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Rapid estimation of small amounts of formaldehyde liberated during periodate oxidation of a sialoglycoprotein
ANALYTICAL
BIOCHEMISTRY
Rapid
Estimation Liberated
G. DURAND,
61, 232-236 (1974)
of Small
Amounts
During Periodate of a Sialoglycoprotein
of Formaldehyde Oxidation
J. FEGER, M. COIGNOUX, AND M. PAYS
J. AGNERAY
Laboratoire
de Biochimie, U, E. R. des Sciences Pharmaceutiques et Biologiques, J. B. Cle’ment, 92290-Chatenay-Malabru-France, (C.N.R.S.: E.R.A. 070-396, Inserm: C.R.L. N” 73.1.110.9) and Laboratoire de Chimie Analytique, U.E.R. des Sciences Pharmaceutiques, 1, rue Vaubenard,l.@OO-Caen-France rue
Received December 7, 1973; accepted March
1, 1974
We describe a method in order to estimate the amount of formaldehyde liberated by mild periodate oxidation. This MBTH method is a rapid, reliable, sensitive, and a rather specific procedure. We obtain these results: 1 mole of formaldehyde is produced when 1 mole of metaperiodate is consumed by 1 mole of N-acetyl-neuraminic acid.
N-acetyl-neuraminic acid (NANA) containing glycoproteins is widely distributed: blood group specific substances, serum glycoproteins, hormones, cell membrane glycoproteins. In removing enzymatic NANA from glycoproteins, we have proved how much it contributes to the physical, chemical, and biological properties of individual glycoproteins. Some of these properties (1,2) often involve 3 carbon polyhydroxy side chain of NANA. To prove it, it was necessary to count the number of moles of metaperiodate taken out and also the number of moles of formic acid and formaldehyde produced. Under very mild conditions (3)) formaldehyde is only liberated from the cleavage between C 9 and C 8 of NANA residues. The primary alcoholic groups yield formaldehyde (Fig. 1). In a recent study (4), we showed that in our conditions of periodate oxidation and after tritiated borohydride reduction, we obtained a radioactive S-carbon analog of NANA. Therefore, the measurement of formaldehyde liberated during periodate oxidation of a sialoglycoprotein is a good way of approaching the establishment of a correlation between structure and biological activity. So this report describes a calorimetric method for the measurement’of formaldehyde liberated from a purified sialoglycoprotein (5). This procedure involves the reaction of formaldehyde with 3-methyl-2232 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
FORMALDEHYDE
$FTER
PERIODATE
233
OXIDATION
-OH + HCOOH
FIG. 1 . Periodate oxidation position of a glycoprotein.
of N-acetyl-neuraminic
acid in nonreducing
terminal
benzothiazolone hydraaone (MBTH) (Fig. 2). A blue formazan cation appears (6,7,8). The reaction is carried out in acid medium (pH 1) and with an oxidizing agent (FeCI,). REAGENTS
I--Period&e
Oxidation
a. 0.01 M periodate solution: prepared daily b. 2% sodium hydrogen carbonate solution c. Sodium arsenite solution N/10 II-Measurement
of Formaldehyde
d. 1O-4 M formaldehyde solution: Prepared daily by decomposition of paraformaldehyde, the solution is analysed by the dimedon graviwetric method e. 2 N hydrochloric acid reagent: Prepared by dilution of hydrochloric acid (d = 1.190) f. 0.154 M 3-methyl-2-benzothiazolone hydrazone reagent MBTHl 276.6 mg; HCI 0.1 N 10 ml
FIG. 2. Reaction
of formaldehyde
with 3-methyl-2-benzothiazolone-hydrazone.
1 MBTH reagent purchased from Eastman Kodak Ref. 8443 (Touzart 3 rue Amyot, Paris 5e) puriss.
et Matignon,
234
DURAND
ET
AL.
g. Aqueous 5 p 109 ferric chloride reagent: h. Dichloromethane puriss.
