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Lysis of glycosides in the presence of l -ascorbic acid
BIOCHIMICA ET BIOPI-IYSICA ACTA
I
BBA 26040 LYSIS OF GLYCOSIDES IN T H E PRESENCE OF L-ASCORBIC ACID J. c. CAYGILL* Rheumatism Research Centre, University of Manchester, Clinical Sciences Building, Manchester, 13 (Great Britain)
(Received July Ist, I968)
SUMMARY The liberation of nitrophenol from eleven nitrophenyl glycosides in acid or neutral solution in the presence of L-ascorbic acid is reported. The conditions under which o-nitrophenol is released from o-nitrophenylfl-D-galactopyranoside are reported. The reaction rate, which is proportional to temperature and glycoside concentration, is maximal between pH 4 and 5, but negligible above pH 8.5, and is increased by Cu~+. At low concentrations of L-ascorbic acid the reaction rate is proportional to L-ascorbic acid concentration, but at higher concentrations the reaction rate is not further increased. Other monosaccharides inhibit the release of nitrophenol.
INTRODUCTION
It has been known for some time that L-ascorbic acid reduces the viscosity of hyaluronic acid sohitionsl, ~, and it has been suggested that this may involve an oxidative-reductive depolymerisation 3. Recently YAMANE, SAKAI AND IKEGUCHI4 reported the dissociation of several low molecular weight o-glucuronides in the presence of L-ascorbic acid. This splitting promoted by L-ascorbic acid has been found to be a general reaction of nitrophenyl glycosides. MATERIALS AND METHODS
L-Ascorbic acid, galactono-i,4-1actone , galactose, glucose and all the nitrophenyl glycosides used in this investigation were obtained from Koch-Light Ltd., except for p-nitrophenyl fl-D-galactopyranoside, which was supplied by B.D.H.Ltd. In all experiments, unless indicated to the contrary, buffered solutions (I.O ml) were incubated in duplicate at 37 ° for a convenient length of time, and the reaction stopped by the addition of 0.2 M sodium borate-NaOH buffer (pH 9.8) (4.0 ml). The liberated o-nitrophenol or p-nitrophenol was estimated by measuring the absorbance in a Mecolab Autocolorimeter (Joyce Loebl) using a 4oo-m/~ interference filter. Standard solutions of recrystallised o- or p-nitrophenol in water were prepared. The addition of 20 ~M Cu *+ did not affect the absorbance of light by the nitrophenol. Appropriate controls lacking L-ascorbic acid or nitrophenyl glycoside were included. ;'3
* Present address: Tropical Products Institute, 56]62, Gray's Inn Road, London, W.C.I, Great Britain. Biockim. Biophys. A~ta, 17o (I968) I- 5
2
J . c . CAYGILL
L-Ascorbic acid dit not give rise to any derivative absorbing at 4oo m/z except at ioo °. The nitrophenyl glycosides all absorbed slightly at this wavelength, and the relevant 'blank' value was subtracted from the readings to give the increase in colour representing liberated nitrophenol. No hydrolysis of the nitrophenyl glycosides used was noted after the addition of 0.2 M borate buffer, but the colorimetric measurement was made as rapidly as practicable after the reaction was stopped (in all cases within 5 h, usually within I h). When it was necessary to delay colorimetric measurement, the tubes were stored in the dark as recommended b y WOOLLEN, HEYWORTH AND WALKER5. The concentrations of the respective components given are the final concentrations in the mixtures incubated at 37 °. For each experiment the nitrophenyl glycoside and L-ascorbic acid were dissolved immediately before use. RESULTS
