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Study of the reaction of formaldehyde with vitamin B12
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
76, 180-187 (1958)
Study of the Reaction of Formaldehyde with Vitamin Blz Pran Vohra, Fayne Lantz and F. H. Kratzer From the Department of Poultry Husbandry, of California, Da&, California
University
Received November 7, 1957
INTRODUCTION A single-carbon compound at the oxidation level of formaldehyde is believed to arise from the a-carbon atom of glycine and further reacts with the a-carbon of glycine to yield serine (1). No free formaldehyde has been obtained yet from the incubation mixture of glycine and whole or fractionated liver or kidney homogenates (2-4). The one-carbon moiety arising from the a-carbon of glycine does not have to be free formaldehyde, however; it can be some “active” formaldehyde derivative. The important role of hydroxymethyltetrahydrofolic acid (CH20HTHFA) in the transformation of glycine to serine supports this hypothesis (5-10). CHzOH-THFA has been prepared non-enzymically, by the action of formaldehyde on tetrahydrofolic acid (11-13). The hydroxymethyl group of this compound is an active precursor of the p-carbon of serine (12, 13). Vitamin Blz is also involved in the biosynthesis of serine from glycine, but the role is less clear (1, 14-17). It has been suggested that it may be involved in the conversion of the o-carbon of glycine into a single carbon unit which can be used both for the synthesis of methyl groups (18, 19) and for the conversion to CHZOH-THFA (18). The structure of vitamin Blz , which was established recently, indicates that the molecule has six amido groups (20). The amido groups can react with formaldehyde to yield hydroxymethyl derivatives, but the possibility of more complex reactions always exists. In view of the importance of CH20H-THFA in the biosynthesis of serine, it would be of interest to investigate the possibility of the synthesis of an analogous hydroxymethyl-vitamin B1z derivative and to study its chemical and biological properties.
VITAMIN
BITFORMALDEHYDE
COMPLEXES
181
In the present study, the reaction of formaldehyde with vitamin B12 has been investigated. The amount of formaldehyde-Cl4 that combines with vitamin B12 was determined by comparing the radioactivity of the vitamin Blz-formaldehyde complex with the radioactivity of the dimedon derivative of the formaldehyde-U4 used in the reaction. EXPERIMENTAL
Preparation of the Vitamin Blz-Formaldehyde
Complexes
Preparation 1. A solution containing 32 mg. crystalline vitamin B& and 110 mg. HWHO in 10 ml. water was allowed to stand for 3 hr. Then nitrogen was bubbled through the solution at room temperature for about 7 hr., and all the HWHO escaping with nitrogen was collected in a 10% alcoholic solution of dimedon. The formaldehyde-dimedon derivative was crystallized from dilute alcohol four times and was designated DI . The vitamin Blz-HCY*HO solution had to be freed of the excess of uncombined formaldehyde. This was done by adding a water-saturated solution of dimedon until no further precipitation occurred. The mixture was kept in the refrigerator for 3 hr., and the precipitate was separated from the deep-red mother liquor containing the vitamin BlrHCFHO. The precipitate was washed with water, dissolved in hot 95% alcohol, decolorized with charcoal, and allowed to crystallize out on cooling. This formaldehyde-dimedon derivative was recrystallized three more times from dilute alcohol and was called Dz . The mother liquor containing the vitamin B12-HCr4H0 was treated with acetone until no further precipitation occurred in the cold. The precipitate was removed and discarded. This freed the mother liquor of any residual dimedon. The red mother liquor, which was about 100 ml. in volume, was freeze-dried to a fluffy purple powder weighing 35 mg. The purple powder was crystallized three times from a solution of 10% water and 90% acetone. The final product (PI) was in the form of lustrous purple needles and weighed 27 mg. Preparation b. A solution containing 10.5 mg. crystalline vitamin Blz and 157 mg. HC”HO in 10 ml. water was left at room temperature for 3 hr. A part of the excess formaldehyde was removed with nitrogen for 5 hr. and collected as the dimedon derivative. The formaldehyde-dimedon derivative was crystallized three times with dilute alcohol and was termed Da . The vitamin Blz-HWHO solution was freeze-dried. During this process, the frozen mass melted a few times, but eventually dried into flakes. These were extracted with water, leaving a faintly colored residue. The water extract was freezedried, redissolved, and freeze-dried again, yielding a powdery product. This was dissolved in a minimum amount of water, and acetone was added until a turbidity appeared. This mixture was cooled overnight, causing purple needles to separate from it. These were centrifuged out and recrystallized three times from 90% acetone, yielding Prep. 2 (Pz).
1 Kindly
supplied by Merck and Company, Rahway, New Jersey.