Prepared with FeCl,*GH,O
PROCEDURE
I-Perioda
te Oxidation
We have added amounts of 0.01 M NaI04 (a) calculated to give ratios of 10,:NANA = 2.6-3.2 and 5.2 to solutions of purified glycoprotein containing 1 mg/ml. The mixtures are kept in the dark at. 4°C for 2 hr. The remaining periodate is measured (9) ; the consumed 10,:NANA ratios are, respectively, 1.24-1.45 and 1.9. We add 0.1 ml of reagent (b) and 0.4 ml of reagent (c) in order to reduce excess periodate to iodate. II-Measurement
of Formaldehyde
a. To 1 ml of each reaction mixture, we add: 0.1 ml of reagent (e) 0.1 ml of reagent (f) 0.1 ml of reagent (g). The mixture is left standing for 1/2 hr. Then 2 ml of reagent (h) are added and the tubes are vigorously shaken and centrifuged. The aqueous excess is eliminated from the organic solvent layer by the addition of a few mgs of anhydrous sodium sulphate. Optical densities of the organic phase are determined at 630 nm against a blank which has been treated in the same manner. Colours are stable for 17 hr. b. In parallel, run a series of standard determinations (O-1 X 10m4 M) of formaldehyde and a determination of solution .containing sialoglycoprotein and all reagents except m-periodate. RESULTS
A
Two assays are carried out with three different ratios IO*-:NANA. Results are shown in Table 1. The amounts of formaldehyde liberated correspond to the amounts ,expected for the oxidation between C 9 and C 8 of N-acetylneuraminic acid. Recoveries of formaldehyde in all cases are: 101.5 + 5.5%. B
Table 2 points out the comparison NANA and of 104-:NANA. (1) When about 1 mole of periodate 1 mole of formaldehyde is liberated.
between the ratios per mole of NANA
of HCHO: is taken out,
FORMALDEHYDE
AFTER
PERIODATE
235
OXIDATIOR‘
TABLE 1 Periodate Oxidation of a Sialoglyceprotein: Comparison of Contents of NANA and of Amounts of HCHO (PM) ; Theoretical Yield of Formaldehyde 1.24
1 .4;i
1.90
NANA (MM) HCHO &M) HCHO (7, of theory)
0.260 0.270 104
0.260 0.2.51 96
0.260 0.260 100
NANA (/AM) HCHO (NM) HCHO (% of theory)
0.297 0.297
0.297 0.300
0.297 0.320 107
Consumed 104:NANA
Correlation
100
101
TABLE 2 Between Consumed 101-:NANA
and HCHO:NANA
(MM)
Ratios
IO,- : NANA
2.6
3.2
5.2
Consumed 104-:NANA
1.24
1.45
1.90
HCHO: NANA
1.04 1
0.96 1.01
1.00 1.07
(2) When more than one of periodate per mole of NANA is taken out, the amount of formaldehyde liberated is always the same. DISCUSSION
These results show that, under mild periodate oxidation, a sialoglycoprotein liberates formaldehyde out of the cleavage between C 9 and C 8 of the N-acetyl-neuraminic units. These results are in accordance with those generally mentioned (10). Moreover the MBTH procedure offers a few advantages when compared to several different methods already used (11). l-The MBTH method is easier to perform than the chromotropic acid (12) and the J acid spectrophotometric (13) ones. 2-The MBTH method is more sensitive than the two methods mentioned above, but less sensitive than the J acid spectrofluorimetric method (11). However, the MBTH method is preferred for its precision, easy manipulation, and quenching limitation. 3-Specificity is now being discussed. a. Under operating conditions, MBTH reacts the same way with formaldehyde (f = 65,006) and glyoxilic acid (14) ; but this latter compound doesnot interfere because it is never produced during periodate oxidation of a sialoglycoprotein. b. MBTH may be also combined with other aliphatic and aromatic
236
DURAND
ET
AL.
