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The depolymerization of “bifidan,” a polysaccharide of Lactobacillus bifidus, by ascorbic acid
ARCHIVES
UF
RIOCHEMISTRY
AND
BIOPHYSICS
The Depolymerization of lactobacillos I\IARIAT\J
M. WANG,2
Received
November
131, 513-520 (1969)
of “Bifidan,” bifidus,
a Polysaccharide
by Ascorbic
K. C. TSOU, 12, 1968; accepted
AND
Acid’
ROBERT
February
F. NORRIS
12, 1969
The viscosity of the bifidan solutions was shown to be irreversibly decreased by ascorbic acid. This viscosity-reducing activity was proportional to the concentration of ascorbic acid and was enhanced by atmospheric oxygen. This activity was inhibited by sodium diethyldithiocarbamatc and peroxidase but only slightly by catalase. HzOz alone had little effect on the viscosity of bifidan solutions. Hydroperoxy radicals may be responsible for the viscosity reduction activity.
Malyoth and Bauer (1) and Norris et al. (2) observed that the highly viscous extracellular polysaccharide of a mucoid strain of Lactobacillus bifidus underwent loss of relative viscosity when exposed to atmospheric conditions. The latter authors ascribed the phenomenon to depolymerization. The extracellular polysaccharide was identified by paper chromatography to be composed of n-glucose, D-galactose, &deoxy-L-talose, and n-galacturonic acid (3). This polysaccharide is now named “bifidan.” Its molecular weight is estimated to be greater than two million based on data obtained from a Sepharose 2B column. An unsuccessful attempt was made to isolate an enzyme which would depolymerize the polysaccharide. The viscosity reducing activity, which was present in the uninoculated culture medium and in the dialyzable fraction of liquid cultures, was found to be ascorbic acid. The present paper describes the viscosity reducing activity of ascorbic acid on bifidan solutions and various factors affecting this activity. MATERIALS
AND
METHODS
Bifidan a-as prepared from a culture “Jackson-mucoid” strain of L. bi$dus 1 This work was supported in part Public Health Grant CA 07339. * Present address: School of Public University of California, Los Angeles, geles, California 90024.
of the by the by U.S. Health, Los An513
method previously reported (3) with the following modifications. Ascorbic acid was found not to be essential for the growth of “Jackson mucoid” and when dictated by the experiment was omitted from t.he medium. Following incubation, treatment of the culture with formalin and sparging with After filtration nitrogen were also omitted. through a thick layer of paper pulp, the culture was filtered a second time through a 0.22.rnp Millipore filter in order to remove all bacteria. The filtrate was next precipitated with 957, ethanol; the precipitated polysaccharide was dissolved in distilled water; and was deproteinized according to the Sevag method (4). Following this procedure, bifidan was precipitated from the solution with propanol (3: 1 v/v), was redispersed in distilled water, and m-as dialyzed for 72 hr. The dialyzed solution of hifidan was next passed through a 0.45.rnb Millipore filter before a final precipitation with propanol. To demonstrate the viscosity-reducing effect of ascorbic acid on solutions of hifidan, the reactions were carried out iu acetate buffer, citrate-phosphate buffer, or phosphate buffer at pH 4.5, the pH of the culture medium, or at pH 7.3 at 37” under atmospheric conditions. A typical mixtrne contained a buffer solution, 1.875 mg bifidan, and various concentrations of ascorbic acid or other red[Lcing agents and inhibitors in a final volume of 3.5 ml. \Vhen inhibitors were used ill the experiments, they were mixed with ascorbic acid before addition to the polysaccharide solrltion. \‘iscometric determinations were made in a Cannon-Zhukov viscometer, size 100. For viscosity measurement, 3.5 ml of the reaction mixtures were introduced il1t.o viscometers kept in a kinematic viscosity bat,h at 37”. The viscosities were meas-
WANG, TSOU, AND NORRIS
514
i
---,
l I I
I
2
.A I
3
1
4
Time (hrs ) FIG. 1. Effect of various concentrations of ascorbic acid on the viscosity of bifidan. Bifidan 0.7 mg/ml, citrate phosphate buffer pH 4.5. Ascorbic acid concentration A = 1.4 mM/liter B = 0.7 mu/liter C = 0.14 mu/liter D = 0.07 m&liter. ured hourly for 2-4 hr. The intrinsic viscosities of bifldan solutions were estimated by plotting Nsp/c vs. concentration (5, 6). To determine the electron paramagnetic resonance, 0.5 ml of 0.05 M solution ascorbic acid was added to 2.5 mg bifidan in 3.0 ml acetate buffer, 0.06 M/liter at pH 4.5. The reaction mixtures were frozen in liquid nitrogen 1 min after mixing and the measurements were then carried out on a Varian V-4500 instrument.3 RESULTS
The effect of increasing concentrations of ascorbic acid on reducing the relative viscosity of bifidan is shown in Fig. 1. It is seen that in 4 hr, the time-limit of the experiment, the reduction in relative viscosity is related to the concentration of the ascorbic acid. 3Ki~dly measured for LIS by Dr. H. Schleyer, Eldridge Reeves Johnson Research Foundation, University of Pennsylvania.
