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Detection and identification of fructose 1-phosphate by paper chromatography
ANALYTICAL
2, 497-501 (1961)
BIOCHEMISTHY
Detection
and
Identification by
Paper KURT
From
the
of
Fructose
1 -Phosphate
Chromatography STEINITZ
Biochemical Section, Rappaport Hospital, Petah Tikva,
Be&son
Received April
Laboratories, Israel
27, 1961
INTRODUCTION
chromatography has not been employed generally for the sepand identification of fructose l-phosphate (FlP),l in part because there has not, been a met,hod for identifying FlP which was specific. Although various solvent systems have been employed (1)) satisfactory separation of FlP, F6P, FDP, and other phosphorylated sugars has not been obtained. A satisfactory separation of FlP from other hexose phosphates can be obtained using the tert-butanol-waterpicric acid system of Hanes and Isherwood (2) in the modification of Sidbury (3) .2 This system is slow, and the picric acid present obviates the possibility of detection other than by hydrolysis and identification of phosphate. Furthermore, satisfactory separation of the three phosphorylated fructose intermediates is not, achieved; hence a modification of the technique which would permit, identification of the individual fructose phosphates is desirable. This has been accomplished by using a solvent free of nonvolatile constituents, which permits greater latitude in the application of different, color reactions. This paper reports a new solvent system and an ultraviolet fluorescence procedure which makes the detection and differentiation of FlP from other hexosephosphates possible. While attempting to develop a method for selectively hydrolyzing the phosphate bond of FlP and then identifying the free fructose or its furfural derivative, we found that, a strong ultraviolet fluorescence was produced by the FlP spot, after dipping the chromatogram in a solution Paper aration
’ Abbreviations used: FlP, fructose l-phosphate; F6P, fructose g-phosphate ; FDP, fructose 1,6-diphosphate ; GIP, glucose l-phosphate ; G6P, glucose bphosphate; Pi, inorganic phosphorus. ‘Also, personal communication from J. B. Sidbury, Jr, 497
498
STEINITZ
containing 6’7% hydrochloric acid and glycerol, followed by heating to 100°C for 10 min. Glycerol had been added in order to retain moisture, but it turned out, that its addition changed the intensity and color of the fluorescence. This fluorescence, after treatment with HCl-glycerol, is the basis for the identification of FlP. The ultraviolet fluorescence is masked when nonvolat,ile substances are used in the solvent system [e.g., picric acid (2)], but hydrochloric acid proved to be a satisfactory substitute. METHODS
Standard samples of FlP, F6P, FDP, GlP, and G6P were chromatographed using S & S 2043b mgl paper with solvent systems containing tert-butanol and hydrochloric acid in different proportions and with certain additions (see Table 1). The chromatograms were dried in air TABLE RF VALUES
Solvents~ GlP 1
2 3 4 5 6
0.37 0.59 0.37 0.48 0.48 0.08
1
OF FRUCTOSE
,PHOSPHATES
G6P
FlP
FDP
F6P
Pi
-0.51 -
0.40 0.61 0.44 0.59 0.53 0.12
0.45 0.61 0.50 0.65 0.60 0.03
0.45 0.69 0.51 0.64 0.60 0.12
0.63 0.87 0.65 1.0
0.72 0.50
36-48 20 16-20 18 18 48
hr, hr, hr, hr, hr, hr,
descend. ascend. ascend. ascend. ascend. descend.
a Solvent 1, tert-butanol-water-picric acid: 80:20:2; solvent 2, tert-hutanol-water-6N HCl, 80:20:2; solvent 3, tert-butanol-0.1 N HCI, 80:20; solvent 4, tert-butanol-O.l N HCI-methanol, 60:20:20; solvent 5, tert-butanol-O.l N HCl-acetone. 60:20:20; Polvent 6, tert-butanol-water-formic acid, 70 : 10 : 20.
