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Ficin-catalyzed hydrolysis of elastin
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Ficin-Catalyzed EMETERIA From the Department
97,
122-127 (1962)
Hydrolysis
YATCO-MANZO
AND
of Elastin J. R. WHITAKER
of Food Science and Technology, University Davis, California’
of California,
Received October 16, 1961 Ficin hydrolyzes elastin quite rapidly with a pH optimum of 5.0-5.5 and a temperature optimum (under conditions used) of 55”. Maximal action of ficin on elastin produces complete solubilization and a splitting of approximately 25% of the peptide bonds involving a-amino acids. Activation energy for the ficin-catalyzed splitting of these bonds is 5.8 kcal./mole. INTRODUCTION
In 1950 Ba16 and Banga (1) first reported the partial purification of a pancreatic elastase and later postulated a possible connection between enzyme level and atherosclerosis (2). Subsequent work indicated the solubilizing action of this enzyme on elastin might be due to its mucolytic activity (3), lipolytic activity (4)) or sulfatase activity (5), although Partridge and Davis (6) concluded that it is due to splitting of peptide bonds. The enzyme was crystallized in 1956 by Lewis et al. (7) and was found to hydrolyze a variety of proteins. The crystalline enzyme was later separated into mucolytic and proteolytic components which were inactive on native elastin separately (8). Elastin-hydrolyzing enzymes have also been isolated from microorganisms (9-11). Mandl and Cohen (10) isolated an elastase from Plauobacterium elastolyticum which appears to be more specific than pancreatic elastase. The plant proteolytic enzymes, papain (7, 11-14)) bromelin, and ficin (1214) also have elastolytic activity. Thomas and Partridge (14) reported that crystalline trypsin, chymotrypsin, pepsin, Rhozyme, and cathepsin from kidney and spleen were inactive on elastin. Elastolytic activity of plant proteolytic enzymes has not been studied in detail. In * This work was supported in part by a research grant, RG-5295, from the National Institutes of Health.
fact, it has not been shown directly that they soluhilize elastin by splitting peptide bonds, as methods which measured only the change in solubility of elastin were used (7, 11-14). The object of the present paper is to define the conditions which influence the elastolytic activity of ficin and to show that it solubilizes elastin by extensive splitting of peptide bonds. MATERIALS
AND METHODS
MATERIALS Elastin was prepared from the ligamentum nuchae of cattle by the method of Partridge, Davis, and Adair (15). The final purified elastin was freeze-dried, mixed with a large amount of Dry Ice, and passed rapidly through a Wiley Mill using a 20-mesh screen. Nitrogen content was 16.33% determined by Method B described by Ballentine (16). Ficin (Merck and Company) was used without fractionation as Thomas and Partridge (14) have found that ammonium sulfate fractionation and chromatography on carboxymethylcellulose failed to show any indication of separating proteolytic activity from elastolytic activity. This material contained 4.94% water and 79.1% protein on a moisture-free basis [biuret method (17)]. Solutions (4%) were prepared as described by Whitaker (18) and stored frozen. Samples were thawed just before use, and the excess was discarded. ASSAY OF ACTIVITY Elastin was weighed on a microbalance into 13 X 100 mm. tubes. Unless the substituent was the variable, each tube contained 20.0 mg. elastin, 1.25 X lo-’ M each cysteine (or mercaptoethanol), and 122
HYDROLYSIS. Versene as activators, 0.2 144,pH 5.5, acetate buffer, and water to a total of 2 ml. when 1.19 mg. enzyme protein was added. After temperature equilibration at 35.0” (except when temperature was variable), enzyme was added to initiate the reaction, and the contents were stirred intermittently throughout the incubation time of 30 min. It was established that amount of stirring did not influence extent of reaction. pH was determined on separate reaction mixtures using a Beckman model G pH meter. Reaction was terminated by addition of 3 ml. of 1 N HCl. Extent of reaction was determined either gravimetrically by the method of Ba16 and Banga (l), by change in absorbance of supernatant liquid at 280 rnp, by nitrogen content of supernatant liquid (19), by ninhydrin assay of supernatant liquid (20), or by a combination of these methods. Reported values are averages of two to five experiments.