The reaction is characterized by a rapid initial phase followed by a phase of slower reduction in viscosity. The intrinsic viscosity of bifidan solution treated with ascorbic acid was also reduced during the course of the reaction. The degree of reduction was likewise related to the concentration of ascorbic acid (Fig. 2). In order to determine whether oxygen exerted an effect on depolymerization, the following experiment was conducted. A small glass tube, closed at one end and open at the other end, and containing 0.0009 g of ascorbic acid, was slid into the viscometer bulb which was partially filled with a solution of bifidan in acetate buffer, 0.06 M/L at pH 4.5. The atmosphere in the viscometer was then evacuated and was replaced by argon which had been passed through an 02 trap of Fieser’s solution (7). Evacuation and re-
control - [VI=
AsA 0.07mMlL
I 0.5 FIG. 2. Change in intrinsic M/liter,
viscosity pH 4.5, temp. 38’, time 2 hr.
I 1.0 of bifidan
19.5
Cql q 9.5
I 1.5 by ascorbic
I 2.0
acid. Acetate
buffer,
0.06
/L d.5
I I
115
h
Time (hrs 1 FIG. 3. Effect of oxygen on ascorbic acid viscosity reducing activity on bifidan. Bifidan 0.54 mg/ml, ascorbic acid 0.0009 g, acetate buffer, 0.06 M/liter, pH 4.5. Under anaerobic conditions, argon replaced the air. 515
516
WANG,
TSOU,
AND
placement with argon was repeated ten times. The viscometer was then stoppered, tilted, and shaken until the ascorbic acid was thoroughly mixed with the bifidan solution. The final concentration of ascorbic acid was 1.4 m/liter. In Fig. 3, it is seen that oxygen accelerates the reduction in viscosity of bifidan. In the presence of argon, presumably free of oxygen, the reaction proceeds at a much slower rate. The viscosity reducing activities of other reducing agents were compared with ascorbic acid (Fig. 4). At the end of 4 hr, ascorbic acid was more active in viscosity reduction than any of the other agents tested. During the first hour of observation, however, ferrous sulfate reduced the viscosity more than did ascorbic acid. When ascorbic acid and
NORRIS
ferrous sulfate were combined the viscosity reduction was greater than with either alone. Glutathione had little if any activity. Sodium diethyldithiocarbamate is an active inhibitor of reduction in viscosity by ascorbic acid (Fig. 5). Although the effect is slight with a concentration of 0.014 mM/liter, progressively greater inhibition is attained with increasing concentrations until almost complete inhibition results with a concentration of 1.4 mqliter. The addition of either 0.2 M NaCl or 0.2 M NaH2P04 diminished but did not completely inhibit the viscosity reducing effect of ascorbic acid (Fig. 6). NaCl inhibited more than NaH2P04. Horse radish catalase showed only a slight initial retardation of the ascorbic acid vis-
l Glutathione reduced
l -0
*
3
2 Time
(hrs
Fe*
4
+AsA
o.‘4mM
)
FIG. 4. Rate of decrease in viscosity of bifidan solution by reducing agents. Bifidan mg/ml, phosphate buffer pH 7.3, temp. 37”, concentration of reducing agents: ascorbic 0.14 mnir/liter and 0.07 mrq’liter, other reducing agents 0.14 mna/liter. Dehydroascorbic prepared by treating a solution of ascorbic acid with norite for 10 min.
0.54 acid acid
DEPOLYMERIZATION 100
OF, “BIFIDAN”
o-..._
517
e-0 DEC 1.4mM/L
80
60
4c l-
control AsA O.O7mM/L 20
I I
I
2
I
3
I
4
Time (hrs ) of the ascorbic acid viscosity reducing activity on bifidan by sodium diet,hyldithiocarbamate (DEC) in concentrations indicated. Bifidan 0.54 mg/ml, phosphate buffer pH 7.3, temp. 37”, ascorbic acid 0.07 mM/liter. FIG. 5. Inhibition
cosity reducing activity at a concentration of 10 pg/ml. At the end of 4 hr, only slight differences in the final viscosity of solutions of bifidan treated with ascorbic acid alone and with ascorbic acid in combination with the various indicated concentrations of catalase were observed (Fig. 7). Horse radish peroxidase, by contrast, did inhibit the viscosity reducing activity of ascorbic acid. Within the limits of the experiment the greater the concentration, the greater the inhibition (Fig. 7). To determine whether Hz02 also affected the viscosity reducing activity of the ascorbic acid, 30% HzOz was added to solutions of bifidan and ascorbic acid as follows: Bifidan 0.54 mg/ml, ascorbic acid 0.07 m&I/liter, Hz04 12.5 mM/liter, or Hz02 0.9 x/liter.