after they were run, and dipped in a freshly prepared solution containing 6 ml of cont. HCl, 3 ml glycerol, and 91 ml acetone (the glycerolacetone mixture may be kept as stock, but the HCI should be added before use. The chromatograms were allowed to stand at room temperature until nearly dry and then heated to 100°C for 5 min. The chromatograms were then viewed with a filtered ultraviolet light source (3600-A region). The heating and ultraviolet scanning were repeated, and the fluorescent areas were marked with a pencil. The chromatograms could then be treated with the molybdate reagent as described by Burrows et al. (4), heated, and irradiated with unfiltered ultraviolet light (2500-A region) to obtain the position of the inorganic phosphate and phosphorylated esters as blue spots. Paper pretreated with HCl-glycerol reagent retains its white color for months after treatment with the
DETECTION
OF
FRUCTOSE
I-PHOSPHATE
499
molybdate reagent, while paper treated only with molybdate reagent gets dark after a short time. The following additional analytical techniques have been applied to chromatograms of the st,andard hexose phosphates : 1. Dipping in a freshly prepared solution of 6% cone. NC1 in acetone, drying superficially, heating for 24 min at lOO”C, and observing under ultraviolet light. 2. The quinine su1fat.e method of R,orem (5). 3. The sulfosalicylic method of Rorem (6). 4. The naphtoresorcinol dip-method as described by Smith (7). 5. The benzidine method as described by Smith (7). RESULTS
AND
DISCUSSION
The RF values found with the different solvents used are reported in Table 1. Solvent No. 3, which contains 80 ml tert-butanol and 20 ml of 0.1 N HCl, compares favorably with the solvent used by Hanes and Isherwood (2) and modified by Sidbury,2 which due to its content of picric acid cannot be used for the detection of ultraviolet fluorescence. The RF values are similar, and the FlP is clearly separated from F6P and FDP. Ascending development with this tert-butanol-HCl solvent ir recommended as the method of choice. Barium, calcium, and magnesium salts of hexose phosphates may be applied directly at the starting line of the chromatogram, as the acidity of the solvent is sufficiently strong to promote migration [see Hinsberg and Lang (8) 1. Solvent No. 6, containing formic acid, does not separate FlP% from F6P and gives RF values similar to solvents C and D used by Hanes and Isherwood (2), also containing formic acid. A higher concentration of HCl (solvent No. 2) does not differentiate between FlP and FDP and partly hydrolyzes GlP. tert-Butanol with much stronger HCl has been used for the separation of nucleotides by Smith and Markham (9). The addition of methanol (solvent No. 4) or of acetone (solvent No. 5) causes an increase of the movement of all phosphoric esters, but the distances between the spots remain essentially unchanged. The results of the ultraviolet fluorescence after the application of the dip reagents are summarized in Table 2. With the HCl-glycerol reagent a typical strong white ultraviolet fluorescence is observed with FlP, and a pale red fluorescence with F6P. The lower limit of detection is about 0.02 pmole for FlP and 0.04 pmole for F6P. If the heating period is prolonged, the red fluorescence of F6P decreases in intensity while the white fluorescence of FlP remains stable for more than 10 min. and
6% HCl and 3% glycerol in acetone
6% HCl in acetone Quinine sulfate (5) Sulfosalicylic acid (6) Naphthoresorcinol (7) Benzidine (7)
2 3 4 5 6
reagent
1
Dip
5 min 100” 10 min 100” 15 min 100” 2-4 min 100” 5 min loo0 As original As original
Heating time and temp.