50-
I
133
OF ELASTIN RESULTS
EFFECTOF PH Effect of pH on hydrolysis of elastin by ficin is shown in Fig. 1. The pH optimum is found at pH 5.0-5.5 by all four methods of assay. It is near pH 7 for casein and gelatin hydrolysis by ficin (21, 22). Grarimetric, nitrogen content, and absorption at 280 mp methods all gave identical results for the amount solubilixed. The ninhydrin assay data indicate a splitting of approximately 18% of the peptide bonds involving a-amino acids of the solubilized elastin at pH 5.5 under conditions used. If an elastase unit (E.U.) is arbitrarily defined as amount of enzyme which will solubilize 1.0 mg. elastin in 30 min. (1) , ficin has a value of I
I
I
8.0
IO
40-
30.!? v) )r 0 0 5
20-
IO-
oLIL 2.0
4.0
6.0
PH
FIG. 1. Effect of pH on hydrolysis of elastin at 35.0" and 30 min. by ficin as measured by ninhydrin method (O), gravimetrically (0 ), supernatant nitrogen content (X), and absorbance of supernatant liquid at 280 rnp (+). Buffer was 0.2 M with respect to each acetate, phosphate, and borate.
124
YATCO-MANZO
AND WHITAKER
TIME (minutes) 00
FIG. 2. Effect of ficin concentration (0, X) and time ( 0 ) on hydrolysis of elastin at pH 5.5 and 35.0”. Activity was measured by ninhydrin method (0, 0 ) and absorbance of supernatant liquid at 280 rnp (X 1.
8.0 E.U./mg. protein at pH 5.5 and 35” and 1.68 E.U./mg. protein at pH 8.0 and 35”. These values will vary with conditions used as the extent of solubilization is not a linear function of ficin concentration (Fig. 2) and is dependent upon the elastin concentration (Fig. 3). Other workers have reported the following values at 3737.5”: crystalline pancreatic elastase, 106-112 E.U./mg. protein at PH 8.7-8.8 (7) and 61.3 E.U./mg. protein at pH 8.7 (23) ; purified Flavobacterium elnstolyticum elastase, 3.1 E.U./mg. enzyme at pH 7.4 (10) ; papain, 3.7 E.U./mg. enzyme at pH 8.8 (7) and 5.0 E.U./mg. enzyme at pH 8.0 (14) ; and ficin, 1.86 E.U./mg. enzyme at pH 8.0 (14).
EFFECT OF FICIN AND ELASTIN CONCENTRATIONS AND RATE OF REACTION
Effect of ficin concentration on hydrolysis of elastin is shown in Fig. 2. At pH 5.5 and 35”, 8.0 mg. enzyme protein is able to completely solubilize 20 mg. elastin in 30 min. Extent of reaction is not a linear function of ficin concentration but is also dependent upon substrate concentration as shown by Fig. 3. There is no indication of the reaction b ecoming independent of substrate concentration up to 75 mg. elastin/l.l9 mg. enzyme protein. Since reaction times of 180 and 240 min. also gave 25% splitting of peptide bonds involving or-amino acids, it would appear this is the maximum degree of hydrolysis of elastin by ficin, a rather exten-
HYDROLYSIS
125
OF ELASTIN
sive degradation. Degree of hydrolysis may be largely correlated with percentage glycine in cattle ligamenturn nuchae elastin [27,0%, ref. (6)] as ficin is known to hydrolyze peptide bonds involving glycine (24). Ficin will also split linkages involving arginine (25)) but, there is only 1.2% arginine in elastin (6). EFFECT OF ACTIVATOR CONCENTRATION Effect of cysteine and mercaptoethanol on hydrolysis of elastin by ficin is shown in Fig. 4. It is surprising to find that these cornpounds produce very little increase in amount of hydrolysis (approx. 20% maximum) as the ficin-catalyzed hydrolysis of casein is increased approximately fivefold by addition of 1.25 x 1O-2 M cysteine to the same ficin used in these experiments. Prior incubation of ficin with 1 x lo-” M iodoacetate for 30 min. completely destroys its elastolytic activity as well as proteolytic activity (casein as substrate). Cysteinc above 1.25 x 1O-2 M markedly inhibits the solubilization of elastin as measured by absorbance at 280 1q.1 but does not affect splitting of peptide bonds as measured by ninhydrin method (Fig. 4).
mg ELASTIN
FIG. 3. Effect of elastin concentration on hydrolysis of ficin at pH 5.5 and 35.0” for 30 min. Activity measuredby ninhydrin method.