The liberation of o- or of p-nitrophenol was found to increase uniformly with time for at least 3 h. In the majority of these experiments it was convenient to estimate aglycone liberation in a suitable time interval (generally I h), as a measure of the initial velocity of the reaction. The results given are expressed in/~moles/h. 2.0 mM solutions of various p-nitrophenyl glycosides were incubated in 0.05 M acetic acid-sodium acetate buffer (pH 4.0 or 5.5) with 4.0 mM L-ascorbic acid and either 20 /~M, 2 /~M or no CuS04. In each case approximately equal amounts of p-nitrophenol were liberated at p H 4.0 or at 5.5, and p-nitrophenol liberation was fastest in the presence of 20 /,M Cu 2+, slower with 2 /,M Cu 2+ and slowest in the absence of Cu e+. The average rates of p-nitrophenol liberation with 2 /~M Cu 2+ are given in Table I. No p-nitrophenol was liberated in the control tubes lacking Lascorbic acid similarly incubated at 37 ° for I h, except in the case of p-nitrophenyl a-D-mannoside, where some hydrolysis occurred. The rate of liberation of p-nitrophenol from p-nitrophenyl fl-D-galactoside incubated at 37 ° in 0.05 M acetate buffer (pH 4.0) with 4.0 mM L-ascorbic acid was found to be proportional to the p-nitrophenyl galactoside concentration over the range 1-16 raM. TABLE I p-NITROPHENOL LIBERATION FROMp-NITROPH]~NYL GLYCOSIDES Average rate of p-nitrophenol liberation from 2.0 mM p-nitrophenyl glycosides incubated for I h at 37 ° in 0.o 5 M acetate buffer (pH 4.0-5.5) with 4.0 mM L-ascorbic acid and 2 #M CuSO 4" 5H20.
Glycoside
p-Nitrophenol liberated (tzmoles/h)
p-Nitrophenyl fl-L-fucoside p-Nitrophenyl fl-D-xyloside p-Nitrophenyl fl-D-galactoside p-Nitrophenyl fl-D-glucuronide p-Nitrophenyl a-L-fucoside p-Nitrophenyl a-D-glucoside p-Nitrophenyl r-D-2 -acetamido-2 -deoxyglucoside p-Nitrophenyl a-n-galactoside p-Nitrophenyl fl-D-glucoside p-Nitrophenyl a-n-mannoside
259 223 218 184 177 148 145 I 18 i 13 lO5
Biochim. Biophys. Acta, 17o (1968) 1-5
GLYCOSIDE LYSIS WITH ASCORBIC ACID
3
When 8 mM o-nitrophenyl fi-D-galactoside was incubated at 37 ° in 0.2 M acetate buffer (pH 5.5) with final concentrations of L-ascorbic acid from I to 25 mM, it was found that o-nitrophenol liberation was proportional to ascorbate concentration up to 4 mM, but at higher ascorbate concentrations the initial rate of o-nitrophenyl liberation was reduced in the absence of Cu 2+, and was not increased in the presence of 20 tzM Cu 2+ (Fig. I). A similar effect was found when 8 mM p-nitrophenyl ~-Dgalactoside was incubated in 0.05 M acetate buffer (pH 4.0) with 0.5 to 20 mM L-ascorbic acid. At these concentrations, L-ascorbic acid did not affect the pH. This indicates inhibition of the reaction rate by excess of ascorbate. A similar inhibition was found in the presence of comparable concentrations of other monosaccharides, o-Nitrophenyl fi-D-galactoside (8 mM) was incubated at 37 ° in o.2 M acetate buffer ~pH 5.5) with 4 mM L-ascorbic acid and either 20 #M, 2 #M or no Cu 2+ in the presence or absence of D-glucose, D-galactose or D-galactono-i, 4lactone. The rate of o-nitrophenol liberation was decreased approx. 20 % with IO mM monosaccharide, IO % with 5 mM monosaccharide, and very slightly by I mM monosaccharide. There was little difference in the percentage inhibition whether Cu~+ was present or not. These monosaccharides inhibited to a similar extent p-nitrophenol liberation from 8 mM p-nitrophenyl fl-D-galactoside by 4 mM L-ascorbic acid in 0.2 M acetate buffer (pH 5.0). ,
2
i
,
!