aldehydes but, in that case, the sensitivity degree (ethanal c = 46,500; propionaldehyde c = 22,060; butynaldehyde c = 12,000) is lower. On the other hand, it is possible to identify MBTH-aldehyde by a thinlayer chromatography on silicagel which is developed in a benzene/ methanol (70: 30 v/v) solvent. c. MBTH gives a coloured diazo compound with proteins (15)) but it is not extracted by dichloromethane. Therefore the polypeptidic moiety of a glycoprotein does not interfere. CONCLUSION
The MBTH procedure is preferred to several different methods of formaldehyde determinations. It is a rapid spectrophotometric method which requires no external heating. It is a precise, sensitive, and a rather specific method of measuring formaldehyde liberated during periodate oxidation. This procedure is very convenient for structural studies of some glycoproteins that can be only obtained in very small quantities. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
SUTTAJIT, M., AND WINZLER, R. (1971) J. Biol. Chem. 246, 3398. SUTTAJIT, M., URBAN, C., AND MCLEAN, R. L. (1971) J. Biol. Chem. 246, 810. KROTOSKI, W. A., AND WEIMER, H. E. (1966) Arch. Biochem. Biophys. 115, 337. sur FEGER, J., CACAN, R., DURAND., AND AGNERAY, J. CoIloque International les Glycoconjugds organid par le C. N. R. S. Villeneuve d’Ascq-21-27 join 1973. FEGER, J. (1972) These Doct. Etat Pharm. Paris. HUNIG, S., AND FRITSCH, K. H. (1957) Liebigs Ann. 609, 172. SAWICKI, E., HAUSER. T. R., STANLEY, T. W., AND ELBERT, W. (1961) Anal. Chem. 33, 93. PAYS, M., MALANGEAU, P., AND BOURDON, R. (1966) Ann. Pharm. FT. 24, 763. MARINETTI, G. V., AND ROUSER, G. (1955) J. Amer. Chem. Sot. 77, 5345. MARSHALL, R. D., AND NEUBERGER,A. (1972) in Glycoproteins (Gottschalk, A., ed.), p. 331, Elsevier Publishing Company, Amsterdam, London, New York. SAWICKI, E.. STANLEY, T. W., AND PFAFF, J. (1963) Anal. Chim. Acta 28, 156. O’DEA, J. F., AND GIBBONS, R. A. (1953) Biochem. J. 55, 580. SAWICKI, E., HAUSER, T. R., AND MCPHERSON, S. (1962) Anal. Chem. 34, 1460. PAYS, M., AND BEWEAN, M. (1970) Ann. Pharm. FT. 28, 153. PAYS, M. (1965) These Doct. Etat Pharm. Paris.
BIOCHEMISTRY
Rapid
Estimation Liberated
G. DURAND,
61, 232-236 (1974)
of Small
Amounts
During Periodate of a Sialoglycoprotein
of Formaldehyde Oxidation
J. FEGER, M. COIGNOUX, AND M. PAYS
J. AGNERAY
Laboratoire
de Biochimie, U, E. R. des Sciences Pharmaceutiques et Biologiques, J. B. Cle’ment, 92290-Chatenay-Malabru-France, (C.N.R.S.: E.R.A. 070-396, Inserm: C.R.L. N” 73.1.110.9) and Laboratoire de Chimie Analytique, U.E.R. des Sciences Pharmaceutiques, 1, rue Vaubenard,l.@OO-Caen-France rue
Received December 7, 1973; accepted March
1, 1974
We describe a method in order to estimate the amount of formaldehyde liberated by mild periodate oxidation. This MBTH method is a rapid, reliable, sensitive, and a rather specific procedure. We obtain these results: 1 mole of formaldehyde is produced when 1 mole of metaperiodate is consumed by 1 mole of N-acetyl-neuraminic acid.
N-acetyl-neuraminic acid (NANA) containing glycoproteins is widely distributed: blood group specific substances, serum glycoproteins, hormones, cell membrane glycoproteins. In removing enzymatic NANA from glycoproteins, we have proved how much it contributes to the physical, chemical, and biological properties of individual glycoproteins. Some of these properties (1,2) often involve 3 carbon polyhydroxy side chain of NANA. To prove it, it was necessary to count the number of moles of metaperiodate taken out and also the number of moles of formic acid and formaldehyde produced. Under very mild conditions (3)) formaldehyde is only liberated from the cleavage between C 9 and C 8 of NANA residues. The primary alcoholic groups yield formaldehyde (Fig. 1). In a recent study (4), we showed that in our conditions of periodate oxidation and after tritiated borohydride reduction, we obtained a radioactive S-carbon analog of NANA. Therefore, the measurement of formaldehyde liberated during periodate oxidation of a sialoglycoprotein is a good way of approaching the establishment of a correlation between structure and biological activity. So this report describes a calorimetric method for the measurement’of formaldehyde liberated from a purified sialoglycoprotein (5). This procedure involves the reaction of formaldehyde with 3-methyl-2232 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
FORMALDEHYDE
$FTER
PERIODATE
233
OXIDATION
-OH + HCOOH
FIG. 1 . Periodate oxidation position of a glycoprotein.