EFFECT OF TEMPERATURE Under the conditions used, ficin has its maximum activity on elastin at pH 5.5 and 55”. Activation energy, E, , for peptide bond splitting of elast’in, is 5.8 kcal./mole over the temperature range of 15-45”. This is in agreement with a reported value of 6.0 kcal./mole for hydrolysis of gelatin by ficin over the range of 40-55” (22). It should be noted that substrate concentration was a limiting factor in these experiments on elastin. Therefore, E, could be a measure of, or involve the effect of temperature on enzyme-substrate combination. Change in temperature would be expected to have no effect on solubility of elastin alone as preparation of elastin involves repeated autoclaving (15). DISCUSSION
In agreement with previous reports (1% 141, ficin is shown to have high elastolytic activity. This is in contrast, t,o a report by Lewis et al. (7) who found that ficin had
oLvz-:-:i 20
4.0
Activator Concenlrolion (Mx I&”
FIG. 4. Effect of cysteine (0, 0) and mercaptoethanol (X) concentration on hydrolysis of elastin by ficin at pH 5.5 and 35.0” for 30 min. Activity measured by ninhydrin method (0, X) and nbsorbance of supernatant liquid at 280 mp. ( 0 ). no elastolytic activity. It is of interest that the pH optimum for solubilization of elastin is identical wit,h the pH optimum for peptide bond hydrolysis in elastin. With gelatin as substrate for ficin, maximum rate of viscosity reduction occurs at pH 7.5 while maximum rate of peptide bond hydrolysis is near pH 5 (22). It would appear from t’he data that the degree of splitting of peptide link-
126
YATCO-MANZO
I .(
AND
WHITAKER
I
Q) z 20.7 -0
0.4 2 FIG. 5. Effect of temperature on hydrolysis measured by ninhydrin method.
ages in elastin by ficin is correlated with the number of linkages involving glycine. Yoneya (26) has reported that synthetic substrates involving glycine are split more rapidly at pH 5.0 than at pH 7.0. Solubilization of elastin by ficin is inhibited by high cysteine concentrations while peptide bond hydrolysis is not inhibited. Comte et al. (27) have also reported that cysteine inhibited the elastolytic activity of a crude Bacillus subtilis protease. Addition of cysteine does not increase the activity of ficin on elastin nearly as much as it does when casein is the substrate (20 vs. 500%). However,+1 x 10e3 M iodoacetate inhibits the actlvlty of ficin on both elastin and casein. These findings may indicate that cysteine decreases the susceptibility of elastin to attack by ficin. Ficin has maximum elastolytic activity at pH 5.5 and 55”. Mandl and Cohen (10) also reported a temperature optimum of 50-55” for the action of Flavobacterium elastolyticum elastase on elastin.
of elastin by ficin at pH 5.5 for 20 min. Activity
REFERENCES 1. BALK, J., AND BANGA, I., Biochem. J. 46, 384 (1950). 2. BALK, J., ARTDBAXGA, I., Acta Physiol. Acad. Sci. Hung. 4,187 (1953). 3. HALL, D. A., REED, R., AND TUNBRIDCE, R. E., Nature 170,264 (1952). 4. LANSING, A. I., ROSENTHAL, T. B., ALEX, M., AND DEMPSEY, E. W., Anat. Record 114, 555 (1952). 5. PEPLER, W. J., AND BRANDT, F. A., Brit. J. Exptl. Pathol. 35,41 (1954). 6. PARTRIDGE,S. M., AND DAVIS, H. F., Biochem. J. 61,21 (1955). 7. LEWIS, U. J., WILLIAMS, D. E., AND BRINK, N. G., J. Biol. Chem. 222,705 (1956). 8. HALL, D. A., Arch. Biochem. Biophys. 67, 366 (1957). 9. NARAYANAN, E. K., DEVI, P., AND MENON, P. S., Indian J. Med. Research 41,295 (1953). 10. MANDL, I., AND COHEN, B. B., Arch. Biochem. Biophgs. 91,47 (1960). 11. EVERETT, A. L., CORDON, T. C., KRAVITZ, E., AND NAGHSKI, J., Stain Technol. 34,325 (1959). 12. MIYADA, D. S., AND TAPPEL, A. L., Food Research 21, 217 (1956).
