEFFICIENTS" E260
x 10-a
(a)
(b)
Compound
pH 4.5
pH 6.7
Thiamine Thiamine-P Thiamine-PP
9.14 10.12
6.89 6.80 7.18
a Solvents were (a) 0.005 M NH,+CH,COO-, pH 4.5, and (b) 0.005 M NH,+CH,COO-, pH 6.7. The E was calculated at A,,, indicated by the arrows in Fig. 3.
a The compounds were quantitated spectroscopically with EtBO determined as described in the text and correlated with total phosphate analysis and labile phosphate. An aliquot of thiamine-PP subjected for the same period was analyzed for labile phosphate. The analysis revealed a decrease of labile phosphate during this period, indicating 10.8% hydrolysis ofthiamine-PP. Adjustment for this change accounts for the increase in thiamine-P and decrease in thiamine-PP.
FORMATION
OF A THIAMINE
ARTIFACT
cm column the flow rate was 24 ml/h and 3.0-ml fractions were collected. A 2.0-ml aliquot containing thiamine, thiamine-P, and thiamine-PP was applied to the 30 x 0.9-cm column. The column was eluted with 0.005 M ammonium acetate, pH 6.5, until the first five fractions were collected and then the linear gradient was established with 250 ml of 0.005 M ammonium acetate, pH 4.5, in the mixing chamber and 250 ml of 0.25 M ammonium acetate, pH 4.5, in the reservoir. All column operations were performed at 4°C. This separation is illustrated in Fig. 2. The column was monitored at 260 nm for the thiamine moiety and for phosphate content using Bartlett’s procedure (9). Total phosphate analyses (9) at these concentrations were not reproducible; however, qualitatively they demonstrated the absence of phosphate in peak I and the presence of phosphate in peaks II and III. The absence of phosphate in peak I in conjunction with the uv spectrum identifies peak I as thiamine. The labile phosphate determination of peak II was negative but for peak III 1 pmol labile phosphate per micromole thiamine compound was found. Hence peak II is identified as thiamine-P and peak III as thiamine-PP. In order to quantitate the thiamine compounds, the extinction coefficients at the conditions of elution were determined and utilized for calculating recovery. The spectra at pH 4.5 and 6.7 are illustrated in Fig. 3 and the extinction coefficients calculated are summarized in Table 1. With this procedure the three thiamine compounds are completely resolved and quantitatively recovered (see Table 2). A review of these data demonstrates a loss of thiamine diphosphate and an increase of thiamine-P during the procedure. Hydrolysis of the labile phosphate of thiamine-PP could account for this exchange. An aliquot of the thiamine-PP solution used to prepare the sample was left standing during the time span of the chromatographic procedure.
149
DURING CHROMATOGRAPHY
When analyzed, it was found that the labile phosphate content had decreased by 10.8%. With this adjustment the material balance for each thiamine compound is between 98 and 102%. CONCLUSION
In this report a simplified single column procedure for the separation of thiamine and the thiamine phosphate esters, which requires routine laboratory facilities and standard techniques, has been described. In addition evidence has been presented demonstrating the production of a thiamine artifact formed when a Dowex-1 formate column and formate buffers were used to separate the thiamine compounds, thus indicating that formate should be avoided when working with these compounds. REFERENCES 1. Rindi, G., and Giuseppe, L. (1961) Biochem. J. 78, 602-606. 2. Matsukama, T., Hirano, H., and Yurugi, S. (1970) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.), Vol. 18A, p. 147, Academic Press, New York. 3. Koike, H., and Yusa, T. (1970) in Methods in Enzymology (McCormick, D. B., and Wright, L. D., eds.), Vol. lSA, pp. 105-108, Academic Press, New York. 4. Suzuoki, Z., Yoneda, M., and Hori, M. (1957) J. Biochem.
44, 783-786.
Koike, H., Wada, T., and Minakami, J. Biochem.
H. (1967)
62, 492-494.
Schellenberger,
A., and Hiibner, G. (1965) Z. 343, 189-192. Siliprandi, D., and Siliprandi, N. (1954) Biochim. Biophys. Acta 14, 52-61. Rossi-Fanelli, A., Ipata, P., and Fasella, P. (1961) Physiol.
Biochem.
Chem.
Biophys.
Res. Commun.
4, 23-27.
9. Bartlett, G. (1959) J. Biol. Chem. 234, 466-468. 10. Fiske, C. H., and Subbarow, Y. (1925) J. Biol. Chem.
66, 375-400.
11. Viscontini, M., Bonnetti, G., and Karrer, P. (1949) Helv. Chim. Acta 32, 1478-1485. 12. Fieser, L., and Fieser, M. (1950) Organic Chemistry, 2nd ed., p. 169, Heath, Boston. 13. Shriner, R., Fuson, R., and Curtin, D. (1956) The systematic Identification of Organic Compounds, 5th ed., p. 129, Wiley, New York.