182
VOHRA,
LANTZ
AND
KRATZER
Preparation S. Forty milligrams vitamin Brz and 699 mg. HCY4H0 were reacted together for 3 hr. in 10 ml. water at room temperature. Nitrogen was then bubbled through the reaction mixture for 18 hr., and the escaping formaldehyde was trapped in an alcoholic solution of dimedon. The formaldehyde-dimedon derivative was collected and designated Db . The addition of water to the first mother liquor yielded another crop of the derivative (DS). The vitamin BrZ-HC14H0 reaction mixture was freeze-dried, and the flakes obtained from this process were dissolved in water. The resulting solution was evaporated in a colored container under very low heat from an infrared lamp. A water aspirator was used in this operation. Nitrogen was bled through the system, and the formaldehyde escaping in the vapors was trapped as the dimedon derivative. The purplish product remaining after drying was dissolved in water and precipitated with acetone. The precipitated needles, which weighed 20.7 mg., were recrystallized three times from 99% acetone containing some cyanide. A sample of the crystalline product was taken and crystallized once more to yield Pa . The rest of the product was crystallized four more times from 90% acetone (P4). The mother liquors of the preparations up to Pa were collected and dried by evaporation under nitrogen to yield a crude product (P6). Preparation 4. The procedure for Prep. 4 was essentially the same as described under Prep. 3. The gases flushing out of the dimedon trap were bubbled through 20 ml. of 0.5 N ceric sulfate in 1 N HzS04 and then 1 ml. of 0.05yo NaOH. The NaOH trap and the condensate obtained during the lyophilization of the vitamin BlZ-HCr4H0 reaction mixture were analyzed for the presence of cyanide. After five recrystallizations from 90% acetone, 36.4 mg. of Prep. 4 (P,) was obtained. A portion of it was recrystallized two more times and designated as P7 . The HWHO-dimedon derivative was recrystallized repeatedly, and samples were collected at the 3rd (De) and 4th recrystallization (DT). Drying of Samples. The samples were dried over P205 at 78°C. under reduced pressure. Microbiological Assay. Vitamin Blz and Preps. PI and Pd were compared for microbiological activity with Lactobacillus leichmannii # 4797 using the Bacto vitamin Brz medium B-366 obtained from the Difco Laboratories, Detroit, Michigan. Cyanide Determinations. A calorimetric method described by Robbie (21) was used for the determination of cyanide in the samples. The sample (1 ml.) was photolyzed (22) in a 12-ml. centrifuge tube cooled on the outside by running water and illuminated for 2 hr. by a spot lamp (150 w., 125 v.) using aluminum foil reflectors. Nitrogen was flushed through the sample during illumination and 1 hr. thereafter. The escaping gases were bubbled through two traps made out of 12-ml. centrifuge tubes connected in series, each containing 1 ml. of 0.05% KOH which absorbed cyanide. The color intensities were measured at 548 w in a Beckman spectrophotometer, model DU. Radioactivity Counting Procedures. To determine specific activity per mole, the samples were subjected to wet combustion (23). The COz was precipitated as on filter-paper pads, and counted in a windowless gas-flow BaCOI , collected counter. Proper corrections were made for self-absorption.
VITAMIN
Blz-FORMALDEHYDE
COMPLEXES
183
In other cases, the samples were pipetted into polyethylene planchets and evaporated. If the samples were colored, they were freeze-dried; otherwise, they were evaporated under an infrared lamp and a fan. The samples, which were elutedfrom paper strips after being submitted to electrophoresis, had some contamination with salts on the planchets. No correction for self-absorption was applied in these cases. Paper Electrophoresis. The samples, placed on paper strips 1 in. wide in buffered solutions, were subjected to electrophoretic study by use of a Spinco apparatus. A constant voltage of 500 v. was used, but the time of the run varied from 7 to 24 hr. The paper strips were dried, cut into sections, and eluted with a 0.01% KCN solution. The buffer systems used (24) without NaCl had a pH range of 4.1-11. pH 6.5 Bu$er. An amount of 4.6 ml. of 0.5 M Na2HPOt was added to 3.3 ml. of 4.0 M NaHzPOd and diluted to 1 1. RESULTS
AND
DISCUSSION
Preliminary plating of the samples PI-P* indicated that they were radioactive. In order to ascertain whether the radioactivity was due to the presence of a trace impurity of HC14H0, the sample P1 was subjected to paper chromatography using a number of solvent systems (25). On elution with water, the deeply colored red spots were found to be radioactive. The association of radioactivity with vitamin Blz spots on paper chromatograms in the sample PI strongly suggests the possibility that when vitamin Blz is treated with HVHO, some derivative may be formed. It has been termed a vitamin Blz-HCHO complex. Using Whatman filter paper No. 4 and set-butanol containing 20 % water and 0.01% KCN as developing solvent, the behavior of an inactive vitamin B,z-HCHO complex was compared with that of pure vitamin B,, . The spots ran parallel on paper and moved the same distance. It was not possible to separate a mixture of vitamin Blz and vitamin B,t-HCHO complex on paper. The microbiological activity of the dry vitamin BlrHCHO complex has been found to be 87 % and 90 % of the pure dry vitamin Blz for the samples Pr and P4 , respectively (Table I). The absorption spectrum of vitamin B12-HCHO in the visible region does not differ from that of vitamin Blz . The extinction coefficients of a 1% solution of sample PI in a l-cm. cell at 278, 361, and 550 rnp are 114, 192, and 59, and for sample P4 the values are 118, 299, and 62; while the corresponding values for vitamin Blz are 115, 204, and 63, respectively. The infrared spectrum of the vitamin BrrHCHO complex has also
184
VOHRA,
LANTZ
AND
KRATZER
TABLE I Comparison of the Microbiological Activity of Vitamin Bla and the samples PI and P4 Sample Pl
P4
ma-.