i
~
'
3o~
~2oo
"~400
,oc z
"~
o
.~
1~
~
l.-Ascorbic acid (mM)
20
25
2
~, pM
Fig. I. Variation in the rate of liberation of o-nitrophenol from 8.0 mM o-nitrophenyl/~-D-galactoside in o.2 M acetate buffer (pH 5.5) with L-ascorbic acid concentration. A k ~ k , 2o ffM Cu2+; O - - O , 2 ffM Cul+; re__m, no Cu 2+. Fig. 2. Variation with p H in the rate of liberation of o-nitrophen01 from 5.0 mM o-nitrophenyl /~-D-galactoside by 4.o mM L-ascorbic acid in citrate-phosphate buffers. ~ k - - , , 20 #M Cu2+; 0 - - 0 , 2 #M CuZ+; l - - l , no Cu z+.
The reaction was found to vary with pH. o-Nitrophenyl fl-D-galactoside (5 aM) was incubated in 0.05 M citric acid-Na2HPO 4 buffers with 4.0 mM L-ascorbic acid, and either 2o ~M, 2 ~M or no Cu 2+. The results are shown in Fig. 2. With the same final concentrations of o-nitrophenyl fl-D-galactoside, ascorbic acid and Cu 2+ in 0.o4 M veronal (HCl-sodium barbitone) buffer, o-nitrophenol was liberated at approximately the same rate in veronal buffer as in citrate phosphate buffer at pH 7.0 to 8.0, but little at pH 8.5 and none at pH 9.0. In no case did the 'blank' value of o-nitrophenyl galactoside (nor any Of: the nitrophenyl glycosides used in these Biochim. Biophy$.Acta, 17o (1968) x-5
4
J . c . CAYGILL
investigations) increase after the addition of borate buffer p H 9.8, confirming that dissociation does not occur above p H 9.0. A similar m a x i m u m rate of liberation at p H 4.5 of p-nitrophenol from 8.0 mM p-nitrophenyl fl-D-galactoside or o-nitrophenol from 8 mM o-nitrophenyl fl-D-galactoside was found when these were incubated with 4 mM L-ascorbic acid with either 20/,M, 2 ~M or no Cu 2+ in a series of 0.05 M acetate buffers of p H 3.5 to 6.0. I t was noted that in all experiments where buffers of various p H values were used, the final p H after the addition of 4.0 ml of 0.2 M borate buffer was p H 9.5-9.7- I t is interesting to note that this apparent 'optimum' at around p H 4.5 is near the pKa of ascorbic acid (4.2). The rate of liberation of o-nitrophenol from 5.0 mM o-nitrophenyl fl-Dgalactoside incubated in 0.05 M acetate buffer (pH 4.6) with 4.0 mM L-ascorbic acid and 20/zM, 2 /zM or no Cu 2+ was found to increase exponentially with temperature over the range o - I o o °. At higher temperatures it was difficult to obtain reproducible results, as the o-nitrophenyl fl-D-galactoside is hydrolysed at an appreciable rate in the absence of L-ascorbic acid. DISCUSSION
In these experiments 1-25 mM L-ascorbic acid was found to liberate nitrophenol from all the nitrophenyl glycosides tested. Lower concentrations of L-ascorbic acid will also liberate nitrophenol, though at a much lower rate (J. C. CAYGILLunpublished observations). This reaction occurs over the range p H 2.0 to 8.0. It is enhanced by the addition of Cu 2+. YAMANE, SAKAI AND IKEGUCHI4 report that the addition 62.5 ~M Cu ~+, Fe 2+, Fe ~+, Co 2+, Mn