12) Korte, F., Sieper, H., Tetrahedron 10, 153 (1960). (13) Leaf, G., Todd, .4. R., Wilkinson, S.,J . C‘hem. SOC.,1942, 185. (14) Loewe, S., Arc,h. Exp. Pathol. Phamaakol. 211, 175 (1950).
(15) Osadchuk, N., Relgional Office, Food and Drug Directorate, 55 St. Clair Ave. E., Toronto, Ont. private communication, June 7, 1962. (16) Powell, G., Salmm, IbI., Bembry, T. H., Walton, R. P., Science 93, 522 (1941)
(17) Schultz, 0. E., Haffner, G., Arch. Pharm. 291/63, 391 (1958). (18) Smith, D. M., Campbell, R. G., Chemist-dnalyst 50, 80 (i961). (19) Taylor, E. C., Strojny, E. J., J. Am. Chem. SOC.82, 5198 (1960). (20) Todd, A. R., Ezperientia 2 , 55 (1946). (21) Wollner, H. J., Matchett, J. R., Levine, J., Loewe S., J . Am. Chem. SOC.64, 26 (1942). (22) Wollner, H. J., Matchett, J. R., Levine, J., Valaer, P., J . Am. Pharm. Assoc. 27, 29 (1938).
(23) Young, J. R., “Tests for Marihuana,’’ Bureau of Internal Revenue, Alcohol Tax Unit, May 1, 1951. Cited in ref. I, p. 564. RECEIVED for review November 15, 1962. Accepted February 25, 1963. Division of Analytical Chemistry, 141st Meeting, ACS, Washingtpn, D. C., March 1962. Work reported in this paper was undertaken as part of the International Scientific Research on Cannabis, pursuant to Resolution 8 (XIV) of the Commission on Narcotic Drugs of the United Nations.
Microscale Identification of Several Sugar Phosphates by Paper Chromatography a nd Electrophoresis R O M A N O PIRAS ancl ENRICO CABIB lnstituto de lnvestigaciones Bioqurinicas “Fvndacibn Campomar” and Faculfad de Ciencias Exacfas y Naturales, Obligado 2490, Buen,x Aires, Argentina
b Severat sugar phosphates have been converted to cyclic phosphates or methyl esters by treatment with dicyclohexylcarbodiimide in methanolic solution. The producis obtained were then separated by paper chromatography with solvents containing cetyltrimethylammonium bromide or by paper electrophoresis in potassium or cetyltrimethylammoriium borate buff ers. The separations obtained were better than those previously described for the untreated esters and the complete procedure could be carried out with less than 0.5 pmole of substance. The compounds obioined in the dicyclohexylcarbodiimide reaction were studied in detail and general rules have been formulated for the prediction of the type of product to be expected in each case. Some correlations between chemical structure and chromatographic or electrophoretic behavior have been observed.
-
A
NUMBER O F PAFERS (11, 18, 28, 29, 33, 34) dealing with the appli-
cation of paper chromatography and electrophoresis to the separation of sugar phosphates and other phosphoric acid esters have appcsared. However, the resolution of diffeIent hexose phosphates achieved by the methods so far described is rather poor and is completely successful only in certain cases. Good results have bem obtained with exchange resin column chromatography (17), but this technique is somewhat cumbersome and is not easily adapted to microscale use. 11, is the purpose of this communication to describe an efficient method for the identification of several sugar monophosphates by
paper chromatography and electrophoresis. Rees has recently shown (27) that it is possible to increase the R, of sugar sulfates by adding a long-chain tetralkylammonium salt to the solvent. I n this way, solvents that are very selective for sugars can also be used for the separation of their sulfates. Attempts t o extend this method to sugar phosphates met with very limited success, apparently because sugar phosphates, unlike the sulfates, possess two dissociable anionic groups. To obtain compounds with a single acid group, phosphate esters were treated with dicyclohexylcarbodiimide (DCC) in methanolic solution, whereby the corresponding cyclic phosphates or methyl esters were formed (15, 16). The products could then be separated by paper chromatography with solvents containing cetyltrimethylammonium (CTA) bromide or by paper electrophoresis with potassium or CTA borate (25)* I n an attempt to ascertain the general applicability of the method, the structure of the derivatives obtained in the DCC reaction was studied in some detail, based on the findings of Khorana and his coworkers (16). EXPERIMENTAL