Estimated vitamin Bu activity mlrg.
0.0457 0.0914 0.1370
0.0360 0.0915 0.1130
79 100 82
87
0.0520 0.0780 0.1043
0.0450 0.0740 0.0920
87 95 88
90
Wa~ce&’
Activity %
Average activity %
been compared with that of vitamin Blz in KBr, and no difference in absorption bands has been found. There was no loss of cyanide during the preparation of B12--HCHO complexes. No cyanide was detected in the alkali or condensate traps of Prepn. 4, and recoveries of 92, 97, and 100 % of the theoretical amount of cyanide were obtained for samples PI , Pd , and vitamin Blz (Table II). The electrophoretic behavior of the vitamin B12-HCY4H0 complex (PI) has been compared with that of vitamin Blz on paper at the following pH values: 4.1, 5.0, 6.3, 7.0, 8.1, 9.0, 10.0, and 11.0. No difference in their mobilities was observed. At every pH, the maximum radioactivity has been found to be associated with the most intensely colored zone of the vitamin Blz complex. The paper strips were cut into sections, and eluted, and the eluates were counted after drying. The results of one such study at pH 6.3 are given in Table III. Information about the electrophoretic behavior of the crude preparation (P6) is included for comparison in the same table. This preparation is of interest because it contains a good proportion of unreacted HCY4H0. The data indicate that most of the activity is conTABLE II Cyanide Content of the Samples PI , PI and Vitamin B,a T$orxe;al :%eof a.
106.8 91.3 104.3
content Pg.
2.05 1.71 1.98
Recovered cyanide Pg.
2.05 1.58 1.92
RC?COWy %
100 92 97
VITAMIN
BITFORMALDEHYDE
TABLE
185
COMPLEXES
III
The Paper-Electrophoretic Behavior of Samples PI and Pg of Vitamin BIZ-HC’~HO Complexes at pH 6.3 Sample Ps
Sample Pl Polarity
Remarks
COUM.S/ min.
Anode
Colorless Faint red Origin Deep red (BH) Faint red Faint red Colorless
9 3 16 318 6 0 2
Cathode
Remarks
COWS/ min.
Colorless Faint red Colorless Origin Faint red Deep red (BH) Faint red Colorless
240 75 29 85,300 1,750 9,900 26 31
centrated at the origin. The radioactivity counts fall in the next section but rise again in the deeply colored zone containing Blz . A few other red zones, more faintly colored, were observed, but they did not contribute much toward the radioactivity. If the radioactivity found in the vitamin BIrHC14H0 complex were due only to the trace impurity of HC14H0, there would have been a continuous fall in the radioactive counts along the paper farther removed from the origin. The close association of the color and the radioactivity peak again suggests the possibility of some complex formation. The various preparations have been subjected to combustion analysis (Table IV) to determine whether the vitamin Bw-HC’~HO complex has a definite composition. This has been attempted using radioactive counts per millimole of the samples combusted as the basis for calculation purposes. The radioactive counts of the duplicate samples of the dimedon derivative of formaldehyde are in close agreement with each other. Vitamin B,, has a molecular weight of 1356. If one mole of vitamin Blz combines with either 1, 2, or 3 moles of HC4H0, the respective molecular weights of the complexes would be 1388, 1420, and 1452. On this basis, the observed counts per mole for the complexes have been calculated. The complex PI appears to have 1 mole of vitamin Blz in combination with 1 mole of HC14H0, while the complex Pz has 1 mole of vitamin Blz in combination with 3 moles of HC4H0. In order to determine whether these complexes could be crystallized to a constant activity, the samples (Pa and P4) from the 4th and 7th crystallizations of the 3rd preparation were combusted. Both of these samples
186
VOHRA,
LANTZ
AND
TABLE
KRATZER
IV
Calculation of Molar Various
Ratios of Vitamin B12 and Forn~aldehyde-C14 in Preparations of a Vitamin B,z-Formaldehyde Complex _.-. -._. --~ -I.--. -- --
Original HCWO
Vitamin Br(HC”HO)r
I
complex Cts./min./mmole
_-..(1)
D7
-
=YF
I
(2)
(3)
172
50,300
172 69
50,250 50,200 20,10020,100
229
67,ONI
225
i65,mcl
(4)
(5) -.