g+ or Zn 2+ but not Mg 2+, Ni ~+, A1s+ or Hg 2+ increases the rate of liberation of p-nitrophenol from p-nitrophenyl fl-D-glucuronide in the presence of L-ascorbic acid. Three questions arise from these experiments. First that if other glycosidic bonds undergo similar cleavage, care must be taken in purifying and analysing compounds such as glycoproteins from sources which m a y contain ascorbic acid. For example WEISS s reported the appearance of an additional component reacting as an enediol or Amadori compound when ascorbic acid was added to a solution of a urinary glycoprotein. Secondly whether L-ascorbic acid can safely be used as a mild hydrolytic agent for glycosides depends upon the identification of the products of the reaction. In these experiments to determine the conditions under which the glycosidic bond was split it was convenient to follow the liberation of nitrophenol from nitrophenyl glycosides. Experiments are in progress to identify the other product of the reaction. Finally this report t h a t L-ascorbic acid promotes the splitting of glycosidic bonds at a physiological p H m a y be a significant clue to the role played b y vitamin C in the body. I t is of interest t h a t tissues such as the connective tissue of skin, which are affected b y vitamin C contain glycosidic bonds in glycosaminoglycans, glycoproteins and collagen. ACKNOWLEDGEMENT I would like to t h a n k Mrs. ZEHRA ALl for her skilled technical assistance. Biochim. Biophy$. Aaa, I7o (i068) I-5
GLYCOSIDE LYSIS WITH ASCORBIC ACID
5
REFERENCES i 2 3 4 5 6
W. VAN B. ROBERTSON, M. W. ROPES AND W. BAUER, Biochem. ]., 35 (1941) 903. W. PIGMAN, Bull. Soc. Chim. Biol., 45 (1963) 185W. PIGMAN,S. RlZVI AND H. L. HOLLEY,A~'thY{ti$ Rheuma., 4 (196I) 24o. Y. YAMANE, K. SAKAI AND K. IK~GUCm, Yakugaku Zasshi, 87 (I967) 227. J. W. WOOLLEN, R. HEYWORTH AND P. G. WALKER, Biochem. J., 78 (I96I) 111. J. B. WExss, M.Sc. Thesis, University of Manchester, Manchester, I966.
Biochim. B$ophys. Aaa, 17° (1968) 1- 5
I
BBA 26040 LYSIS OF GLYCOSIDES IN T H E PRESENCE OF L-ASCORBIC ACID J. c. CAYGILL* Rheumatism Research Centre, University of Manchester, Clinical Sciences Building, Manchester, 13 (Great Britain)
(Received July Ist, I968)
SUMMARY The liberation of nitrophenol from eleven nitrophenyl glycosides in acid or neutral solution in the presence of L-ascorbic acid is reported. The conditions under which o-nitrophenol is released from o-nitrophenylfl-D-galactopyranoside are reported. The reaction rate, which is proportional to temperature and glycoside concentration, is maximal between pH 4 and 5, but negligible above pH 8.5, and is increased by Cu~+. At low concentrations of L-ascorbic acid the reaction rate is proportional to L-ascorbic acid concentration, but at higher concentrations the reaction rate is not further increased. Other monosaccharides inhibit the release of nitrophenol.