DIP
+ + + + + + -
U~~wi$
REAGENTS
2
Gray-brown Gray-brown Gray Gray-brown
Background
FRUCTOSE
TABLE FOR
White White Faint white Yellow + -
FIP
PHOSPHATES FDP
Pale red Faint red Trace Yellow f + Red Faint gray-white Red +
FBP
White -
GlP
Trace +
GGP
DETECTION OF FRUCTOSE ~-PHOSPHATE
501
decreases only later. The fluorescent spot,s are stable and can be detected on the paper after weeks. No other react.ion on paper chromatograms has been described which distinguishes the different fructose phosphates. The HCl reagent without glycerol produces a yellow fluorescence with bot,h FlP and F6P after a short heating. The spot of F6P has a weak reddish tinge, but the difference between the two spots is not obvious. The other color reagents mentioned in Table 2 can be used for additional confirmation. The quinine sulfate reagent (7) gives a strong white fluorescence with all three fructose phosphates. The sulfosalicylic reagent (5) gives no color with FlP but a red fluorescence wit,h F6P. SUMMARY
A specific procedure is described for the select,ive detection of fructose l-phosphate (FlP). The chromatograms are developed in tcrtbutanol-0.1 N HCl (4: 1). After complete drying, the chromatograms are dipped in a solution of HCl and glycerol in acetone, dried, and heated for specified periods. Viewed under ulbraviolet light, FlP shows a typical white and fructose 6-phosphate (F6P) a pale red fluorescence. After marking the spots, the chromatograms arc treated with molybdatc reagent and all phosphates are located. Other reagents for the detection of fructose phosphates are used for comparison. REFERENCES B~NIVJKS~I, R. S., AND AXELROD, R., J. Bid. Chem. 193, 405 (1951) HANES, C. S., AND ISHERWOOD, F. A., hrature 164, 1107 (1949). SIDBURY, J. B., JR., Biochem. Preparations 7. 61 H. A. Lardy, Editor (1960). BURROWS, S., GRYLLS, E. M. S., AND H.+RRISOS, J. S., Nnture 170, 800 (1952). ROREM, E. S., Nature 183, 1739 (1959). ROREM, E. S., Anal. Biochem. 1, 218 (1960). SMITH, I., “Chromatographic Techniques,” p. 168. Heinemann, London, 1958. HINSBERG, Ii., ASD LANG, K., “Medizinisrhe Chemie,” p, 106. TJrhan & f$phwarzenberg, Miinchen, 1957. 9. SMITH, J. 11.. .\ND M.WKH.W, R., Hiochcm. .I. 46, 509 (1950).
1. 2. 3. 4. 5. 6. 7. 8.
2, 497-501 (1961)
BIOCHEMISTHY
Detection
and
Identification by
Paper KURT
From
the
of
Fructose
1 -Phosphate
Chromatography STEINITZ
Biochemical Section, Rappaport Hospital, Petah Tikva,
Be&son
Received April
Laboratories, Israel
27, 1961
INTRODUCTION
chromatography has not been employed generally for the sepand identification of fructose l-phosphate (FlP),l in part because there has not, been a met,hod for identifying FlP which was specific. Although various solvent systems have been employed (1)) satisfactory separation of FlP, F6P, FDP, and other phosphorylated sugars has not been obtained. A satisfactory separation of FlP from other hexose phosphates can be obtained using the tert-butanol-waterpicric acid system of Hanes and Isherwood (2) in the modification of Sidbury (3) .2 This system is slow, and the picric acid present obviates the possibility of detection other than by hydrolysis and identification of phosphate. Furthermore, satisfactory separation of the three phosphorylated fructose intermediates is not, achieved; hence a modification of the technique which would permit, identification of the individual fructose phosphates is desirable. This has been accomplished by using a solvent free of nonvolatile constituents, which permits greater latitude in the application of different, color reactions. This paper reports a new solvent system and an ultraviolet fluorescence procedure which makes the detection and differentiation of FlP from other hexosephosphates possible. While attempting to develop a method for selectively hydrolyzing the phosphate bond of FlP and then identifying the free fructose or its furfural derivative, we found that, a strong ultraviolet fluorescence was produced by the FlP spot, after dipping the chromatogram in a solution Paper aration