Materials. All sugar esters used belong t o the D-series. The l-phosphates of glucose, galactose, xylose, and mannose are t h e a-anomers. T h e phosphate group is abbreviated P , as in glucose 1-P. M o s t sugar phosphates used were commercial samples. A sample of galactose 1-P was prepared as described by Hansen, Rutter, and Krichevsky ( l a ) ,
and one of xylose 1-P was obtained according to Meagher and Hassid (23). The purity of the esters was investigated by submitting the corresponding sugars to paper electrophoresis after hydrolysis with dialyzed alkaline phosphatase (Armour Research Division, Chicago, Ill.). The phosphatase preparation was free from phosphohexoisomerases, as shown by the fact that crystalline glucoje 6-P gave rise to glucose only. Both fructose 6-P and mannose 6-P samples liberated some glucose, indicating a contamination, probably with glucose 6-P. I n addition, both mannose 6-P and galactose 6-P samples gave a fasterrunning, reducing spot in solvent A (see below). They were therefore purified by chromatography with this solvent on Whatman KO.3 paper, in the manner already described ( 6 ) . Fructose 6-P was purified in the same fashion, but using solvent F (see below). Chromotropic acid (Eaqtman Organic Chemicals, Rochester, N. Y.) was purified by fractional precipitation with ethyl ether from an aqueous ethanolic solution and was stored under reduced pressure. Dowex 50 (H+) resin of 20- to 50mesh and 8% cross-linkage was used. General Procedure for t h e Treatment of Sugar Phosphates with DCC. The technique adopted was similar to that described by Khorana et al. (16). To prevent the formation of ,V-phosphoryl ureas the reaction was carried out with the addition of triethylamine and in the absence of water. Five micromoles of the ester (sodium salt) in 0.4 ml. of water were passed three times through a Dowex 50 (H+) resin column 5 em. long and 0.4 em. in diameter. The columns were flushed with 0.4 ml. of water. a 13- X 100-mm. test tube being used to collect the effluents. After adding 0.2 ml. of pyridine, the tube contents were brought to dryVOL. 35, NO. 6, M A Y 1963
* 755
ness under reduced pressure in a rotatory evaporator at 30" C. One milliliter of anhydrous pyridine was added and the tube contents were again brought to dryness. This operation was repeated with two successive 1-ml. additions of anhydrous pyridine. Then 1 ml. of anhydrous methyl alcohol was added, followed by 0.004 ml. of triethylamine and 20 mg. of DCC, and the tube was tightly stoppered. After 16 hours a t 37" C., the reaction mixture was concentrated t o dryness, 0.3 ml. of water was added, and three extractions, each with 1 ml. of ether, were carried out. The solution of the DCC product obtained was kept at -10" C. and aliquots were used directly for paper chromatography or electrophoresis. When very small amounts of sugar phosphate were used (0.3 to 1.0 pmole) all the volumes were reduced. The DCC/triethylamine/phosphoric ester ratio was alxays kept constant. Chromatography. Whatman paper S o . 1, 3 or 4 hIM, was washed as described previously (6). Chromatograms were developed by the descending method. The solvents used were as follows: A, n-butanol-pyridine-water (6:4: 3) ( 1 4 ) ; B, solvent A containing 2.5y0 w./v. of CTA bromide; C, solvent -4 saturated with potassium borate; D , solvent A containing 2.5% w:/v. of CTA bromide and saturated with potassium borate; E, isopropyl alcoholconcentrated ammonia-water (7 : 1:2 ) (3); and F, ethyl alcohol-l-VI ammonium acetate at pH 7.5 ( 7 : 5 : 3 )(24). Solvents C and D were prepared as A and B, respectively, but using a saturated solution of potassium borate instead of water; after seeding with crystals of potassium borate, the mixtures were shaken during a few minutes in an ice bath. The excess salt crystallized out, and the clear supernatant solutions were decanted. The paper to be used with these solvents &-as dipped in 0.01N potassium borate and dried at room temperature. The running time for solvents A , B , C, and D was 32, 24, 42, and 24 hours, respectively, a t room temperature (about 20" C.). To counteract the tendency of several compounds to give elongated spots with solvents