13. WASG,
H.,
WEIR,
C. E.,
HYDROLYSIS
OF ELASTIN
M. L.,
20. MOORE,
BIRKKER,
AXD
GIKGER, B., Food Research 23, 423 (1958). 14. THOMAS, J., AND PARTRIDGE, S. M., Biochem. J.
74,600 (1960). 15. PARTRIDGE,
H. F., AND ADAIR, G. S., Biochem. J. 61,ll (1955). 16. BALLENTINE, R., in “Methods in Enzymology” (Colowick and Kaplan, eds.), Vol. 3, pp. 98791. Academic Press, New York, 1957. 17. LAYR‘E, E., in “Methods in Enzymology” (Colowick and Kaplan, eds.), Vol. 3, pp. 450-l. Academic Press, New York, 1957. 18. WHITAKER, J. R., Food Technol. 13, 86 (1959). 19. JOHNSON, M. J., J. Biol. Chem. 137,575 (1941). S.
M.,
DAVIS,
127 S., AND STEIK,
W.
H., J. Biol.
Chem.
211,907 (1954). 21.
WHITAKER,
22. WHITAKER,
J. R., Food Research 22,483 (1957). J. R., Food Research 22, 468
(1957).
23. HALL, D. A., AND CZERXAWSKI, 5.73,356 (1959).
J. W., Biockem.
T., J. Biochem. (Tokyo) 37, 105 (1950). 25. IRVIKG, G. W., JR., FRUTON, J. S., AND BERGMANN, M., J. Biol. Chem. 138,231 (1941). 26. YONEYA, T., Acta Schol. Med. Univ. Kioto 35,186 (1958). 27. COMTE, P., BAZIN, S., AND DELAUNAY, A., Ann. inst. Pasteur 101,185 (1961).
24. YONEYA,
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Ficin-Catalyzed EMETERIA From the Department
97,
122-127 (1962)
Hydrolysis
YATCO-MANZO
AND
of Elastin J. R. WHITAKER
of Food Science and Technology, University Davis, California’
of California,
Received October 16, 1961 Ficin hydrolyzes elastin quite rapidly with a pH optimum of 5.0-5.5 and a temperature optimum (under conditions used) of 55”. Maximal action of ficin on elastin produces complete solubilization and a splitting of approximately 25% of the peptide bonds involving a-amino acids. Activation energy for the ficin-catalyzed splitting of these bonds is 5.8 kcal./mole. INTRODUCTION
In 1950 Ba16 and Banga (1) first reported the partial purification of a pancreatic elastase and later postulated a possible connection between enzyme level and atherosclerosis (2). Subsequent work indicated the solubilizing action of this enzyme on elastin might be due to its mucolytic activity (3), lipolytic activity (4)) or sulfatase activity (5), although Partridge and Davis (6) concluded that it is due to splitting of peptide bonds. The enzyme was crystallized in 1956 by Lewis et al. (7) and was found to hydrolyze a variety of proteins. The crystalline enzyme was later separated into mucolytic and proteolytic components which were inactive on native elastin separately (8). Elastin-hydrolyzing enzymes have also been isolated from microorganisms (9-11). Mandl and Cohen (10) isolated an elastase from Plauobacterium elastolyticum which appears to be more specific than pancreatic elastase. The plant proteolytic enzymes, papain (7, 11-14)) bromelin, and ficin (1214) also have elastolytic activity. Thomas and Partridge (14) reported that crystalline trypsin, chymotrypsin, pepsin, Rhozyme, and cathepsin from kidney and spleen were inactive on elastin. Elastolytic activity of plant proteolytic enzymes has not been studied in detail. In * This work was supported in part by a research grant, RG-5295, from the National Institutes of Health.