’ Abbreviations used: FlP, fructose l-phosphate; F6P, fructose g-phosphate ; FDP, fructose 1,6-diphosphate ; GIP, glucose l-phosphate ; G6P, glucose bphosphate; Pi, inorganic phosphorus. ‘Also, personal communication from J. B. Sidbury, Jr, 497
498
STEINITZ
containing 6’7% hydrochloric acid and glycerol, followed by heating to 100°C for 10 min. Glycerol had been added in order to retain moisture, but it turned out, that its addition changed the intensity and color of the fluorescence. This fluorescence, after treatment with HCl-glycerol, is the basis for the identification of FlP. The ultraviolet fluorescence is masked when nonvolat,ile substances are used in the solvent system [e.g., picric acid (2)], but hydrochloric acid proved to be a satisfactory substitute. METHODS
Standard samples of FlP, F6P, FDP, GlP, and G6P were chromatographed using S & S 2043b mgl paper with solvent systems containing tert-butanol and hydrochloric acid in different proportions and with certain additions (see Table 1). The chromatograms were dried in air TABLE RF VALUES
Solvents~ GlP 1
2 3 4 5 6
0.37 0.59 0.37 0.48 0.48 0.08
1
OF FRUCTOSE
,PHOSPHATES
G6P
FlP
FDP
F6P
Pi
-0.51 -
0.40 0.61 0.44 0.59 0.53 0.12
0.45 0.61 0.50 0.65 0.60 0.03
0.45 0.69 0.51 0.64 0.60 0.12
0.63 0.87 0.65 1.0
0.72 0.50
36-48 20 16-20 18 18 48
hr, hr, hr, hr, hr, hr,
descend. ascend. ascend. ascend. ascend. descend.
a Solvent 1, tert-butanol-water-picric acid: 80:20:2; solvent 2, tert-hutanol-water-6N HCl, 80:20:2; solvent 3, tert-butanol-0.1 N HCI, 80:20; solvent 4, tert-butanol-O.l N HCI-methanol, 60:20:20; solvent 5, tert-butanol-O.l N HCl-acetone. 60:20:20; Polvent 6, tert-butanol-water-formic acid, 70 : 10 : 20.
after they were run, and dipped in a freshly prepared solution containing 6 ml of cont. HCl, 3 ml glycerol, and 91 ml acetone (the glycerolacetone mixture may be kept as stock, but the HCI should be added before use. The chromatograms were allowed to stand at room temperature until nearly dry and then heated to 100°C for 5 min. The chromatograms were then viewed with a filtered ultraviolet light source (3600-A region). The heating and ultraviolet scanning were repeated, and the fluorescent areas were marked with a pencil. The chromatograms could then be treated with the molybdate reagent as described by Burrows et al. (4), heated, and irradiated with unfiltered ultraviolet light (2500-A region) to obtain the position of the inorganic phosphate and phosphorylated esters as blue spots. Paper pretreated with HCl-glycerol reagent retains its white color for months after treatment with the
DETECTION
OF
FRUCTOSE
I-PHOSPHATE
499
molybdate reagent, while paper treated only with molybdate reagent gets dark after a short time. The following additional analytical techniques have been applied to chromatograms of the st,andard hexose phosphates : 1. Dipping in a freshly prepared solution of 6% cone. NC1 in acetone, drying superficially, heating for 24 min at lOO”C, and observing under ultraviolet light. 2. The quinine su1fat.e method of R,orem (5). 3. The sulfosalicylic method of Rorem (6). 4. The naphtoresorcinol dip-method as described by Smith (7). 5. The benzidine method as described by Smith (7). RESULTS
AND
DISCUSSION