A. B, C, and D, the paper m s cut in the manner described by Matthias (22). Electrophoresis. Strips of washed Whatman KO.1 paper, 50 em. long, were used in the apparatus described by Markham and Smith (Wl), with toluene as the refrigerant. The buffers and the technique were as already reported (2!). The paper was treated with collodion only when using CTA electrolytes. The conditions of the runs were as follows: ammonium acetate, 14 v. per cm. during 2.5 hours, potassium borate, 20 v. per em. during 2 hours: CTA borate, 20 v. per em. during 5 hours; and CTA carbonate, 20 v. per em. during 4 hours. I n quantitative experiments, duplicate samples of the compounds were run on parallel strips. One strip was 756
ANALYTICAL CHEMISTRY
treated with compound-locating reagent and the area of the other strip corresponding t o the spot on the treated strip was cut out and eluted with water. Detection of Spots. The l-phosphates and their derivatives were revealed according t o Burrows, Grylls, and Harrison ( 5 ) , while the 5- or 6phosphates and derivatives were detected with a silver nitrate reagent (SI). In the latter case, the development of color was inhibited by high concentrations of CTA bromide. At the 2.5% concentration used, good re-
fl-
Ru b ber bu I b
later. The pellet was resuspended in water and dissolved by stepwise addition of Dowex 50 (H+) resin. The liquid was then passed through a column of the same resin and the sample was treated with DCC as described above in General Procedure. In another experiment only 0.5 pmole of glucose 1-P was used and the amounts of most of the other reagents were halved. Formation of S-Phosphoryl Ureas. An aliquot (0.4 pinole) of the derivative obtained with DCC in the General Procedure was passed through a Dowex 50 (H+) resin column, 3 em. long and 0.4 cm. in diameter. The effluent was neutralized with pyridine, concentrated t o dryness, and 0.02 ml. of pyridinewater (4:1), containing 52 mg. per ml. of DCC, was added. The tube was qealed and kept for 48 hours a t room temperature, after which the contents were transferred to a strip of LThatman paper S o . 4, and wbmitted to chromatography n ith solvent E . Investigation of Methyl Esters. Since it has been shonn (16) that amannose 1-P cannot give a cyclic phosphate in the DCC reaction, the formation of a methyl ester was indicated. Therefore the DCC product of 0-mannose 1-P was chosen as model compound for the investigation of methyl eqters. The hydrolysis of the compound was carried out in two steps. In the first, the substance was heated a t 100" C. in 1iV HC1 for 1 hour By analogy nith the behavior of mannose 1-P, it was presumed that the bond between the phosphate group and the sugar would be split by this treatment. In fact, paper chromatograms of the hydrolysis products in qolvent E and acid in tert-butylalcohol-water-picric (80:20:4 gram.) (11'1 shoned, when sprayed with a molybdate reagent ( 5 ) , only t n o spots-a main spot corresponding to methyl phosphate (32) and a very faint one corresponding to inorganic phosphate ( P t ) . Since the rate of hvdrolvsis of methvl phosphate is maximal i t pH 4 (4), the second step m s carried out a t this pH. The liberated methyl alcohol &as then distilled and assayed with an adaptation of Boos's technirrue (2)that is, by Oxidation with potassium permanganate in acid solution, followed by a spectrophotoinet'ric determination of formaldehyde with chromotropic acid. The amount of P, liberated in the second step of hydrolysig was equivalent to that of methanol, and mi: about 75qi', of the tot,al, as expected according to the data of Bunton et al. (4). The general method finally adopted is now described. It was first necessary to eliminate the traces of met'hyl alcohol carried over from the DCC reaction mixture. For this purpose the solution of the DCC product was streaked across a strip of Whatman S o . 1 filter paper and, after drying, the paper was washed several times with ethyl ether by capillary ascent. The substance was then eluted with water, passed through Don-ex 50 ( H i ) resin: and neutralized.
i-k tubei I/ I Rubber stopper
Test 13 x 100 mm.
4.0-ml. mark
1.5-ml. mark l.&m I. mark
! ---
Water
Air bubble NaHS03
sol.