INTRODUCTION
It has been known for some time that L-ascorbic acid reduces the viscosity of hyaluronic acid sohitionsl, ~, and it has been suggested that this may involve an oxidative-reductive depolymerisation 3. Recently YAMANE, SAKAI AND IKEGUCHI4 reported the dissociation of several low molecular weight o-glucuronides in the presence of L-ascorbic acid. This splitting promoted by L-ascorbic acid has been found to be a general reaction of nitrophenyl glycosides. MATERIALS AND METHODS
L-Ascorbic acid, galactono-i,4-1actone , galactose, glucose and all the nitrophenyl glycosides used in this investigation were obtained from Koch-Light Ltd., except for p-nitrophenyl fl-D-galactopyranoside, which was supplied by B.D.H.Ltd. In all experiments, unless indicated to the contrary, buffered solutions (I.O ml) were incubated in duplicate at 37 ° for a convenient length of time, and the reaction stopped by the addition of 0.2 M sodium borate-NaOH buffer (pH 9.8) (4.0 ml). The liberated o-nitrophenol or p-nitrophenol was estimated by measuring the absorbance in a Mecolab Autocolorimeter (Joyce Loebl) using a 4oo-m/~ interference filter. Standard solutions of recrystallised o- or p-nitrophenol in water were prepared. The addition of 20 ~M Cu *+ did not affect the absorbance of light by the nitrophenol. Appropriate controls lacking L-ascorbic acid or nitrophenyl glycoside were included. ;'3
* Present address: Tropical Products Institute, 56]62, Gray's Inn Road, London, W.C.I, Great Britain. Biockim. Biophys. A~ta, 17o (I968) I- 5
2
J . c . CAYGILL
L-Ascorbic acid dit not give rise to any derivative absorbing at 4oo m/z except at ioo °. The nitrophenyl glycosides all absorbed slightly at this wavelength, and the relevant 'blank' value was subtracted from the readings to give the increase in colour representing liberated nitrophenol. No hydrolysis of the nitrophenyl glycosides used was noted after the addition of 0.2 M borate buffer, but the colorimetric measurement was made as rapidly as practicable after the reaction was stopped (in all cases within 5 h, usually within I h). When it was necessary to delay colorimetric measurement, the tubes were stored in the dark as recommended b y WOOLLEN, HEYWORTH AND WALKER5. The concentrations of the respective components given are the final concentrations in the mixtures incubated at 37 °. For each experiment the nitrophenyl glycoside and L-ascorbic acid were dissolved immediately before use. RESULTS
The liberation of o- or of p-nitrophenol was found to increase uniformly with time for at least 3 h. In the majority of these experiments it was convenient to estimate aglycone liberation in a suitable time interval (generally I h), as a measure of the initial velocity of the reaction. The results given are expressed in/~moles/h. 2.0 mM solutions of various p-nitrophenyl glycosides were incubated in 0.05 M acetic acid-sodium acetate buffer (pH 4.0 or 5.5) with 4.0 mM L-ascorbic acid and either 20 /~M, 2 /~M or no CuS04. In each case approximately equal amounts of p-nitrophenol were liberated at p H 4.0 or at 5.5, and p-nitrophenol liberation was fastest in the presence of 20 /,M Cu 2+, slower with 2 /,M Cu 2+ and slowest in the absence of Cu e+. The average rates of p-nitrophenol liberation with 2 /~M Cu 2+ are given in Table I. No p-nitrophenol was liberated in the control tubes lacking Lascorbic acid similarly incubated at 37 ° for I h, except in the case of p-nitrophenyl a-D-mannoside, where some hydrolysis occurred. The rate of liberation of p-nitrophenol from p-nitrophenyl fl-D-galactoside incubated at 37 ° in 0.05 M acetate buffer (pH 4.0) with 4.0 mM L-ascorbic acid was found to be proportional to the p-nitrophenyl galactoside concentration over the range 1-16 raM. TABLE I p-NITROPHENOL LIBERATION FROMp-NITROPH]~NYL GLYCOSIDES Average rate of p-nitrophenol liberation from 2.0 mM p-nitrophenyl glycosides incubated for I h at 37 ° in 0.o 5 M acetate buffer (pH 4.0-5.5) with 4.0 mM L-ascorbic acid and 2 #M CuSO 4" 5H20.