Ninety
minutes
after
addition
of
ascorbic acid and H202, the specific viscosities were:
&P Control Ascorbic acid 0.07 mM/liter Hz02 12.5 mM/liter Hz02 0.9 M/liter
1.81 0.45
1.75 1.14
Thus, Hz02 has very little direct viscosity reducing effect on bifidan. The electron paramagnetic resonance spectrum indicated that only a very low concentration of free radical was present in the reaction mixture. The free radical concentration was estimated to be less than 2 X lo-’ 11.Since the concentration of ascorbic acid was 1.5 X 1O-3 M, the ratio of free radical to ascorbic acid was less than 10-4. DISCUSSIOPJ
Under the experimental conditions used in this study, ascorbic acid was found to be a
WANG, TSOU, AND NORRIS
80.
1
I 2
I 3
I 4
Time (hrs) FIG. 6. Reduction of viscosity of bifidan by ascorbic acid in the presence of chloride ions and phosphate ions. Bifidan 0.54 mg/ml, 0.2 M sodium chloride, 0.2 M NaHgPOd, ascorbic acid 0.7 m&liter, acetate buffer, 0.06 M/liter, pH 4.5, temp. 37”.
strong degrading agent of bifidan. This is evident from the increasingly greater reduction in specific viscosity of the polysaccharide solution as the concentration of ascorbic acid is increased. Herp et al. (8) have recently reported that L-ascorbic acid depolymerized some polysaccharides and synthetic polymers. The presence of carboxyl groups or ester groups in the macromolecule appeared to be necessary for the ascorbic acid to degrade the polysaccharide. Since bifidan contains galacturonic acid and therefore carboxyl groups, depolymerization by ascorbic acid may be effected by a similar reaction. The viscosity reducing activity of ascorbic acid observed in this study also resembles the oxidative-reductive depolymerization of hyaluronic acid described earlier by Pigman et al. (9). The main
features of their observation were: (1) Participation of a small amount of O2 in the reaction; (2) loss of activity of the reducing agents due to oxidation; (3) decreasing inhibitory effect with the addition of halides, I > Br > Cl, and (4) acceleration of the reaction by phosphate and fluoride ions. Like hyaluronic acid the viscosity reducing activity of ascorbic acid on bifidan was inhibited by diethyldithiocarbamate and to a less extent by chloride ions. In contrast to hyaluronic acid, phosphate ions were also slightly inhibitory. Hale (10) likewise reported that inorganic phosphate had little effect on the viscosity reduction of hyaluronic acid by ascorbic acid. Depolymerization of hyaluronic acid by ascorbic acid appears to be dependent upon the presence of oxygen. According to Pigman
DEPOLYMERIZATION
et al. (11) the effect of oxygen can be demonstrated with as little as 2 to 3 parts per million. In the present study, ascorbic acid also depolymerized bifidan in the presence of oxygen, but did so more slowly when air was replaced with argon. Although argon was used instead of nitrogen because of the belief that it is less likely to be contaminated with oxygen, the presence of small quantities of oxygen cannot be excluded. It has not been proved, therefore, that depolymerization of bifidan by ascorbic acid can occur in the a.bsence of oxygen. At pH 7.2 practically all of the ascorbic acid is present as the monovalent ion which reacts with molecular O2 at a finite rate and above pH 7.2 the divalent ion is responsible for the oxygen absorption. The reaction of the monovalent ion is independent of the 02
519
OF “BIFIDAN”
pressure. Weissberger et al. (12) previously suggested that the monovalent ion of ascorbic acid must undergo an ill-defined process before it reacts with molecular OZ. This ratedetermining step not involving O2 may be the key to the viscosity-reducing activity of ascorbic acid under anaerobic or microaerophilic conditions. Ascorbic acid is a well known autoxidant. The mechanism of autoxidation by reducing agents in aqueous media is generally considered to be that of a free radical reaction (11). The participation of free radical in the depolymerization of some polysaccharides by ascorbic acid has also been postulated (8, 13). Therefore, it was appropriate for us to examine the electron spin resonance spectrum of a reaction mixture of ascorbic acid and bifidan. The study indicated the A-*--A o---O e-0
control cotalase IOug/ml catalase 5ug/ml HRP20ug/ml HRP IOug/ml HRP 5ug/ml
m-m 0 -0 m--m
I
I I
I
2
I
3
r
4
Time (hrs 1 FIG. 7. Effect of catalase and horse radish peroxidase (HRP) on ascorbic reducing activity. Bifidan 0.54 mg/ml, ascorbic acid 7 mM/liter, phosphate temp. 37”. Catalase and HRP in concentrations indicated.