ample
+
xidant Figure 1. Assembly for oxidation of small quantities of methanol to formaldehyde s d t s R ere obtained by adhering strictly to the directions given previously for trehalose (7). Phosphate Determinations. Samples were analyzed for phosphate by the Fiske and SubbaRow procedure (9)'
Determination of Esters in Mixtures with Cellular Extracts. Dried brewers' yeast (0.5 gram) was autolyzed with 2 ml. of water for 1 hour at 37" C. After centrifuging for 10 minutes a t 10,000 X G, the precipitate was discarded. T o 0.2 ml. of the wpernatant liquid, 5 wmoles of galactose 1-P and 5 pmoles of glucose I-P nere added, and the volume was made up to 0.5 ml. with water. Then 0.5 nil. of 10% trichloroacetic acid wap added and the tube was centrifuged at 25,000 X G for 10 minutes. The precipitate mas washed twice with 0.25 ml. of 5% trichloroacetic acid, and the conibinerl supernat? and ashings were extracted four times with ether. Then 0.2 ml. of 0.51M glycine a t pH 10 was added, followed by 0.1 ml. of 1M barium acetate. The mixture nas kept in the cold until the precipitate began to settle, and then centrifuged for 5 minutes a t 1,700 X G. The precipitate was washed twice with 0.15 ml. of 0.01Jf glycine a t pH 10. To the combined supernate and washings 8 ml. of cold ethanol were added and the precipitate that formed was centrifuged 1 hour
I
SOLVENT B SOLVENT C DaaDer Wh.NO1 Paper Wh.NO1
SOLVENT 0 'aper Wh. N O 4
borate
I
CTA borate T a q i Z p - treated esters es ers esters
10
4
treated
I
-GI*
:o Figure 2. R.vG values of esters and their DCC products in different solvents
11
Figure 3. Mobilities of esters and their DCC products in different electrolytes
Abbreviations: G1, glucore 1 -P: G6, glucose 6-P; Gal, galactose 1 -P; Ga6 galactose 6-P; M1, mannose 1 -P; M6, mannose 6-P; F1, fructose 1 -P; F6, fructose 6-P; R5, ribose 5-P; X1, xylose 1 -P. DCC products indicated same way, with asterisk. Two spots given b y DCC products of fructose 1 -P: F1 *I, F1 *II
Between 1.0 and 2 5 fimoles of each DCC product were heated at 100" C. in a sealed tube in 0 3 ml. of 1N HCl for 1 hour. After adjusting t o p H 4 with 10N NaOH, the volume was brought up to 1 ml. with 0.07.W potassium acid phthalate. The tube was sealed again and th2n heated in an autoclave at 1.05 a t n . (122" C.) for 4 hours. A similar amount of the same DCC product, submitted to the same treatment except for no heating, was used as a blank in each case. Each sample nas transferred quantitatively to a 50-1111. round-bottom flask, water was added up to a total volume of 3 ml., and the solution was distilled with a mirrodistillation assembly, the first milliliter of distillate being collected in a ter.t tube. To each tube 0.1 ml. of 3y0 potassium permanganate solution containing 7.5% phosphoric acid mas added, and the tube was immediate1,rr stoppered with the assembly shown in Figure 1 to avoid losses of formaldehyde. The pipet (2-mm. i. d.) contained a freshly prepared saturated solution of sodium bisulfite, in slight e x c w of the amount neceqcary to reduce t i e permanganate, and an equal volume of water (about 0.06 ml. of each). After the tubes were shaken horizontally for 30 minutes at 37" C., the excess permanganate n a s destroyed by expel1 ng the bisulfite contained in the pipet The tubes n-ere then immersed for 5 minutes in ice water, the water contihed in the pipet Bas expelled, and 0.2 ml. of 2% chromotropic acid was added, followed by n t e r up t o the 1.5-ml. mark. Concentrated sulfuric acid \vas then added up to the 4.0-ml. mark. The tubes nere shaken, then heated for 5 minutes a t 100" C. and the absorbance was read in the Coleman Junior spectrophotometer a t 580 mp. Standards were run with amounts
of methyl alcohol ranging from 0.2 t o 1.0 pmole. The same standard curve was obtained whether the methyl alcohol was determined directly or after distillation. RESULTS
Optimal Conditions for t h e DCC Reaction. The time course of t h e reaction was followed by submitting aliquots of the reaction mixture t o paper electrophoresis with ammonium acetate. The reaction products always showed a lower mobility than the original phosphates. At room temperature, or 30" C., the reaction was not complete after 60 hours. At 37" C., after 16 hours, the original sugar phosphate spot had disappeared in all cases. At 67" C., with galactose 6-P or fructose 1-P,the reaction wis almost total after 30 minutes and complete after 1 hour. The temperature finally chosen, 37" C., affords a reasonable reaction time, under mild conditions, suitable for unstable compounds. Chromatography. The DCC products showed higher R f ' s than the original rqters, and the addition of CTA bromide t o the solvent further increased the mobilities, as qhonn in Figure 2 . The impregnation of the paper with boric acid or its salts, a method m-hich has given good results in similar cases (8, I S ) , was studird next. When CTA borate a t p H 9.4 as used, all the substances moved with the solvent front. With boric acid and potassium borate a t different p H values, some separations were improved. The best results were obtained by impregnating the paper with 0.01.1.1 potassium borate. The