fact, it has not been shown directly that they soluhilize elastin by splitting peptide bonds, as methods which measured only the change in solubility of elastin were used (7, 11-14). The object of the present paper is to define the conditions which influence the elastolytic activity of ficin and to show that it solubilizes elastin by extensive splitting of peptide bonds. MATERIALS
AND METHODS
MATERIALS Elastin was prepared from the ligamentum nuchae of cattle by the method of Partridge, Davis, and Adair (15). The final purified elastin was freeze-dried, mixed with a large amount of Dry Ice, and passed rapidly through a Wiley Mill using a 20-mesh screen. Nitrogen content was 16.33% determined by Method B described by Ballentine (16). Ficin (Merck and Company) was used without fractionation as Thomas and Partridge (14) have found that ammonium sulfate fractionation and chromatography on carboxymethylcellulose failed to show any indication of separating proteolytic activity from elastolytic activity. This material contained 4.94% water and 79.1% protein on a moisture-free basis [biuret method (17)]. Solutions (4%) were prepared as described by Whitaker (18) and stored frozen. Samples were thawed just before use, and the excess was discarded. ASSAY OF ACTIVITY Elastin was weighed on a microbalance into 13 X 100 mm. tubes. Unless the substituent was the variable, each tube contained 20.0 mg. elastin, 1.25 X lo-’ M each cysteine (or mercaptoethanol), and 122
HYDROLYSIS. Versene as activators, 0.2 144,pH 5.5, acetate buffer, and water to a total of 2 ml. when 1.19 mg. enzyme protein was added. After temperature equilibration at 35.0” (except when temperature was variable), enzyme was added to initiate the reaction, and the contents were stirred intermittently throughout the incubation time of 30 min. It was established that amount of stirring did not influence extent of reaction. pH was determined on separate reaction mixtures using a Beckman model G pH meter. Reaction was terminated by addition of 3 ml. of 1 N HCl. Extent of reaction was determined either gravimetrically by the method of Ba16 and Banga (l), by change in absorbance of supernatant liquid at 280 rnp, by nitrogen content of supernatant liquid (19), by ninhydrin assay of supernatant liquid (20), or by a combination of these methods. Reported values are averages of two to five experiments.
50-
I
133
OF ELASTIN RESULTS
EFFECTOF PH Effect of pH on hydrolysis of elastin by ficin is shown in Fig. 1. The pH optimum is found at pH 5.0-5.5 by all four methods of assay. It is near pH 7 for casein and gelatin hydrolysis by ficin (21, 22). Grarimetric, nitrogen content, and absorption at 280 mp methods all gave identical results for the amount solubilixed. The ninhydrin assay data indicate a splitting of approximately 18% of the peptide bonds involving a-amino acids of the solubilized elastin at pH 5.5 under conditions used. If an elastase unit (E.U.) is arbitrarily defined as amount of enzyme which will solubilize 1.0 mg. elastin in 30 min. (1) , ficin has a value of I
I
I
8.0
IO
40-
30.!? v) )r 0 0 5
20-
IO-
oLIL 2.0
4.0
6.0
PH
FIG. 1. Effect of pH on hydrolysis of elastin at 35.0" and 30 min. by ficin as measured by ninhydrin method (O), gravimetrically (0 ), supernatant nitrogen content (X), and absorbance of supernatant liquid at 280 rnp (+). Buffer was 0.2 M with respect to each acetate, phosphate, and borate.
124
YATCO-MANZO
AND WHITAKER
TIME (minutes) 00
FIG. 2. Effect of ficin concentration (0, X) and time ( 0 ) on hydrolysis of elastin at pH 5.5 and 35.0”. Activity was measured by ninhydrin method (0, 0 ) and absorbance of supernatant liquid at 280 rnp (X 1.
8.0 E.U./mg. protein at pH 5.5 and 35” and 1.68 E.U./mg. protein at pH 8.0 and 35”. These values will vary with conditions used as the extent of solubilization is not a linear function of ficin concentration (Fig. 2) and is dependent upon the elastin concentration (Fig. 3). Other workers have reported the following values at 3737.5”: crystalline pancreatic elastase, 106-112 E.U./mg. protein at PH 8.7-8.8 (7) and 61.3 E.U./mg. protein at pH 8.7 (23) ; purified Flavobacterium elnstolyticum elastase, 3.1 E.U./mg. enzyme at pH 7.4 (10) ; papain, 3.7 E.U./mg. enzyme at pH 8.8 (7) and 5.0 E.U./mg. enzyme at pH 8.0 (14) ; and ficin, 1.86 E.U./mg. enzyme at pH 8.0 (14).