acid viscosity buffer pH 7.3,
520
WANG,
TSOU,
presence of only a very low concentration of free radicals. In spite of this finding, the formation of very short-lived radicals as hydroxyl radicals or hydroperoxy radicals, not detectable under the experimental conditions of this study, is possible. Ascorbic acid itself showed a very low concentration of free radicals in acetate buffer pH 4.5. This was in agreement with the observation by Yamazaki and Piette (14) that no free radical was detected from the mixture of ascorbate and dehydroascorbate at pH 4.8. These authors found that ascorbic acid free radicals are formed during the peroxidase reaction in the presence of HzOz. If ascorbic acid free radicals are involved in the reaction, the peroxidase experiments should demonstrate an acceleration of the viscosity reducing activity of ascorbic acid. Our results showed, however, that peroxidase inhibited the viscosity-reducing activity. It is unlikely, therefore, that ascorbic acid free radical is involved in this system. Ascorbic acid autoxidizes to form HzOz. Since catalase has been found to be a strong inhibitor of the ascorbic acid system, the participation of HzOz in the degradation reaction was suggested (13, 15). Our results show that catalase has very little effect on the viscosity-reducing activity of ascorbic acid. The catalase activity probably was inhibited by ascorbic acid. It has been demonstrated that Na ascorbate completely inhibits the in vitro decomposition of HzOz by catalase at a low substrate concentration but the peroxidase-like activity of the catalase is retained (16). However, HzOz plays only a very minor part in reducing the viscosity of bifidan. Robertson et al. (15) reported that Hz02 accelerates the breakdown of mucin by ascorbic acid, but HzOz, when present without ascorbic acid, caused no change in mucin. Mucin has a protein component, however, as well as the polysaccharide moiety. Alexander and Fox (17) found very little if any degradation of polymethacrylic acid by ascorbic acid in the absence of oxygen. They favor the view that the .OOH radical rather than the OH radical is responsible for degradation of the polymer. Norman and Radda (18) showed that a mixture of ferrous ion, oxygen, and ascorbic
AND
NORRIS
acid employed to simulate metabolic hydroxylations yields hydroperoxy radicals and not hydroxy radicals. Rickards, Herp, and Pigman (19), on the other hand, cited evidence that the diminished viscosity of hyaluronic acid in the presence of ascorbic acid is also due to a free radical, but this is most likely the OH radical. In the present experiments the lack of inhibition by catalase and, in contrast, the strong inhibition of the viscosity-reducing activity of ascorbic acid by peroxidase favors .OOH as the active radical. REFERENCES 1. MALYOTH, G., AND BAUER, A., 2. Biol. 104, 404 (1951). 2. NORRIS, R. F., DE SIPIN, M., ZILLIKEN, F. W. HARVEY, T. S., AND GY~RGY, P., J. Bacterial. 67, 159 (1954). 3. WANG, M., STEERS, E., AND NORRIS, R. F., J. Bacterial. 86, 898 (1963). 4. STAUB, A. M. in “Methods in Carbohydrate Chemistry” (R. L. Whistler, ed.), Vol. 5, p. 5. Academic Press, New York (1965). HUGGINS, M. L., J. Am. Chem. Sot. 64, 2716 (1942). SUNDBLAD, L., Acta Sot. Med. Upsalien. 68, 113 (1953). FIESER, L. F., J. Am. Chem. Sot. 46, 2639 (1924). HERP, A., RICIURDS, T., MATSUMURA, G., JAKOSALEM, L. B., AND PIGMAN, W., Carbohydrate Res. 4. 63 (1967). 9. PIGMAN, W., RIZVI, S., AND HOLLEY, H. L., Arthritis Rheumat. 4, 240 (1961). 10. HALE, C. W., Biochem. J. 38, 362 (1944). 11. PIGMAN, W., MATSUMURA, G., AND HERP, A., in “Symposium on Biorheology” (A. L. Copley, ed.), p. 505, Wiley, New York (1965). 12. WEISSBERGER, A., LUVALLE, J. E., AND THOMAS, D. S., JR., J. Am. Chem. Sot. 66, 1934 (1943). 13. MATSIJMKJRA, G., AND PIGMAN, W., Arch. Biochem. Biophys. 110, 526 (1965). 14. YAMAZAKI, I., AND PIETTE, L. H., Biochem. Biophys. Acta 60, 62 (1961). 15. ROBERTSON, W. V. B., ROPES, M. W., AND BAUER, W., Biochem. J. 36, 903 (1941). 16. KOVACS, E., AND MAZAREAN, H. H., Arzneimiftel-Forsch. 17, 761 (1967). 17. ALEXANDER, P., AND Fox, M., J. Polymer Sci. 33, 493 (1958). 18. NORMAN, R. 0. C., AND RADDA, G. K., Proc. Chem. Sot. 1962, 138 (1962). 19. RICKARDS, T., HERP, A., .\ND PIGMAN, W., J. Polymer Sci. 6, 931 (1967).