(6)
(7)
Observed* --
(8) .~
(9)
PI
36.9
1
50,250
51,300
1’2
42.5
2 3 4
40,200 60,300 80,400
60,400 61,800 63,100
66,300 Pa (4th cryst.) P, (7th tryst.)
25.8
0.5 1 0.5 1
33,150 66,300 33,150 66,300
35,400 35,600 31,500 31,800
162 147’W~48,500j l’s (5th 172 tryst.) I’, (7th tryst.)
73.7
1 2 1 2
48,500 97,000 48,500 97,000
103,000 105,000 110,000 112,000
22.9
79.1
o Calculated from col. 4. * Calculated from col. 6. c Rounded to whole numbers.
have radioactive counts of the same order. However, the data indicate that in this preparation 1 mole of HCY4H0 links 2 moles of vitamin B12 . Also, the radioactivities of the samples PB and P7 agree reasonably among themselves; however, they contain 1 mole of vitamin B12 in combination with 2 moles of HC”H0. The results obtained in the present study show that when HC!r4H0 is allowed to react with vitamin BIs , some complexes are formed whose structure varies in different preparations. This is not unexpected in view of the similar findings for CHZOH-THFA (12). The biological role of these complexes needs further study and is under investigation.
VITAMIN
BlrFORMALDEHYDE
COMPLEXES
187
SUMMARY
Formaldehyde-C4 reacts with vitamin BIz in vitro to form complexes that cannot be separated from vitamin Blz by crystallization, paper chromatography, or paper electrophoresis at several pH values. No differences were noted in the absorption spectra in the visible, ultraviolet, and infrared regions between vitamin BIZ reacted with formaldehyde and unreacted vitamin Blz . Four samples prepared by slightly different methods contained 0.5, 1, 2, and 3 moles of formaldehyde-C4 combined with each mole of vitamin Blz . REFERENCES 1. ARNSTEIN, H. R. V., Biochem. Sot. Symposia No. 13,92 (1955). 2. MACKENZIE, C. G., J. Biol. Chem. 186,351 (1950). 3. MACKENZIE, C. G., JOHNSTON, J. M., AND FRISELL, W. R., J. Biol. Chem. 203, 743 (1953). 4. MACKENZIE, C. G., AND ABELES, R. H., J. Biol. Chem. 222,145 (1956). 5. ELWYN, D., WEISSBACH, A., HENRY, S. S., AND SPRINSON, D. B., J. Biol. Chem. 213, 281 (1955). 6. KISLIUK, R. L., AND SAKAMI, W., J. Biol. Chem. 214, 47 (1955). 7. ALEXANDER, N., AND GREENBERG, D. M., J. Biol. Chem. 214, 821 (1955). 8. SAKAMI, W., in “A Symposium on Amino Acid Metabolism” (McElroy, W. D., and Glass, B., eds.), p. 685. The Johns Hopkins Press, Baltimore, 1955. 9. BLAKLEY, R. L., Biochem. J. 63,448 (1954). 10. BLAKLEY, R. L., Biochem. J. 66, 331 (1967). 11. JAENICKE, L., Federation Proc. 16, 281 (1956). 12. BLAKLEY, R. L., Biochim. et Biophys. Acta 23, 654 (1957). 13. KISLIUK, R. L., J. Biol. Chem. 227, 806 (1957). 14. STEKOL, J. A., WEISS, S., AND WEISS, K. W., Arch. Biochem. Biophys. 36, 5 (1952). 15. ARNSTEIN, H. R. V., AND NEUBERGER, A., Biochem. J. 60, xxxviii (1952). 16, ARNSTEIN, H. R. V., AND NEUBERGER, A., Biochem. J. 66,259,271 (1953). 17. ARNSTEIN, H. R. V., Biochem. J. 60, xii (1955). 18. VOHRA, P., LANTZ, F. H., AND KRATZER, F. H., J. Biol. Chem. 221,501 (1966). 19. CHANG, I., AND JOHNSON, B. C., Arch. Biochem. Biophys. 66, 151 (1955). 20. HODGKIN, D. C., PICKWORTH, J., ROBERTSON, J. H., TRUEBLOOD, K. N., PROSEN, R. J,, WHITE, J. G., BONNET, R., CANNON, J. R., JOHNSON, A. W., SUTHERLAND, I., TODD, A. R., AND SMITH, E. L., Nature 176,325 (1955). 21. ROBBIE, W. A., Arch. Biochem. 6, 49 (1945). 22. BOXER, G. E., AND RICHARDS, J. C., Arch. Biochem. 30, 372 (1951). 23. VAN SLYKE, D. D., AND FOLCH, J., J. Biol. Chem. 136, 509 (1940). 24. MILLER, G. L., AND GOLDER, R. H., Arch. Biochem. 29, 420 (1950). 26. KON, S. K., Biochem. Sot. Symposia No. 13, 17 (1955).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
76, 180-187 (1958)
Study of the Reaction of Formaldehyde with Vitamin Blz Pran Vohra, Fayne Lantz and F. H. Kratzer From the Department of Poultry Husbandry, of California, Da&, California
University
Received November 7, 1957