solvent was saturated with the same salt to prevent double fronting. The results, given in Figure 2, are expressed in the form R,,~Q(distance traveled by ester)/(distance traveled by 8-methylglucoside). 8-Alethylglucoside, because of its inability to form a complex with borate ( I O ) , gives practically the same R, in the four solvents shown in the figure (0.47, 0.50, 0.45, and 0.50 in solvents A , B, C, and D, respectively). The RWGof the esters and their derivatives may thus be compared directly. With solvents C and D, the untreated sugar phosphates did not niove from the starting line. With solvent A, several compounds showed tailing and sometimes gave multiple spots, which appear to be artifacts. With all the other solvents a single spot was obtained for each compound tested, with t n o exceptions: the DCC product of fructose 1-P gave rise in all cases to two spots of about the same intensity, and that of galactose 6-P showed a second component in solvent E . In one experiment, P , was treated with DCC in the same inanner as the esters. Upon chromatography of the reaction product a single SIJOt n a s found, with a n R j similar to that of dimethyl phosphate. Electrophoresis. Thc mobilities of the original sugar pho-pliatrs and the DCC product> are shown in Figure 3. T h r value- are. a? in the C ~ S P of p a p w chromatography, the mean of a t leait fire determinations and the intiiridual deviation5 from the mean were rrithin =t87G. It is clear that only with borate buffers can the DCC products or original esters be separated. Here, too, the DCC product from fructose 1-P gave 2 spots, while that from galactose 6-P gave a minor component with potassium borate only. This spot, not shown in Figure 3, VOL. 35, NO. 6,
MAY 1 9 6 3
e
757
Table 1.
Quantitative Determination of Sugar Phosphates
Step After precipitation from yeast extract
Amount Substances added, treated pmoles Glucose 1-P+ Galactose 1-P 5 5 Glucose 1-P 0.44 Glucose 1-P+ Glucose 6-P+ ... Mannose 6-P Mannose 6-P ... a Of the mixture.
+
Amount recovered, pmoles
10" (100%) 0.435 (99a/o)
ANALYTICAL CHEMISTRY
9.6" 0.395
+ 5.9 + 4.5
...
4.6
...
20.8
moved about 29 em. from the starting line. Separations. By taking advantage of their differing electrophoretic and chromatographic mobilities, i t is possible to separate any pair of the D C C products, and thus identify any of the compounds tested thus far. It is possible to extend the present work by using other solvents previously employed in paper chromatography of sugars. Preliminary experiments with n-butanol-ethanol-water (5: 1:4),ethyl acetate-acetic acid-water (3 :1:I), and (4:1: 5 ) n-butanol-acetic acid-water (19), all of them containing 2.5% CTA bromide, gave very promising results. The last two solvents interfered with the silver nitrate spot locating reagent, and a benzidine reagent (1) had to be used instead. The possibility of the formation of secondary products, resulting from the condensation of different esters between themselves, was investigated by submitting a mixture of glucose 1-P, glucose f3-P and mannose 1-P to the DCC treatment. After paper chromatography and electrophoresis, only those spots were found Khich are given by these substances when tested individually. Mixtures of Phosphoric Esters with Natural Extracts. To study possible interference by substances contained in cellular extracts, some sugar phosphates were added t o a yeast autolyzate, later recovered as the barium salts and then treated with DCC (see Experimental). T h e results were similar t o those obtained with pure solutions of the phosphoric esters. Prior elimination of Pi avoids the formation of dimethyl phosphate in the DCC reaction. Quantitative Applications. The recovery of the phosphorylated compound, based on total phosphate determinations, was studied after each of the following steps: precipitation as barium salts; DCC treat-
758
After DCC Amount Amount added, recovered, pmoles pmoles
Amount added, pmoles
After electrophoresis with K2B40,and elution hmount recovered, pmoles
__
9.15 (95%) 0.37 (94%)
0.29'
0.137
+ 0.141 (96%)
14.4'(96%)
0.26"
0.07s
+ 0.096 + 0.073 (95%)
19.7 (95%)
0.16
0.154 (9670)