UF
RIOCHEMISTRY
AND
BIOPHYSICS
The Depolymerization of lactobacillos I\IARIAT\J
M. WANG,2
Received
November
131, 513-520 (1969)
of “Bifidan,” bifidus,
a Polysaccharide
by Ascorbic
K. C. TSOU, 12, 1968; accepted
AND
Acid’
ROBERT
February
F. NORRIS
12, 1969
The viscosity of the bifidan solutions was shown to be irreversibly decreased by ascorbic acid. This viscosity-reducing activity was proportional to the concentration of ascorbic acid and was enhanced by atmospheric oxygen. This activity was inhibited by sodium diethyldithiocarbamatc and peroxidase but only slightly by catalase. HzOz alone had little effect on the viscosity of bifidan solutions. Hydroperoxy radicals may be responsible for the viscosity reduction activity.
Malyoth and Bauer (1) and Norris et al. (2) observed that the highly viscous extracellular polysaccharide of a mucoid strain of Lactobacillus bifidus underwent loss of relative viscosity when exposed to atmospheric conditions. The latter authors ascribed the phenomenon to depolymerization. The extracellular polysaccharide was identified by paper chromatography to be composed of n-glucose, D-galactose, &deoxy-L-talose, and n-galacturonic acid (3). This polysaccharide is now named “bifidan.” Its molecular weight is estimated to be greater than two million based on data obtained from a Sepharose 2B column. An unsuccessful attempt was made to isolate an enzyme which would depolymerize the polysaccharide. The viscosity reducing activity, which was present in the uninoculated culture medium and in the dialyzable fraction of liquid cultures, was found to be ascorbic acid. The present paper describes the viscosity reducing activity of ascorbic acid on bifidan solutions and various factors affecting this activity. MATERIALS
AND
METHODS
Bifidan a-as prepared from a culture “Jackson-mucoid” strain of L. bi$dus 1 This work was supported in part Public Health Grant CA 07339. * Present address: School of Public University of California, Los Angeles, geles, California 90024.
of the by the by U.S. Health, Los An513
method previously reported (3) with the following modifications. Ascorbic acid was found not to be essential for the growth of “Jackson mucoid” and when dictated by the experiment was omitted from t.he medium. Following incubation, treatment of the culture with formalin and sparging with After filtration nitrogen were also omitted. through a thick layer of paper pulp, the culture was filtered a second time through a 0.22.rnp Millipore filter in order to remove all bacteria. The filtrate was next precipitated with 957, ethanol; the precipitated polysaccharide was dissolved in distilled water; and was deproteinized according to the Sevag method (4). Following this procedure, bifidan was precipitated from the solution with propanol (3: 1 v/v), was redispersed in distilled water, and m-as dialyzed for 72 hr. The dialyzed solution of hifidan was next passed through a 0.45.rnb Millipore filter before a final precipitation with propanol. To demonstrate the viscosity-reducing effect of ascorbic acid on solutions of hifidan, the reactions were carried out iu acetate buffer, citrate-phosphate buffer, or phosphate buffer at pH 4.5, the pH of the culture medium, or at pH 7.3 at 37” under atmospheric conditions. A typical mixtrne contained a buffer solution, 1.875 mg bifidan, and various concentrations of ascorbic acid or other red[Lcing agents and inhibitors in a final volume of 3.5 ml. \Vhen inhibitors were used ill the experiments, they were mixed with ascorbic acid before addition to the polysaccharide solrltion. \‘iscometric determinations were made in a Cannon-Zhukov viscometer, size 100. For viscosity measurement, 3.5 ml of the reaction mixtures were introduced il1t.o viscometers kept in a kinematic viscosity bat,h at 37”. The viscosities were meas-
WANG, TSOU, AND NORRIS
514
i
---,
l I I
I
2
.A I
3
1
4
Time (hrs ) FIG. 1. Effect of various concentrations of ascorbic acid on the viscosity of bifidan. Bifidan 0.7 mg/ml, citrate phosphate buffer pH 4.5. Ascorbic acid concentration A = 1.4 mM/liter B = 0.7 mu/liter C = 0.14 mu/liter D = 0.07 m&liter. ured hourly for 2-4 hr. The intrinsic viscosities of bifldan solutions were estimated by plotting Nsp/c vs. concentration (5, 6). To determine the electron paramagnetic resonance, 0.5 ml of 0.05 M solution ascorbic acid was added to 2.5 mg bifidan in 3.0 ml acetate buffer, 0.06 M/liter at pH 4.5. The reaction mixtures were frozen in liquid nitrogen 1 min after mixing and the measurements were then carried out on a Varian V-4500 instrument.3 RESULTS
The effect of increasing concentrations of ascorbic acid on reducing the relative viscosity of bifidan is shown in Fig. 1. It is seen that in 4 hr, the time-limit of the experiment, the reduction in relative viscosity is related to the concentration of the ascorbic acid. 3Ki~dly measured for LIS by Dr. H. Schleyer, Eldridge Reeves Johnson Research Foundation, University of Pennsylvania.