INTRODUCTION A single-carbon compound at the oxidation level of formaldehyde is believed to arise from the a-carbon atom of glycine and further reacts with the a-carbon of glycine to yield serine (1). No free formaldehyde has been obtained yet from the incubation mixture of glycine and whole or fractionated liver or kidney homogenates (2-4). The one-carbon moiety arising from the a-carbon of glycine does not have to be free formaldehyde, however; it can be some “active” formaldehyde derivative. The important role of hydroxymethyltetrahydrofolic acid (CH20HTHFA) in the transformation of glycine to serine supports this hypothesis (5-10). CHzOH-THFA has been prepared non-enzymically, by the action of formaldehyde on tetrahydrofolic acid (11-13). The hydroxymethyl group of this compound is an active precursor of the p-carbon of serine (12, 13). Vitamin Blz is also involved in the biosynthesis of serine from glycine, but the role is less clear (1, 14-17). It has been suggested that it may be involved in the conversion of the o-carbon of glycine into a single carbon unit which can be used both for the synthesis of methyl groups (18, 19) and for the conversion to CHZOH-THFA (18). The structure of vitamin Blz , which was established recently, indicates that the molecule has six amido groups (20). The amido groups can react with formaldehyde to yield hydroxymethyl derivatives, but the possibility of more complex reactions always exists. In view of the importance of CH20H-THFA in the biosynthesis of serine, it would be of interest to investigate the possibility of the synthesis of an analogous hydroxymethyl-vitamin B1z derivative and to study its chemical and biological properties.
VITAMIN
BITFORMALDEHYDE
COMPLEXES
181
In the present study, the reaction of formaldehyde with vitamin B12 has been investigated. The amount of formaldehyde-Cl4 that combines with vitamin B12 was determined by comparing the radioactivity of the vitamin Blz-formaldehyde complex with the radioactivity of the dimedon derivative of the formaldehyde-U4 used in the reaction. EXPERIMENTAL
Preparation of the Vitamin Blz-Formaldehyde
Complexes
Preparation 1. A solution containing 32 mg. crystalline vitamin B& and 110 mg. HWHO in 10 ml. water was allowed to stand for 3 hr. Then nitrogen was bubbled through the solution at room temperature for about 7 hr., and all the HWHO escaping with nitrogen was collected in a 10% alcoholic solution of dimedon. The formaldehyde-dimedon derivative was crystallized from dilute alcohol four times and was designated DI . The vitamin Blz-HCY*HO solution had to be freed of the excess of uncombined formaldehyde. This was done by adding a water-saturated solution of dimedon until no further precipitation occurred. The mixture was kept in the refrigerator for 3 hr., and the precipitate was separated from the deep-red mother liquor containing the vitamin BlrHCFHO. The precipitate was washed with water, dissolved in hot 95% alcohol, decolorized with charcoal, and allowed to crystallize out on cooling. This formaldehyde-dimedon derivative was recrystallized three more times from dilute alcohol and was called Dz . The mother liquor containing the vitamin B12-HCr4H0 was treated with acetone until no further precipitation occurred in the cold. The precipitate was removed and discarded. This freed the mother liquor of any residual dimedon. The red mother liquor, which was about 100 ml. in volume, was freeze-dried to a fluffy purple powder weighing 35 mg. The purple powder was crystallized three times from a solution of 10% water and 90% acetone. The final product (PI) was in the form of lustrous purple needles and weighed 27 mg. Preparation b. A solution containing 10.5 mg. crystalline vitamin Blz and 157 mg. HC”HO in 10 ml. water was left at room temperature for 3 hr. A part of the excess formaldehyde was removed with nitrogen for 5 hr. and collected as the dimedon derivative. The formaldehyde-dimedon derivative was crystallized three times with dilute alcohol and was termed Da . The vitamin Blz-HWHO solution was freeze-dried. During this process, the frozen mass melted a few times, but eventually dried into flakes. These were extracted with water, leaving a faintly colored residue. The water extract was freezedried, redissolved, and freeze-dried again, yielding a powdery product. This was dissolved in a minimum amount of water, and acetone was added until a turbidity appeared. This mixture was cooled overnight, causing purple needles to separate from it. These were centrifuged out and recrystallized three times from 90% acetone, yielding Prep. 2 (Pz).
1 Kindly
supplied by Merck and Company, Rahway, New Jersey.