ment; and paper electrophoresis of the products, followed by elution. As indicated in Table I, after each step a portion of the product obtained was taken for the next one. The results of Table I show that the over-all recovery was about 90%. Determination of Structure of the DCC Products. Taking into account the composition of the reaction mixture, it was expected (16, 16) t h a t the reaction products should be cyclic phosphates and/or methyl esters of the sugar phosphates. The cyclic compounds would be formed as the result of internal esterification between the phosphate group and a conveniently placed hydroxyl of the sugar moiety. The type of ring formed (five-membered, six-membered, etc.) can be determined on the basis of certain typical properties (16). The compounds with five-membered rings give the corresponding N-phosphoryl ureas by treatment with DCC in aqueous pyridine (16). In addition, the five-membered rings are quantitatively opened by hydrolysis with 0.1N HC1 during 4 hours a t 30" C. (20). The six-membered rings remain unaltered under both conditions. The formation of N-phosphoryl ureas can be detected by submitting the reaction products to paper chromatography with solvent E , where these compounds give spots with R, ~ 0 . 9 . A positive test was given by the DCC products of fructose 1-P, glucose 1-P, galactose 1-P,and xylose 1-P. The product from galactose 6-P gave a faint N-phosphoryl urea spot. These results were confirmed by acid treatment of the DCC products. I n this case, each five-membered ring can give rise to two different compounds, depending on which of the two linkages of the phosphate group with the sugar is broken. Accordingly, when the DCC products of glucose 1-P, galactose 1-P and xylose 1-P were treated with acid, lyophilized, and then submitted to paper electrophoresis with borate buffers,
two spots corresponding to the 1- and 2-phosphates were found in each case. A spot of P,, originated from further hydrolysis of the 1-ester also appeared (see Figure 4). From these results, we conclude that these DCC products are 1,2-cyclic phosphates. With the derivatives from fructose I-P, only Pi and a fructose 1-P spot were found. The two DCC products of fructose 1-P have been studied (86) and shown t o be the 1,2-cyclic phosphates of the pyranose and of the furanose form. It has also been shown (26) that the fructose 2-phosphates obtained are extremely labile in acid, which would explain why only P, and fructose 1-P appear in the electrophoresis shown in Figure 4. The minor component of the galactose 6-P DCC product also disappeared after the acid treatment, giving rise to a spot of galactose 6-P and another one, which probably corresponds to galactose 5-P (see Discussion). The presence of methyl esters in the DCC products was then investigated. Those which had already been identified as five-membered cyclic phosphates gave a negative test, while with all the others the test was positive, as shown in Table 11. The DCC product of mannose 1-P gave the highest amount of liberated methanol. Since the other compounds are derivatives of sugar 5- or 6-phosphates, this result may be ascribed to their greater resistance to acid hydrolysis. It was, however, conceivable that these compounds were not methyl esters, but that methanol mas formed as a decomposition product during the acid treatment. To discard this possibility, the 5- and &phosphates were treated with DCC, using as solvent aqueous pyridine rather than methanol. The reaction mixture was as described by Khorana et al. (16) plus 0.016 ml. of triethylamine per ml. of aqueous pyridine, and the reaction time was 72 hours. In this case the products gave
CH20H
Electrophoresis with potassium borate
CH2OH
Standards G I * Hydrol.
0
0
rOOO+- 0 0
0
FI* Hydrol. X I* Hydrol. Standards
FIT Xl*FI*Il
X I F1
Pi
Electrophoresis with CTA borate Gal Gal* Pi Standards
Figure 4. Electropihoretic submitted to acid hydrolysis
pattern
of DCC products
different mobilities from those formed in methanol and must be cyclic phosphates. mThen submitted to the determination of methyl esters they gave a negative test (see Table 11).
' OH cl-DMannose -1 met hy I- P I
DISCUSSION