The reaction is characterized by a rapid initial phase followed by a phase of slower reduction in viscosity. The intrinsic viscosity of bifidan solution treated with ascorbic acid was also reduced during the course of the reaction. The degree of reduction was likewise related to the concentration of ascorbic acid (Fig. 2). In order to determine whether oxygen exerted an effect on depolymerization, the following experiment was conducted. A small glass tube, closed at one end and open at the other end, and containing 0.0009 g of ascorbic acid, was slid into the viscometer bulb which was partially filled with a solution of bifidan in acetate buffer, 0.06 M/L at pH 4.5. The atmosphere in the viscometer was then evacuated and was replaced by argon which had been passed through an 02 trap of Fieser’s solution (7). Evacuation and re-
control - [VI=
AsA 0.07mMlL
I 0.5 FIG. 2. Change in intrinsic M/liter,
viscosity pH 4.5, temp. 38’, time 2 hr.
I 1.0 of bifidan
19.5
Cql q 9.5
I 1.5 by ascorbic
I 2.0
acid. Acetate
buffer,
0.06
/L d.5
I I
115
h
Time (hrs 1 FIG. 3. Effect of oxygen on ascorbic acid viscosity reducing activity on bifidan. Bifidan 0.54 mg/ml, ascorbic acid 0.0009 g, acetate buffer, 0.06 M/liter, pH 4.5. Under anaerobic conditions, argon replaced the air. 515
516
WANG,
TSOU,
AND
placement with argon was repeated ten times. The viscometer was then stoppered, tilted, and shaken until the ascorbic acid was thoroughly mixed with the bifidan solution. The final concentration of ascorbic acid was 1.4 m/liter. In Fig. 3, it is seen that oxygen accelerates the reduction in viscosity of bifidan. In the presence of argon, presumably free of oxygen, the reaction proceeds at a much slower rate. The viscosity reducing activities of other reducing agents were compared with ascorbic acid (Fig. 4). At the end of 4 hr, ascorbic acid was more active in viscosity reduction than any of the other agents tested. During the first hour of observation, however, ferrous sulfate reduced the viscosity more than did ascorbic acid. When ascorbic acid and
NORRIS
ferrous sulfate were combined the viscosity reduction was greater than with either alone. Glutathione had little if any activity. Sodium diethyldithiocarbamate is an active inhibitor of reduction in viscosity by ascorbic acid (Fig. 5). Although the effect is slight with a concentration of 0.014 mM/liter, progressively greater inhibition is attained with increasing concentrations until almost complete inhibition results with a concentration of 1.4 mqliter. The addition of either 0.2 M NaCl or 0.2 M NaH2P04 diminished but did not completely inhibit the viscosity reducing effect of ascorbic acid (Fig. 6). NaCl inhibited more than NaH2P04. Horse radish catalase showed only a slight initial retardation of the ascorbic acid vis-
l Glutathione reduced
l -0
*
3
2 Time
(hrs
Fe*
4
+AsA
o.‘4mM
)
FIG. 4. Rate of decrease in viscosity of bifidan solution by reducing agents. Bifidan mg/ml, phosphate buffer pH 7.3, temp. 37”, concentration of reducing agents: ascorbic 0.14 mnir/liter and 0.07 mrq’liter, other reducing agents 0.14 mna/liter. Dehydroascorbic prepared by treating a solution of ascorbic acid with norite for 10 min.
0.54 acid acid
DEPOLYMERIZATION 100
OF, “BIFIDAN”
o-..._
517
e-0 DEC 1.4mM/L
80
60
4c l-
control AsA O.O7mM/L 20
I I
I
2
I
3
I
4
Time (hrs ) of the ascorbic acid viscosity reducing activity on bifidan by sodium diet,hyldithiocarbamate (DEC) in concentrations indicated. Bifidan 0.54 mg/ml, phosphate buffer pH 7.3, temp. 37”, ascorbic acid 0.07 mM/liter. FIG. 5. Inhibition
cosity reducing activity at a concentration of 10 pg/ml. At the end of 4 hr, only slight differences in the final viscosity of solutions of bifidan treated with ascorbic acid alone and with ascorbic acid in combination with the various indicated concentrations of catalase were observed (Fig. 7). Horse radish peroxidase, by contrast, did inhibit the viscosity reducing activity of ascorbic acid. Within the limits of the experiment the greater the concentration, the greater the inhibition (Fig. 7). To determine whether Hz02 also affected the viscosity reducing activity of the ascorbic acid, 30% HzOz was added to solutions of bifidan and ascorbic acid as follows: Bifidan 0.54 mg/ml, ascorbic acid 0.07 m&I/liter, Hz04 12.5 mM/liter, or Hz02 0.9 x/liter.