182
VOHRA,
LANTZ
AND
KRATZER
Preparation S. Forty milligrams vitamin Brz and 699 mg. HCY4H0 were reacted together for 3 hr. in 10 ml. water at room temperature. Nitrogen was then bubbled through the reaction mixture for 18 hr., and the escaping formaldehyde was trapped in an alcoholic solution of dimedon. The formaldehyde-dimedon derivative was collected and designated Db . The addition of water to the first mother liquor yielded another crop of the derivative (DS). The vitamin BrZ-HC14H0 reaction mixture was freeze-dried, and the flakes obtained from this process were dissolved in water. The resulting solution was evaporated in a colored container under very low heat from an infrared lamp. A water aspirator was used in this operation. Nitrogen was bled through the system, and the formaldehyde escaping in the vapors was trapped as the dimedon derivative. The purplish product remaining after drying was dissolved in water and precipitated with acetone. The precipitated needles, which weighed 20.7 mg., were recrystallized three times from 99% acetone containing some cyanide. A sample of the crystalline product was taken and crystallized once more to yield Pa . The rest of the product was crystallized four more times from 90% acetone (P4). The mother liquors of the preparations up to Pa were collected and dried by evaporation under nitrogen to yield a crude product (P6). Preparation 4. The procedure for Prep. 4 was essentially the same as described under Prep. 3. The gases flushing out of the dimedon trap were bubbled through 20 ml. of 0.5 N ceric sulfate in 1 N HzS04 and then 1 ml. of 0.05yo NaOH. The NaOH trap and the condensate obtained during the lyophilization of the vitamin BlZ-HCr4H0 reaction mixture were analyzed for the presence of cyanide. After five recrystallizations from 90% acetone, 36.4 mg. of Prep. 4 (P,) was obtained. A portion of it was recrystallized two more times and designated as P7 . The HWHO-dimedon derivative was recrystallized repeatedly, and samples were collected at the 3rd (De) and 4th recrystallization (DT). Drying of Samples. The samples were dried over P205 at 78°C. under reduced pressure. Microbiological Assay. Vitamin Blz and Preps. PI and Pd were compared for microbiological activity with Lactobacillus leichmannii # 4797 using the Bacto vitamin Brz medium B-366 obtained from the Difco Laboratories, Detroit, Michigan. Cyanide Determinations. A calorimetric method described by Robbie (21) was used for the determination of cyanide in the samples. The sample (1 ml.) was photolyzed (22) in a 12-ml. centrifuge tube cooled on the outside by running water and illuminated for 2 hr. by a spot lamp (150 w., 125 v.) using aluminum foil reflectors. Nitrogen was flushed through the sample during illumination and 1 hr. thereafter. The escaping gases were bubbled through two traps made out of 12-ml. centrifuge tubes connected in series, each containing 1 ml. of 0.05% KOH which absorbed cyanide. The color intensities were measured at 548 w in a Beckman spectrophotometer, model DU. Radioactivity Counting Procedures. To determine specific activity per mole, the samples were subjected to wet combustion (23). The COz was precipitated as on filter-paper pads, and counted in a windowless gas-flow BaCOI , collected counter. Proper corrections were made for self-absorption.
VITAMIN
Blz-FORMALDEHYDE
COMPLEXES
183
In other cases, the samples were pipetted into polyethylene planchets and evaporated. If the samples were colored, they were freeze-dried; otherwise, they were evaporated under an infrared lamp and a fan. The samples, which were elutedfrom paper strips after being submitted to electrophoresis, had some contamination with salts on the planchets. No correction for self-absorption was applied in these cases. Paper Electrophoresis. The samples, placed on paper strips 1 in. wide in buffered solutions, were subjected to electrophoretic study by use of a Spinco apparatus. A constant voltage of 500 v. was used, but the time of the run varied from 7 to 24 hr. The paper strips were dried, cut into sections, and eluted with a 0.01% KCN solution. The buffer systems used (24) without NaCl had a pH range of 4.1-11. pH 6.5 Bu$er. An amount of 4.6 ml. of 0.5 M Na2HPOt was added to 3.3 ml. of 4.0 M NaHzPOd and diluted to 1 1. RESULTS
AND
DISCUSSION
Preliminary plating of the samples PI-P* indicated that they were radioactive. In order to ascertain whether the radioactivity was due to the presence of a trace impurity of HC14H0, the sample P1 was subjected to paper chromatography using a number of solvent systems (25). On elution with water, the deeply colored red spots were found to be radioactive. The association of radioactivity with vitamin Blz spots on paper chromatograms in the sample PI strongly suggests the possibility that when vitamin Blz is treated with HVHO, some derivative may be formed. It has been termed a vitamin Blz-HCHO complex. Using Whatman filter paper No. 4 and set-butanol containing 20 % water and 0.01% KCN as developing solvent, the behavior of an inactive vitamin B,z-HCHO complex was compared with that of pure vitamin B,, . The spots ran parallel on paper and moved the same distance. It was not possible to separate a mixture of vitamin Blz and vitamin B,t-HCHO complex on paper. The microbiological activity of the dry vitamin BlrHCHO complex has been found to be 87 % and 90 % of the pure dry vitamin Blz for the samples Pr and P4 , respectively (Table I). The absorption spectrum of vitamin B12-HCHO in the visible region does not differ from that of vitamin Blz . The extinction coefficients of a 1% solution of sample PI in a l-cm. cell at 278, 361, and 550 rnp are 114, 192, and 59, and for sample P4 the values are 118, 299, and 62; while the corresponding values for vitamin Blz are 115, 204, and 63, respectively. The infrared spectrum of the vitamin BrrHCHO complex has also
184
VOHRA,
LANTZ
AND
KRATZER
TABLE I Comparison of the Microbiological Activity of Vitamin Bla and the samples PI and P4 Sample Pl
P4
ma-.