The 10 sugar monophosphates studied in the present investigation can all be identified by using one or the other of the chromatographic solvents or electrophoretic conditions dmribed. I n particular, any DCC product from a 1-phosphate can be separated from that of the corresponding &phosphate by paper chromatography with solvent B. This represents a definite improvement on the previously described methods that were applied to the unmodified phosphoric esters. A bidimensional technique cannot, howver, be used in this case, because it s not easy to remove the CTA or potassium salts from the paper after the first run. Good results were obtained by first submitting the sample to electrophoresis with CTA or potassium borate, followed by elution of the spot and eliminzkion of the borate (56). The substance could now be chromatographed on paper with solvent B, for instance, or again be submitted to electrophoresis undtlr different conditions. As little as 0.3 pmole of ester was carried through the whole procedure, including the DCC treatment. The DCC products were studied in detail, to gain some insight into the general applicability O F the method and to correlate separations with chemical structure. The conclusions obtained are illustrated in Flgure 5. When aqueous pyridine is used as solvent in the DCC treatment, cyclic phosphates are formed when a con\.eniently oriented hydroxyl group is available (16). This hydroxyl may or may not be in a position adjacent to the phosphate (cases a and b). With methyl alcohol as
d-O.Mannose.1-P
Figure 5. Reaction of sugar phosphates with DCC under different conditions
solvent, however, only five-membered rings are formed; that is, only a neighboring hydroxyl of the sugar can react with the phosphate group (case a). When no neighboring hydroxyl is available for reaction (5- or &phosphates, case b ) or when it is in an unfavorable orientation (a-mannose 1-P, case c), only methyl esters are obtained. Galactose 6-P gave, in addition to the methyl ester, a minor product which behaved like a five-membered cyclic phosphate. Since the galactose derivatives show some tendency to exist in the furanose form (SO), the &hydroxyl might become partially available for reaction in this case, thus giving rise to some galactose 5,6-cyclic phosphate. I n all cases, the elimination of one of the dissociated groups of the sugar phosphate brings about an increase in Rj values which is further increased by the addition of CTA salts to the solvent. The CTA ion probably combines with the remaining charge on the phosphate group, to form an undissociated molecule which behaves not unlike a neutral sugar. I n fact, the Rj values of the 5or 6-phosphate products (methyl esters) in solvent A or B increase in the same order as do those of the corresponding sugars. The importance of the 1-hydroxyl in the formation of sugar-borate cornplexes (10) is reflected in the greater
mobility of the DCC products of 5or &phosphates in electrophoresis with potassium borate, as compared with that of 1-phosphate derivatives. I n paper chromatography with solvent D, the effect is reversed, since in this case the highest borate concentration is in
Table II. Investigation of Methyl Esters in DCC Products
Methanol liberated5 pmoles/pmole of substance treated
Substance Mannose ~ ~ ~ ~ . 0,005 - . Mannose 1-P 0,oos DCC Products: mannose I-P 0.73; 0.78 mannose 6-P 0 . 6 0 ; 0.64; 0 . 4 8 glucose 1-P 0.05: 0.03 glucose 6-P 0 . 5 5 , 0.54 galactose I-P 0 . 0 4 ; 0.04 galactose 6-P 0.60; 0.56 fructose I-P 0.03 fructose 6-P 0 . 6 7 ; 0.62 ribose 5-P 0.38 xylose I-P 0 DCC Products obtained in aqueous pyridine* 0 a Different data for same substance are given when more than one experiment nas carried out. Products from glucose 6-P, fructose 6-P, mannose 6-P, and galactose 6-P were analyzed, with same results in all cases. ~~
VOL. 35, NO. 6 , M A Y 1963
759
the immobile phase and greater complex formation corresponds to lower R, values. The similarity of the respective behavior of glucose, xylose, and fructose 1,Zcyclic phosphates, in both solvents B and D is a reflection of their similarity in configurations and their inability to form borate complexes. Galactose 1,2-cyclic phosphate has an afinity for borate because of its two cis hydroxyls, and accordingly its mobility is considerably decreased in solvent D. The inverted order of mobility in CTA borate as compared Kith potassium borate can be ascribed to an effect discussed in a previous publication ($6). I n this case the correlation coefficient is even greater. That CTA buffers in certain cases allow better separations than do solutions of alkali salts (%), is confirmed by the greater resolution observed in electrophoresis with C T d borate and CTA carbonate, as compared to that with potassium borate and ammonium acetate, respectively. Finally, it should be noted that by carrying out the DCC reaction in aqueous pyridine, instead of in methyl alcohol, it is often possible to obtain different products (see Figure 5) whose separation may be useful in the identification of a particular compound. ACKNOWLEDGMENT
The authors express their gratitude
to C. E. Cardini, K6lida Gonzklez,
Aldo hlitta, and Eduardo Recondo for gifts of some sugar phosphate samples. LITERATURE CITED
(1) Bacon, J. S. D., Edelman, J., Biochem.