EFFECT OF FICIN AND ELASTIN CONCENTRATIONS AND RATE OF REACTION
Effect of ficin concentration on hydrolysis of elastin is shown in Fig. 2. At pH 5.5 and 35”, 8.0 mg. enzyme protein is able to completely solubilize 20 mg. elastin in 30 min. Extent of reaction is not a linear function of ficin concentration but is also dependent upon substrate concentration as shown by Fig. 3. There is no indication of the reaction b ecoming independent of substrate concentration up to 75 mg. elastin/l.l9 mg. enzyme protein. Since reaction times of 180 and 240 min. also gave 25% splitting of peptide bonds involving or-amino acids, it would appear this is the maximum degree of hydrolysis of elastin by ficin, a rather exten-
HYDROLYSIS
125
OF ELASTIN
sive degradation. Degree of hydrolysis may be largely correlated with percentage glycine in cattle ligamenturn nuchae elastin [27,0%, ref. (6)] as ficin is known to hydrolyze peptide bonds involving glycine (24). Ficin will also split linkages involving arginine (25)) but, there is only 1.2% arginine in elastin (6). EFFECT OF ACTIVATOR CONCENTRATION Effect of cysteine and mercaptoethanol on hydrolysis of elastin by ficin is shown in Fig. 4. It is surprising to find that these cornpounds produce very little increase in amount of hydrolysis (approx. 20% maximum) as the ficin-catalyzed hydrolysis of casein is increased approximately fivefold by addition of 1.25 x 1O-2 M cysteine to the same ficin used in these experiments. Prior incubation of ficin with 1 x lo-” M iodoacetate for 30 min. completely destroys its elastolytic activity as well as proteolytic activity (casein as substrate). Cysteinc above 1.25 x 1O-2 M markedly inhibits the solubilization of elastin as measured by absorbance at 280 1q.1 but does not affect splitting of peptide bonds as measured by ninhydrin method (Fig. 4).
mg ELASTIN
FIG. 3. Effect of elastin concentration on hydrolysis of ficin at pH 5.5 and 35.0” for 30 min. Activity measuredby ninhydrin method.
EFFECT OF TEMPERATURE Under the conditions used, ficin has its maximum activity on elastin at pH 5.5 and 55”. Activation energy, E, , for peptide bond splitting of elast’in, is 5.8 kcal./mole over the temperature range of 15-45”. This is in agreement with a reported value of 6.0 kcal./mole for hydrolysis of gelatin by ficin over the range of 40-55” (22). It should be noted that substrate concentration was a limiting factor in these experiments on elastin. Therefore, E, could be a measure of, or involve the effect of temperature on enzyme-substrate combination. Change in temperature would be expected to have no effect on solubility of elastin alone as preparation of elastin involves repeated autoclaving (15). DISCUSSION
In agreement with previous reports (1% 141, ficin is shown to have high elastolytic activity. This is in contrast, t,o a report by Lewis et al. (7) who found that ficin had
oLvz-:-:i 20
4.0
Activator Concenlrolion (Mx I&”
FIG. 4. Effect of cysteine (0, 0) and mercaptoethanol (X) concentration on hydrolysis of elastin by ficin at pH 5.5 and 35.0” for 30 min. Activity measured by ninhydrin method (0, X) and nbsorbance of supernatant liquid at 280 mp. ( 0 ). no elastolytic activity. It is of interest that the pH optimum for solubilization of elastin is identical wit,h the pH optimum for peptide bond hydrolysis in elastin. With gelatin as substrate for ficin, maximum rate of viscosity reduction occurs at pH 7.5 while maximum rate of peptide bond hydrolysis is near pH 5 (22). It would appear from t’he data that the degree of splitting of peptide link-
126
YATCO-MANZO
I .(
AND
WHITAKER
I
Q) z 20.7 -0
0.4 2 FIG. 5. Effect of temperature on hydrolysis measured by ninhydrin method.