Estimated vitamin Bu activity mlrg.
0.0457 0.0914 0.1370
0.0360 0.0915 0.1130
79 100 82
87
0.0520 0.0780 0.1043
0.0450 0.0740 0.0920
87 95 88
90
Wa~ce&’
Activity %
Average activity %
been compared with that of vitamin Blz in KBr, and no difference in absorption bands has been found. There was no loss of cyanide during the preparation of B12--HCHO complexes. No cyanide was detected in the alkali or condensate traps of Prepn. 4, and recoveries of 92, 97, and 100 % of the theoretical amount of cyanide were obtained for samples PI , Pd , and vitamin Blz (Table II). The electrophoretic behavior of the vitamin B12-HCY4H0 complex (PI) has been compared with that of vitamin Blz on paper at the following pH values: 4.1, 5.0, 6.3, 7.0, 8.1, 9.0, 10.0, and 11.0. No difference in their mobilities was observed. At every pH, the maximum radioactivity has been found to be associated with the most intensely colored zone of the vitamin Blz complex. The paper strips were cut into sections, and eluted, and the eluates were counted after drying. The results of one such study at pH 6.3 are given in Table III. Information about the electrophoretic behavior of the crude preparation (P6) is included for comparison in the same table. This preparation is of interest because it contains a good proportion of unreacted HCY4H0. The data indicate that most of the activity is conTABLE II Cyanide Content of the Samples PI , PI and Vitamin B,a T$orxe;al :%eof a.
106.8 91.3 104.3
content Pg.
2.05 1.71 1.98
Recovered cyanide Pg.
2.05 1.58 1.92
RC?COWy %
100 92 97
VITAMIN
BITFORMALDEHYDE
TABLE
185
COMPLEXES
III
The Paper-Electrophoretic Behavior of Samples PI and Pg of Vitamin BIZ-HC’~HO Complexes at pH 6.3 Sample Ps
Sample Pl Polarity
Remarks
COUM.S/ min.
Anode
Colorless Faint red Origin Deep red (BH) Faint red Faint red Colorless
9 3 16 318 6 0 2
Cathode
Remarks
COWS/ min.
Colorless Faint red Colorless Origin Faint red Deep red (BH) Faint red Colorless
240 75 29 85,300 1,750 9,900 26 31
centrated at the origin. The radioactivity counts fall in the next section but rise again in the deeply colored zone containing Blz . A few other red zones, more faintly colored, were observed, but they did not contribute much toward the radioactivity. If the radioactivity found in the vitamin BIrHC14H0 complex were due only to the trace impurity of HC14H0, there would have been a continuous fall in the radioactive counts along the paper farther removed from the origin. The close association of the color and the radioactivity peak again suggests the possibility of some complex formation. The various preparations have been subjected to combustion analysis (Table IV) to determine whether the vitamin Bw-HC’~HO complex has a definite composition. This has been attempted using radioactive counts per millimole of the samples combusted as the basis for calculation purposes. The radioactive counts of the duplicate samples of the dimedon derivative of formaldehyde are in close agreement with each other. Vitamin B,, has a molecular weight of 1356. If one mole of vitamin Blz combines with either 1, 2, or 3 moles of HC4H0, the respective molecular weights of the complexes would be 1388, 1420, and 1452. On this basis, the observed counts per mole for the complexes have been calculated. The complex PI appears to have 1 mole of vitamin Blz in combination with 1 mole of HC14H0, while the complex Pz has 1 mole of vitamin Blz in combination with 3 moles of HC4H0. In order to determine whether these complexes could be crystallized to a constant activity, the samples (Pa and P4) from the 4th and 7th crystallizations of the 3rd preparation were combusted. Both of these samples
186
VOHRA,
LANTZ
AND
TABLE
KRATZER
IV
Calculation of Molar Various
Ratios of Vitamin B12 and Forn~aldehyde-C14 in Preparations of a Vitamin B,z-Formaldehyde Complex _.-. -._. --~ -I.--. -- --
Original HCWO
Vitamin Br(HC”HO)r
I
complex Cts./min./mmole
_-..(1)
D7
-
=YF
I
(2)
(3)
172
50,300
172 69
50,250 50,200 20,10020,100
229
67,ONI
225
i65,mcl
(4)
(5) -.