J . 48, 114 (1951). (2) Boos, R. N., AXAL.CHEM.20, 964 (1948). (3) Brown, D. M., Todd, A. R., J . Chem. Soc., 1953, 2040. (4) Bunton, C. A., Llewell n, D. R., A., Zbid.. Oldham, K. G., Vernon, 1958.3574. (5) Buirows, S.,Grylls, F. S. M., Harrison, J. s.,Nature 170, 800 (1952). (6) Cabib, E., Carminatti, H., J. Biol. Chem., 236,883 (1961). (7) Cabib, E., Leloir, L. F., Zbid., 231, 259 (1958). (8) Cohen. S. S.. McXair Scott. D. B.. Science i l l , 543 (1950). (9) Fiske, C. H., SubbaRow, Y., J . Biol. Chem. 66, 375 (1925). (10) Foster, A. B., Advances i n Carbohydrate Chem. 12, 81 (1957). (11) Hanes, C. S., Isherwood, F. A,, Kature 164, 1107 (1949). (12) Hansen, R. G., Rutter, W. J., Krichevsky, P., Biochem. Preparations 4, l(1955). (13) Harrap, F. E. G., hrature 182, 876 (1958). (14) Jeanes, A , , Wise, C. S., Dimler, R. J., ANAL.CHEM.23, 415 (1951). (15) Khorana, H. G., J . 4 m . Chem. SOC. 81, 4657 (1959). (16) Khorana, H. G., Tener, G. M., Wright, R. S., Moffatt, J. G., Zbid., 79, 430 (1957). (17) Khym, J. X., Cohn, W. E., Zbid., 75, 1153 (1953). (18) Lederer, E., Lederer, M., “Chromatography,” 2nd ed., p. 226, Elsevier, Amsterdam, 1957. (19) Lederer, E., Lederer, M., Ibid., p. 245. (20) Xarkham, R., “Methods in Enzy-
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(26) Pontis, H. G., Fischer, C., Instituto de Investigaciones Bioquimicas, Fundaci6n Campomar, Buenos Aires, Argentina, private communication, 1962. (27) Rees, D. A., Nature 185, 309 (1960). (28) Runeckles, V. C., Krotkov, G., Arch. Bzochem. Baophys. 70, 442 (1957). (29) Schwimmer, S., Bevenue, A., Weston, W.J., Ibzd., 60, 279 (1956). (30) Sowden, J. C., “The Carbohydrates,” W. Pigman, ed., p. 88, Academic Press, Sew York, 1957. (31) Trevelyan, W. E., Procter, D . P., Harrison, J. S., Suture 166, 444 (1953). (32) Ukita, T., Nagasawa, K., Irie, RI., J . Am. Chem. SOC.80, 1373 (1958). . (33) Wawszkiewicz, E. J., A N ~ LCHEV. 33, 252 (1961). (34) Wood, T., J . Chromatog. 6, 142 (1961). (35) Zill, L. P., Khym, J. X., Cheniae, AI., J . Am. Chem. SOC.75, 1339 (1953). RECEIVEDfor review August 6, 1962. Accepted January 7 , 1963. Taken from a thesis to be submitted by Romano Piras t o the University of Buenos Aires in fulfillment of the requirements for the degree of Doctor in Biochemistry. One of us (R. P.) is a fellow of the Consejo Sacional de Investigaciones CientiGcas y TBcnicas. This investigation was supported in part by a research grant (No. (2-3442) from the National Institutes of Health, U. S. Public Health Service, and by the Rockefeller Foundation.
Determination of Free and Esterified Cholesterol by a Modified Digitonin-Anthrone Method JOSEPH R. GOODMAN, LELAND P. JARNAGIN, RITA M. MEIER,’
and IRMA A. SHONlEYl
Veterans Administration Hospital, San Francisco, Calif.
b The digitonin-anthrone indirect determination of cholesterol was revised to produce more reliable data and to provide separate, final aliquots on all fractions that were suitable for C14 counting. Modification was made in the method as reported by Vahouny, et a/. Minor changes were made in the procedure for free and total cholesterol. Ester cholesterol was determined as an original value, thus providing primary values of both free and ester cholesterol, the sum of which was equal to the total cholesterol value. This internal check in the method strengthens the reliability of the data. Final aliquots were obtained on all fractions for C14 counting in a liquid scintillation counter. Primary values on 760
ANALYTICAL CHEMISTRY
all fractions for both concentration and activity increased the reliability of specific activity calculations. The procedure gave data that was in close agreement with the Sperry-Webb method on a group of sera. It was further checked by determining recovery of added pure cholesterol.
T
indirect determination of cholesterol by Feichtmeir and Bergerman ( 1 ) has recently been revised by Vahouny, et al. (3, 4). Their refinements have materially improved the quantitative character of the method. The rapid precipitation and recrystallization steps have decreased the time required for HE DIGITOKIN-ASTHRONE
precipitation. The stability of the color produced by the digitonin-anthrone reaction and the sensitivity of the method are iti chief advantages. These factors and an increased reliability nould make it attractive as a method in research. It nould also he more useful in procedures involving CI4 cholesterol if the ester fraction were isolated for both analysis and counting. Since cholesterol levels in animals are usually loner than in man, and the volume of blood available for analysis is usually smaller, such a method would be of interest in animal research. 1 Present address, Veterans Administration Hospital, Long Beach, Calif.