of N-acetyl-neuraminic
acid in nonreducing
terminal
benzothiazolone hydraaone (MBTH) (Fig. 2). A blue formazan cation appears (6,7,8). The reaction is carried out in acid medium (pH 1) and with an oxidizing agent (FeCI,). REAGENTS
I--Period&e
Oxidation
a. 0.01 M periodate solution: prepared daily b. 2% sodium hydrogen carbonate solution c. Sodium arsenite solution N/10 II-Measurement
of Formaldehyde
d. 1O-4 M formaldehyde solution: Prepared daily by decomposition of paraformaldehyde, the solution is analysed by the dimedon graviwetric method e. 2 N hydrochloric acid reagent: Prepared by dilution of hydrochloric acid (d = 1.190) f. 0.154 M 3-methyl-2-benzothiazolone hydrazone reagent MBTHl 276.6 mg; HCI 0.1 N 10 ml
FIG. 2. Reaction
of formaldehyde
with 3-methyl-2-benzothiazolone-hydrazone.
1 MBTH reagent purchased from Eastman Kodak Ref. 8443 (Touzart 3 rue Amyot, Paris 5e) puriss.
et Matignon,
234
DURAND
ET
AL.
g. Aqueous 5 p 109 ferric chloride reagent: h. Dichloromethane puriss.
Prepared with FeCl,*GH,O
PROCEDURE
I-Perioda
te Oxidation
We have added amounts of 0.01 M NaI04 (a) calculated to give ratios of 10,:NANA = 2.6-3.2 and 5.2 to solutions of purified glycoprotein containing 1 mg/ml. The mixtures are kept in the dark at. 4°C for 2 hr. The remaining periodate is measured (9) ; the consumed 10,:NANA ratios are, respectively, 1.24-1.45 and 1.9. We add 0.1 ml of reagent (b) and 0.4 ml of reagent (c) in order to reduce excess periodate to iodate. II-Measurement
of Formaldehyde
a. To 1 ml of each reaction mixture, we add: 0.1 ml of reagent (e) 0.1 ml of reagent (f) 0.1 ml of reagent (g). The mixture is left standing for 1/2 hr. Then 2 ml of reagent (h) are added and the tubes are vigorously shaken and centrifuged. The aqueous excess is eliminated from the organic solvent layer by the addition of a few mgs of anhydrous sodium sulphate. Optical densities of the organic phase are determined at 630 nm against a blank which has been treated in the same manner. Colours are stable for 17 hr. b. In parallel, run a series of standard determinations (O-1 X 10m4 M) of formaldehyde and a determination of solution .containing sialoglycoprotein and all reagents except m-periodate. RESULTS
A
Two assays are carried out with three different ratios IO*-:NANA. Results are shown in Table 1. The amounts of formaldehyde liberated correspond to the amounts ,expected for the oxidation between C 9 and C 8 of N-acetylneuraminic acid. Recoveries of formaldehyde in all cases are: 101.5 + 5.5%. B
Table 2 points out the comparison NANA and of 104-:NANA. (1) When about 1 mole of periodate 1 mole of formaldehyde is liberated.