(6)
(7)
Observed* --
(8) .~
(9)
PI
36.9
1
50,250
51,300
1’2
42.5
2 3 4
40,200 60,300 80,400
60,400 61,800 63,100
66,300 Pa (4th cryst.) P, (7th tryst.)
25.8
0.5 1 0.5 1
33,150 66,300 33,150 66,300
35,400 35,600 31,500 31,800
162 147’W~48,500j l’s (5th 172 tryst.) I’, (7th tryst.)
73.7
1 2 1 2
48,500 97,000 48,500 97,000
103,000 105,000 110,000 112,000
22.9
79.1
o Calculated from col. 4. * Calculated from col. 6. c Rounded to whole numbers.
have radioactive counts of the same order. However, the data indicate that in this preparation 1 mole of HCY4H0 links 2 moles of vitamin B12 . Also, the radioactivities of the samples PB and P7 agree reasonably among themselves; however, they contain 1 mole of vitamin B12 in combination with 2 moles of HC”H0. The results obtained in the present study show that when HC!r4H0 is allowed to react with vitamin BIs , some complexes are formed whose structure varies in different preparations. This is not unexpected in view of the similar findings for CHZOH-THFA (12). The biological role of these complexes needs further study and is under investigation.
VITAMIN
BlrFORMALDEHYDE
COMPLEXES
187
SUMMARY
Formaldehyde-C4 reacts with vitamin BIz in vitro to form complexes that cannot be separated from vitamin Blz by crystallization, paper chromatography, or paper electrophoresis at several pH values. No differences were noted in the absorption spectra in the visible, ultraviolet, and infrared regions between vitamin BIZ reacted with formaldehyde and unreacted vitamin Blz . Four samples prepared by slightly different methods contained 0.5, 1, 2, and 3 moles of formaldehyde-C4 combined with each mole of vitamin Blz . REFERENCES 1. ARNSTEIN, H. R. V., Biochem. Sot. Symposia No. 13,92 (1955). 2. MACKENZIE, C. G., J. Biol. Chem. 186,351 (1950). 3. MACKENZIE, C. G., JOHNSTON, J. M., AND FRISELL, W. R., J. Biol. Chem. 203, 743 (1953). 4. MACKENZIE, C. G., AND ABELES, R. H., J. Biol. Chem. 222,145 (1956). 5. ELWYN, D., WEISSBACH, A., HENRY, S. S., AND SPRINSON, D. B., J. Biol. Chem. 213, 281 (1955). 6. KISLIUK, R. L., AND SAKAMI, W., J. Biol. Chem. 214, 47 (1955). 7. ALEXANDER, N., AND GREENBERG, D. M., J. Biol. Chem. 214, 821 (1955). 8. SAKAMI, W., in “A Symposium on Amino Acid Metabolism” (McElroy, W. D., and Glass, B., eds.), p. 685. The Johns Hopkins Press, Baltimore, 1955. 9. BLAKLEY, R. L., Biochem. J. 63,448 (1954). 10. BLAKLEY, R. L., Biochem. J. 66, 331 (1967). 11. JAENICKE, L., Federation Proc. 16, 281 (1956). 12. BLAKLEY, R. L., Biochim. et Biophys. Acta 23, 654 (1957). 13. KISLIUK, R. L., J. Biol. Chem. 227, 806 (1957). 14. STEKOL, J. A., WEISS, S., AND WEISS, K. W., Arch. Biochem. Biophys. 36, 5 (1952). 15. ARNSTEIN, H. R. V., AND NEUBERGER, A., Biochem. J. 60, xxxviii (1952). 16, ARNSTEIN, H. R. V., AND NEUBERGER, A., Biochem. J. 66,259,271 (1953). 17. ARNSTEIN, H. R. V., Biochem. J. 60, xii (1955). 18. VOHRA, P., LANTZ, F. H., AND KRATZER, F. H., J. Biol. Chem. 221,501 (1966). 19. CHANG, I., AND JOHNSON, B. C., Arch. Biochem. Biophys. 66, 151 (1955). 20. HODGKIN, D. C., PICKWORTH, J., ROBERTSON, J. H., TRUEBLOOD, K. N., PROSEN, R. J,, WHITE, J. G., BONNET, R., CANNON, J. R., JOHNSON, A. W., SUTHERLAND, I., TODD, A. R., AND SMITH, E. L., Nature 176,325 (1955). 21. ROBBIE, W. A., Arch. Biochem. 6, 49 (1945). 22. BOXER, G. E., AND RICHARDS, J. C., Arch. Biochem. 30, 372 (1951). 23. VAN SLYKE, D. D., AND FOLCH, J., J. Biol. Chem. 136, 509 (1940). 24. MILLER, G. L., AND GOLDER, R. H., Arch. Biochem. 29, 420 (1950). 26. KON, S. K., Biochem. Sot. Symposia No. 13, 17 (1955).