The RF values found with the different solvents used are reported in Table 1. Solvent No. 3, which contains 80 ml tert-butanol and 20 ml of 0.1 N HCl, compares favorably with the solvent used by Hanes and Isherwood (2) and modified by Sidbury,2 which due to its content of picric acid cannot be used for the detection of ultraviolet fluorescence. The RF values are similar, and the FlP is clearly separated from F6P and FDP. Ascending development with this tert-butanol-HCl solvent ir recommended as the method of choice. Barium, calcium, and magnesium salts of hexose phosphates may be applied directly at the starting line of the chromatogram, as the acidity of the solvent is sufficiently strong to promote migration [see Hinsberg and Lang (8) 1. Solvent No. 6, containing formic acid, does not separate FlP% from F6P and gives RF values similar to solvents C and D used by Hanes and Isherwood (2), also containing formic acid. A higher concentration of HCl (solvent No. 2) does not differentiate between FlP and FDP and partly hydrolyzes GlP. tert-Butanol with much stronger HCl has been used for the separation of nucleotides by Smith and Markham (9). The addition of methanol (solvent No. 4) or of acetone (solvent No. 5) causes an increase of the movement of all phosphoric esters, but the distances between the spots remain essentially unchanged. The results of the ultraviolet fluorescence after the application of the dip reagents are summarized in Table 2. With the HCl-glycerol reagent a typical strong white ultraviolet fluorescence is observed with FlP, and a pale red fluorescence with F6P. The lower limit of detection is about 0.02 pmole for FlP and 0.04 pmole for F6P. If the heating period is prolonged, the red fluorescence of F6P decreases in intensity while the white fluorescence of FlP remains stable for more than 10 min. and
6% HCl and 3% glycerol in acetone
6% HCl in acetone Quinine sulfate (5) Sulfosalicylic acid (6) Naphthoresorcinol (7) Benzidine (7)
2 3 4 5 6
reagent
1
Dip
5 min 100” 10 min 100” 15 min 100” 2-4 min 100” 5 min loo0 As original As original
Heating time and temp.
DIP
+ + + + + + -
U~~wi$
REAGENTS
2
Gray-brown Gray-brown Gray Gray-brown
Background
FRUCTOSE
TABLE FOR
White White Faint white Yellow + -
FIP
PHOSPHATES FDP
Pale red Faint red Trace Yellow f + Red Faint gray-white Red +
FBP
White -
GlP
Trace +
GGP
DETECTION OF FRUCTOSE ~-PHOSPHATE
501
decreases only later. The fluorescent spot,s are stable and can be detected on the paper after weeks. No other react.ion on paper chromatograms has been described which distinguishes the different fructose phosphates. The HCl reagent without glycerol produces a yellow fluorescence with bot,h FlP and F6P after a short heating. The spot of F6P has a weak reddish tinge, but the difference between the two spots is not obvious. The other color reagents mentioned in Table 2 can be used for additional confirmation. The quinine sulfate reagent (7) gives a strong white fluorescence with all three fructose phosphates. The sulfosalicylic reagent (5) gives no color with FlP but a red fluorescence wit,h F6P. SUMMARY
A specific procedure is described for the select,ive detection of fructose l-phosphate (FlP). The chromatograms are developed in tcrtbutanol-0.1 N HCl (4: 1). After complete drying, the chromatograms are dipped in a solution of HCl and glycerol in acetone, dried, and heated for specified periods. Viewed under ulbraviolet light, FlP shows a typical white and fructose 6-phosphate (F6P) a pale red fluorescence. After marking the spots, the chromatograms arc treated with molybdatc reagent and all phosphates are located. Other reagents for the detection of fructose phosphates are used for comparison. REFERENCES B~NIVJKS~I, R. S., AND AXELROD, R., J. Bid. Chem. 193, 405 (1951) HANES, C. S., AND ISHERWOOD, F. A., hrature 164, 1107 (1949). SIDBURY, J. B., JR., Biochem. Preparations 7. 61 H. A. Lardy, Editor (1960). BURROWS, S., GRYLLS, E. M. S., AND H.+RRISOS, J. S., Nnture 170, 800 (1952). ROREM, E. S., Nature 183, 1739 (1959). ROREM, E. S., Anal. Biochem. 1, 218 (1960). SMITH, I., “Chromatographic Techniques,” p. 168. Heinemann, London, 1958. HINSBERG, Ii., ASD LANG, K., “Medizinisrhe Chemie,” p, 106. TJrhan & f$phwarzenberg, Miinchen, 1957. 9. SMITH, J. 11.. .\ND M.WKH.W, R., Hiochcm. .I. 46, 509 (1950).
1. 2. 3. 4. 5. 6. 7. 8.