Ninety
minutes
after
addition
of
ascorbic acid and H202, the specific viscosities were:
&P Control Ascorbic acid 0.07 mM/liter Hz02 12.5 mM/liter Hz02 0.9 M/liter
1.81 0.45
1.75 1.14
Thus, Hz02 has very little direct viscosity reducing effect on bifidan. The electron paramagnetic resonance spectrum indicated that only a very low concentration of free radical was present in the reaction mixture. The free radical concentration was estimated to be less than 2 X lo-’ 11.Since the concentration of ascorbic acid was 1.5 X 1O-3 M, the ratio of free radical to ascorbic acid was less than 10-4. DISCUSSIOPJ
Under the experimental conditions used in this study, ascorbic acid was found to be a
WANG, TSOU, AND NORRIS
80.
1
I 2
I 3
I 4
Time (hrs) FIG. 6. Reduction of viscosity of bifidan by ascorbic acid in the presence of chloride ions and phosphate ions. Bifidan 0.54 mg/ml, 0.2 M sodium chloride, 0.2 M NaHgPOd, ascorbic acid 0.7 m&liter, acetate buffer, 0.06 M/liter, pH 4.5, temp. 37”.
strong degrading agent of bifidan. This is evident from the increasingly greater reduction in specific viscosity of the polysaccharide solution as the concentration of ascorbic acid is increased. Herp et al. (8) have recently reported that L-ascorbic acid depolymerized some polysaccharides and synthetic polymers. The presence of carboxyl groups or ester groups in the macromolecule appeared to be necessary for the ascorbic acid to degrade the polysaccharide. Since bifidan contains galacturonic acid and therefore carboxyl groups, depolymerization by ascorbic acid may be effected by a similar reaction. The viscosity reducing activity of ascorbic acid observed in this study also resembles the oxidative-reductive depolymerization of hyaluronic acid described earlier by Pigman et al. (9). The main
features of their observation were: (1) Participation of a small amount of O2 in the reaction; (2) loss of activity of the reducing agents due to oxidation; (3) decreasing inhibitory effect with the addition of halides, I > Br > Cl, and (4) acceleration of the reaction by phosphate and fluoride ions. Like hyaluronic acid the viscosity reducing activity of ascorbic acid on bifidan was inhibited by diethyldithiocarbamate and to a less extent by chloride ions. In contrast to hyaluronic acid, phosphate ions were also slightly inhibitory. Hale (10) likewise reported that inorganic phosphate had little effect on the viscosity reduction of hyaluronic acid by ascorbic acid. Depolymerization of hyaluronic acid by ascorbic acid appears to be dependent upon the presence of oxygen. According to Pigman
DEPOLYMERIZATION
et al. (11) the effect of oxygen can be demonstrated with as little as 2 to 3 parts per million. In the present study, ascorbic acid also depolymerized bifidan in the presence of oxygen, but did so more slowly when air was replaced with argon. Although argon was used instead of nitrogen because of the belief that it is less likely to be contaminated with oxygen, the presence of small quantities of oxygen cannot be excluded. It has not been proved, therefore, that depolymerization of bifidan by ascorbic acid can occur in the a.bsence of oxygen. At pH 7.2 practically all of the ascorbic acid is present as the monovalent ion which reacts with molecular O2 at a finite rate and above pH 7.2 the divalent ion is responsible for the oxygen absorption. The reaction of the monovalent ion is independent of the 02
519
OF “BIFIDAN”
pressure. Weissberger et al. (12) previously suggested that the monovalent ion of ascorbic acid must undergo an ill-defined process before it reacts with molecular OZ. This ratedetermining step not involving O2 may be the key to the viscosity-reducing activity of ascorbic acid under anaerobic or microaerophilic conditions. Ascorbic acid is a well known autoxidant. The mechanism of autoxidation by reducing agents in aqueous media is generally considered to be that of a free radical reaction (11). The participation of free radical in the depolymerization of some polysaccharides by ascorbic acid has also been postulated (8, 13). Therefore, it was appropriate for us to examine the electron spin resonance spectrum of a reaction mixture of ascorbic acid and bifidan. The study indicated the A-*--A o---O e-0
control cotalase IOug/ml catalase 5ug/ml HRP20ug/ml HRP IOug/ml HRP 5ug/ml
m-m 0 -0 m--m
I
I I
I
2
I
3
r
4
Time (hrs 1 FIG. 7. Effect of catalase and horse radish peroxidase (HRP) on ascorbic reducing activity. Bifidan 0.54 mg/ml, ascorbic acid 7 mM/liter, phosphate temp. 37”. Catalase and HRP in concentrations indicated.