Glycoside
p-Nitrophenol liberated (tzmoles/h)
p-Nitrophenyl fl-L-fucoside p-Nitrophenyl fl-D-xyloside p-Nitrophenyl fl-D-galactoside p-Nitrophenyl fl-D-glucuronide p-Nitrophenyl a-L-fucoside p-Nitrophenyl a-D-glucoside p-Nitrophenyl r-D-2 -acetamido-2 -deoxyglucoside p-Nitrophenyl a-n-galactoside p-Nitrophenyl fl-D-glucoside p-Nitrophenyl a-n-mannoside
259 223 218 184 177 148 145 I 18 i 13 lO5
Biochim. Biophys. Acta, 17o (1968) 1-5
GLYCOSIDE LYSIS WITH ASCORBIC ACID
3
When 8 mM o-nitrophenyl fi-D-galactoside was incubated at 37 ° in 0.2 M acetate buffer (pH 5.5) with final concentrations of L-ascorbic acid from I to 25 mM, it was found that o-nitrophenol liberation was proportional to ascorbate concentration up to 4 mM, but at higher ascorbate concentrations the initial rate of o-nitrophenyl liberation was reduced in the absence of Cu 2+, and was not increased in the presence of 20 tzM Cu 2+ (Fig. I). A similar effect was found when 8 mM p-nitrophenyl ~-Dgalactoside was incubated in 0.05 M acetate buffer (pH 4.0) with 0.5 to 20 mM L-ascorbic acid. At these concentrations, L-ascorbic acid did not affect the pH. This indicates inhibition of the reaction rate by excess of ascorbate. A similar inhibition was found in the presence of comparable concentrations of other monosaccharides, o-Nitrophenyl fi-D-galactoside (8 mM) was incubated at 37 ° in o.2 M acetate buffer ~pH 5.5) with 4 mM L-ascorbic acid and either 20 #M, 2 #M or no Cu 2+ in the presence or absence of D-glucose, D-galactose or D-galactono-i, 4lactone. The rate of o-nitrophenol liberation was decreased approx. 20 % with IO mM monosaccharide, IO % with 5 mM monosaccharide, and very slightly by I mM monosaccharide. There was little difference in the percentage inhibition whether Cu~+ was present or not. These monosaccharides inhibited to a similar extent p-nitrophenol liberation from 8 mM p-nitrophenyl fl-D-galactoside by 4 mM L-ascorbic acid in 0.2 M acetate buffer (pH 5.0). ,
2
i
,
!
i
~
'
3o~
~2oo
"~400
,oc z
"~
o
.~
1~
~
l.-Ascorbic acid (mM)
20
25
2
~, pM
Fig. I. Variation in the rate of liberation of o-nitrophenol from 8.0 mM o-nitrophenyl/~-D-galactoside in o.2 M acetate buffer (pH 5.5) with L-ascorbic acid concentration. A k ~ k , 2o ffM Cu2+; O - - O , 2 ffM Cul+; re__m, no Cu 2+. Fig. 2. Variation with p H in the rate of liberation of o-nitrophen01 from 5.0 mM o-nitrophenyl /~-D-galactoside by 4.o mM L-ascorbic acid in citrate-phosphate buffers. ~ k - - , , 20 #M Cu2+; 0 - - 0 , 2 #M CuZ+; l - - l , no Cu z+.