ages in elastin by ficin is correlated with the number of linkages involving glycine. Yoneya (26) has reported that synthetic substrates involving glycine are split more rapidly at pH 5.0 than at pH 7.0. Solubilization of elastin by ficin is inhibited by high cysteine concentrations while peptide bond hydrolysis is not inhibited. Comte et al. (27) have also reported that cysteine inhibited the elastolytic activity of a crude Bacillus subtilis protease. Addition of cysteine does not increase the activity of ficin on elastin nearly as much as it does when casein is the substrate (20 vs. 500%). However,+1 x 10e3 M iodoacetate inhibits the actlvlty of ficin on both elastin and casein. These findings may indicate that cysteine decreases the susceptibility of elastin to attack by ficin. Ficin has maximum elastolytic activity at pH 5.5 and 55”. Mandl and Cohen (10) also reported a temperature optimum of 50-55” for the action of Flavobacterium elastolyticum elastase on elastin.
of elastin by ficin at pH 5.5 for 20 min. Activity
REFERENCES 1. BALK, J., AND BANGA, I., Biochem. J. 46, 384 (1950). 2. BALK, J., ARTDBAXGA, I., Acta Physiol. Acad. Sci. Hung. 4,187 (1953). 3. HALL, D. A., REED, R., AND TUNBRIDCE, R. E., Nature 170,264 (1952). 4. LANSING, A. I., ROSENTHAL, T. B., ALEX, M., AND DEMPSEY, E. W., Anat. Record 114, 555 (1952). 5. PEPLER, W. J., AND BRANDT, F. A., Brit. J. Exptl. Pathol. 35,41 (1954). 6. PARTRIDGE,S. M., AND DAVIS, H. F., Biochem. J. 61,21 (1955). 7. LEWIS, U. J., WILLIAMS, D. E., AND BRINK, N. G., J. Biol. Chem. 222,705 (1956). 8. HALL, D. A., Arch. Biochem. Biophys. 67, 366 (1957). 9. NARAYANAN, E. K., DEVI, P., AND MENON, P. S., Indian J. Med. Research 41,295 (1953). 10. MANDL, I., AND COHEN, B. B., Arch. Biochem. Biophgs. 91,47 (1960). 11. EVERETT, A. L., CORDON, T. C., KRAVITZ, E., AND NAGHSKI, J., Stain Technol. 34,325 (1959). 12. MIYADA, D. S., AND TAPPEL, A. L., Food Research 21, 217 (1956).
13. WASG,
H.,
WEIR,
C. E.,
HYDROLYSIS
OF ELASTIN
M. L.,
20. MOORE,
BIRKKER,
AXD
GIKGER, B., Food Research 23, 423 (1958). 14. THOMAS, J., AND PARTRIDGE, S. M., Biochem. J.
74,600 (1960). 15. PARTRIDGE,
H. F., AND ADAIR, G. S., Biochem. J. 61,ll (1955). 16. BALLENTINE, R., in “Methods in Enzymology” (Colowick and Kaplan, eds.), Vol. 3, pp. 98791. Academic Press, New York, 1957. 17. LAYR‘E, E., in “Methods in Enzymology” (Colowick and Kaplan, eds.), Vol. 3, pp. 450-l. Academic Press, New York, 1957. 18. WHITAKER, J. R., Food Technol. 13, 86 (1959). 19. JOHNSON, M. J., J. Biol. Chem. 137,575 (1941). S.
M.,
DAVIS,
127 S., AND STEIK,
W.
H., J. Biol.
Chem.
211,907 (1954). 21.
WHITAKER,
22. WHITAKER,
J. R., Food Research 22,483 (1957). J. R., Food Research 22, 468
(1957).
23. HALL, D. A., AND CZERXAWSKI, 5.73,356 (1959).
J. W., Biockem.
T., J. Biochem. (Tokyo) 37, 105 (1950). 25. IRVIKG, G. W., JR., FRUTON, J. S., AND BERGMANN, M., J. Biol. Chem. 138,231 (1941). 26. YONEYA, T., Acta Schol. Med. Univ. Kioto 35,186 (1958). 27. COMTE, P., BAZIN, S., AND DELAUNAY, A., Ann. inst. Pasteur 101,185 (1961).
24. YONEYA,