acid viscosity buffer pH 7.3,
520
WANG,
TSOU,
presence of only a very low concentration of free radicals. In spite of this finding, the formation of very short-lived radicals as hydroxyl radicals or hydroperoxy radicals, not detectable under the experimental conditions of this study, is possible. Ascorbic acid itself showed a very low concentration of free radicals in acetate buffer pH 4.5. This was in agreement with the observation by Yamazaki and Piette (14) that no free radical was detected from the mixture of ascorbate and dehydroascorbate at pH 4.8. These authors found that ascorbic acid free radicals are formed during the peroxidase reaction in the presence of HzOz. If ascorbic acid free radicals are involved in the reaction, the peroxidase experiments should demonstrate an acceleration of the viscosity reducing activity of ascorbic acid. Our results showed, however, that peroxidase inhibited the viscosity-reducing activity. It is unlikely, therefore, that ascorbic acid free radical is involved in this system. Ascorbic acid autoxidizes to form HzOz. Since catalase has been found to be a strong inhibitor of the ascorbic acid system, the participation of HzOz in the degradation reaction was suggested (13, 15). Our results show that catalase has very little effect on the viscosity-reducing activity of ascorbic acid. The catalase activity probably was inhibited by ascorbic acid. It has been demonstrated that Na ascorbate completely inhibits the in vitro decomposition of HzOz by catalase at a low substrate concentration but the peroxidase-like activity of the catalase is retained (16). However, HzOz plays only a very minor part in reducing the viscosity of bifidan. Robertson et al. (15) reported that Hz02 accelerates the breakdown of mucin by ascorbic acid, but HzOz, when present without ascorbic acid, caused no change in mucin. Mucin has a protein component, however, as well as the polysaccharide moiety. Alexander and Fox (17) found very little if any degradation of polymethacrylic acid by ascorbic acid in the absence of oxygen. They favor the view that the .OOH radical rather than the OH radical is responsible for degradation of the polymer. Norman and Radda (18) showed that a mixture of ferrous ion, oxygen, and ascorbic
AND
NORRIS
acid employed to simulate metabolic hydroxylations yields hydroperoxy radicals and not hydroxy radicals. Rickards, Herp, and Pigman (19), on the other hand, cited evidence that the diminished viscosity of hyaluronic acid in the presence of ascorbic acid is also due to a free radical, but this is most likely the OH radical. In the present experiments the lack of inhibition by catalase and, in contrast, the strong inhibition of the viscosity-reducing activity of ascorbic acid by peroxidase favors .OOH as the active radical. REFERENCES 1. MALYOTH, G., AND BAUER, A., 2. Biol. 104, 404 (1951). 2. NORRIS, R. F., DE SIPIN, M., ZILLIKEN, F. W. HARVEY, T. S., AND GY~RGY, P., J. Bacterial. 67, 159 (1954). 3. WANG, M., STEERS, E., AND NORRIS, R. F., J. Bacterial. 86, 898 (1963). 4. STAUB, A. M. in “Methods in Carbohydrate Chemistry” (R. L. Whistler, ed.), Vol. 5, p. 5. Academic Press, New York (1965). HUGGINS, M. L., J. Am. Chem. Sot. 64, 2716 (1942). SUNDBLAD, L., Acta Sot. Med. Upsalien. 68, 113 (1953). FIESER, L. F., J. Am. Chem. Sot. 46, 2639 (1924). HERP, A., RICIURDS, T., MATSUMURA, G., JAKOSALEM, L. B., AND PIGMAN, W., Carbohydrate Res. 4. 63 (1967). 9. PIGMAN, W., RIZVI, S., AND HOLLEY, H. L., Arthritis Rheumat. 4, 240 (1961). 10. HALE, C. W., Biochem. J. 38, 362 (1944). 11. PIGMAN, W., MATSUMURA, G., AND HERP, A., in “Symposium on Biorheology” (A. L. Copley, ed.), p. 505, Wiley, New York (1965). 12. WEISSBERGER, A., LUVALLE, J. E., AND THOMAS, D. S., JR., J. Am. Chem. Sot. 66, 1934 (1943). 13. MATSIJMKJRA, G., AND PIGMAN, W., Arch. Biochem. Biophys. 110, 526 (1965). 14. YAMAZAKI, I., AND PIETTE, L. H., Biochem. Biophys. Acta 60, 62 (1961). 15. ROBERTSON, W. V. B., ROPES, M. W., AND BAUER, W., Biochem. J. 36, 903 (1941). 16. KOVACS, E., AND MAZAREAN, H. H., Arzneimiftel-Forsch. 17, 761 (1967). 17. ALEXANDER, P., AND Fox, M., J. Polymer Sci. 33, 493 (1958). 18. NORMAN, R. 0. C., AND RADDA, G. K., Proc. Chem. Sot. 1962, 138 (1962). 19. RICKARDS, T., HERP, A., .\ND PIGMAN, W., J. Polymer Sci. 6, 931 (1967).