The reaction was found to vary with pH. o-Nitrophenyl fl-D-galactoside (5 aM) was incubated in 0.05 M citric acid-Na2HPO 4 buffers with 4.0 mM L-ascorbic acid, and either 2o ~M, 2 ~M or no Cu 2+. The results are shown in Fig. 2. With the same final concentrations of o-nitrophenyl fl-D-galactoside, ascorbic acid and Cu 2+ in 0.o4 M veronal (HCl-sodium barbitone) buffer, o-nitrophenol was liberated at approximately the same rate in veronal buffer as in citrate phosphate buffer at pH 7.0 to 8.0, but little at pH 8.5 and none at pH 9.0. In no case did the 'blank' value of o-nitrophenyl galactoside (nor any Of: the nitrophenyl glycosides used in these Biochim. Biophy$.Acta, 17o (1968) x-5
4
J . c . CAYGILL
investigations) increase after the addition of borate buffer p H 9.8, confirming that dissociation does not occur above p H 9.0. A similar m a x i m u m rate of liberation at p H 4.5 of p-nitrophenol from 8.0 mM p-nitrophenyl fl-D-galactoside or o-nitrophenol from 8 mM o-nitrophenyl fl-D-galactoside was found when these were incubated with 4 mM L-ascorbic acid with either 20/,M, 2 ~M or no Cu 2+ in a series of 0.05 M acetate buffers of p H 3.5 to 6.0. I t was noted that in all experiments where buffers of various p H values were used, the final p H after the addition of 4.0 ml of 0.2 M borate buffer was p H 9.5-9.7- I t is interesting to note that this apparent 'optimum' at around p H 4.5 is near the pKa of ascorbic acid (4.2). The rate of liberation of o-nitrophenol from 5.0 mM o-nitrophenyl fl-Dgalactoside incubated in 0.05 M acetate buffer (pH 4.6) with 4.0 mM L-ascorbic acid and 20/zM, 2 /zM or no Cu 2+ was found to increase exponentially with temperature over the range o - I o o °. At higher temperatures it was difficult to obtain reproducible results, as the o-nitrophenyl fl-D-galactoside is hydrolysed at an appreciable rate in the absence of L-ascorbic acid. DISCUSSION
In these experiments 1-25 mM L-ascorbic acid was found to liberate nitrophenol from all the nitrophenyl glycosides tested. Lower concentrations of L-ascorbic acid will also liberate nitrophenol, though at a much lower rate (J. C. CAYGILLunpublished observations). This reaction occurs over the range p H 2.0 to 8.0. It is enhanced by the addition of Cu 2+. YAMANE, SAKAI AND IKEGUCHI4 report that the addition 62.5 ~M Cu ~+, Fe 2+, Fe ~+, Co 2+, Mn g+ or Zn 2+ but not Mg 2+, Ni ~+, A1s+ or Hg 2+ increases the rate of liberation of p-nitrophenol from p-nitrophenyl fl-D-glucuronide in the presence of L-ascorbic acid. Three questions arise from these experiments. First that if other glycosidic bonds undergo similar cleavage, care must be taken in purifying and analysing compounds such as glycoproteins from sources which m a y contain ascorbic acid. For example WEISS s reported the appearance of an additional component reacting as an enediol or Amadori compound when ascorbic acid was added to a solution of a urinary glycoprotein. Secondly whether L-ascorbic acid can safely be used as a mild hydrolytic agent for glycosides depends upon the identification of the products of the reaction. In these experiments to determine the conditions under which the glycosidic bond was split it was convenient to follow the liberation of nitrophenol from nitrophenyl glycosides. Experiments are in progress to identify the other product of the reaction. Finally this report t h a t L-ascorbic acid promotes the splitting of glycosidic bonds at a physiological p H m a y be a significant clue to the role played b y vitamin C in the body. I t is of interest t h a t tissues such as the connective tissue of skin, which are affected b y vitamin C contain glycosidic bonds in glycosaminoglycans, glycoproteins and collagen. ACKNOWLEDGEMENT I would like to t h a n k Mrs. ZEHRA ALl for her skilled technical assistance. Biochim. Biophy$. Aaa, I7o (i068) I-5
GLYCOSIDE LYSIS WITH ASCORBIC ACID
5
REFERENCES i 2 3 4 5 6
W. VAN B. ROBERTSON, M. W. ROPES AND W. BAUER, Biochem. ]., 35 (1941) 903. W. PIGMAN, Bull. Soc. Chim. Biol., 45 (1963) 185W. PIGMAN,S. RlZVI AND H. L. HOLLEY,A~'thY{ti$ Rheuma., 4 (196I) 24o. Y. YAMANE, K. SAKAI AND K. IK~GUCm, Yakugaku Zasshi, 87 (I967) 227. J. W. WOOLLEN, R. HEYWORTH AND P. G. WALKER, Biochem. J., 78 (I96I) 111. J. B. WExss, M.Sc. Thesis, University of Manchester, Manchester, I966.
Biochim. B$ophys. Aaa, 17° (1968) 1- 5