between the ratios per mole of NANA
of HCHO: is taken out,
FORMALDEHYDE
AFTER
PERIODATE
235
OXIDATIOR‘
TABLE 1 Periodate Oxidation of a Sialoglyceprotein: Comparison of Contents of NANA and of Amounts of HCHO (PM) ; Theoretical Yield of Formaldehyde 1.24
1 .4;i
1.90
NANA (MM) HCHO &M) HCHO (7, of theory)
0.260 0.270 104
0.260 0.2.51 96
0.260 0.260 100
NANA (/AM) HCHO (NM) HCHO (% of theory)
0.297 0.297
0.297 0.300
0.297 0.320 107
Consumed 104:NANA
Correlation
100
101
TABLE 2 Between Consumed 101-:NANA
and HCHO:NANA
(MM)
Ratios
IO,- : NANA
2.6
3.2
5.2
Consumed 104-:NANA
1.24
1.45
1.90
HCHO: NANA
1.04 1
0.96 1.01
1.00 1.07
(2) When more than one of periodate per mole of NANA is taken out, the amount of formaldehyde liberated is always the same. DISCUSSION
These results show that, under mild periodate oxidation, a sialoglycoprotein liberates formaldehyde out of the cleavage between C 9 and C 8 of the N-acetyl-neuraminic units. These results are in accordance with those generally mentioned (10). Moreover the MBTH procedure offers a few advantages when compared to several different methods already used (11). l-The MBTH method is easier to perform than the chromotropic acid (12) and the J acid spectrophotometric (13) ones. 2-The MBTH method is more sensitive than the two methods mentioned above, but less sensitive than the J acid spectrofluorimetric method (11). However, the MBTH method is preferred for its precision, easy manipulation, and quenching limitation. 3-Specificity is now being discussed. a. Under operating conditions, MBTH reacts the same way with formaldehyde (f = 65,006) and glyoxilic acid (14) ; but this latter compound doesnot interfere because it is never produced during periodate oxidation of a sialoglycoprotein. b. MBTH may be also combined with other aliphatic and aromatic
236
DURAND
ET
AL.
aldehydes but, in that case, the sensitivity degree (ethanal c = 46,500; propionaldehyde c = 22,060; butynaldehyde c = 12,000) is lower. On the other hand, it is possible to identify MBTH-aldehyde by a thinlayer chromatography on silicagel which is developed in a benzene/ methanol (70: 30 v/v) solvent. c. MBTH gives a coloured diazo compound with proteins (15)) but it is not extracted by dichloromethane. Therefore the polypeptidic moiety of a glycoprotein does not interfere. CONCLUSION
The MBTH procedure is preferred to several different methods of formaldehyde determinations. It is a rapid spectrophotometric method which requires no external heating. It is a precise, sensitive, and a rather specific method of measuring formaldehyde liberated during periodate oxidation. This procedure is very convenient for structural studies of some glycoproteins that can be only obtained in very small quantities. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.
SUTTAJIT, M., AND WINZLER, R. (1971) J. Biol. Chem. 246, 3398. SUTTAJIT, M., URBAN, C., AND MCLEAN, R. L. (1971) J. Biol. Chem. 246, 810. KROTOSKI, W. A., AND WEIMER, H. E. (1966) Arch. Biochem. Biophys. 115, 337. sur FEGER, J., CACAN, R., DURAND., AND AGNERAY, J. CoIloque International les Glycoconjugds organid par le C. N. R. S. Villeneuve d’Ascq-21-27 join 1973. FEGER, J. (1972) These Doct. Etat Pharm. Paris. HUNIG, S., AND FRITSCH, K. H. (1957) Liebigs Ann. 609, 172. SAWICKI, E., HAUSER. T. R., STANLEY, T. W., AND ELBERT, W. (1961) Anal. Chem. 33, 93. PAYS, M., MALANGEAU, P., AND BOURDON, R. (1966) Ann. Pharm. FT. 24, 763. MARINETTI, G. V., AND ROUSER, G. (1955) J. Amer. Chem. Sot. 77, 5345. MARSHALL, R. D., AND NEUBERGER,A. (1972) in Glycoproteins (Gottschalk, A., ed.), p. 331, Elsevier Publishing Company, Amsterdam, London, New York. SAWICKI, E.. STANLEY, T. W., AND PFAFF, J. (1963) Anal. Chim. Acta 28, 156. O’DEA, J. F., AND GIBBONS, R. A. (1953) Biochem. J. 55, 580. SAWICKI, E., HAUSER, T. R., AND MCPHERSON, S. (1962) Anal. Chem. 34, 1460. PAYS, M., AND BEWEAN, M. (1970) Ann. Pharm. FT. 28, 153. PAYS, M. (1965) These Doct. Etat Pharm. Paris.