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Irradiated pectin as a substrate for pectic enzymes
Radiation Botany, 1971, Vol. 11, pp. 383 to 387. Pergamon Press. Printed in Great Britain.
I R R A D I A T E D P E C T I N AS A S U B S T R A T E F O R P E C T I C ENZYMES* a . j. ROMANZ, L. P. SOMOGYI I" and JUANITA M.ANALO~ Department of Pomology, University of California, Davis, California, U.S.A.
(Received 24 May 1971) ROM.~'¢~R.J., SOMOO~aL. P. and I~IA_,NALOJUANITO.Irradiatedpectin as a substratefor pectic enzymes. tL~aTXON BOTANY11, 383--387, 1971.--Gamma irradiation of a 1 per cent pectin solution with doses up to 250 krad increased the susceptibility of the pectin to pectin methylesterase and decreased its susceptibility to pectin-polygalacturonase and pectin-transeliminase. Major physicochemical changes occurred in the 500--1000 krad dose range as revealed by a rapid decrease in susceptibility to all three enzymes. Irradiation under anoxic conditions did not significantly alter the radiation effect. Uniquely altered interactions of the enzymes with irradiated substrate offer a diagnostic tool for the study of both the nature of radiation damage and the mode of enzymic attack. INTRODUCTION
IRRADIATION of pectin results in significant degradation and loss of viscosity.(9, as) Since natural softening of fruit is also accompanied by degradative changes in pectic constituents,0S) softening of irradiated fruit is often attributed to a radiation scission of the pectins. However, analyses of pectins extracted from irradiated fruit have led to conflicting opinions as to whether observed radiation damage to the pectins is adequate(S, 1°) or inadequate(is) to account for the gross tissue changes. Moreover, an assessment of causal factors must take into account the dynamic, physiological responses of the tissues themselves to irradiation stress. Indeed, evidence has been presented that PMEll activity in cherriesCa s) and in expressed juice from irradiated oranges(2) actually increased after doses in the order of 300 krad. In a review of this general subject ROMAmI14) called attention to the possibility that both radiation scission and crosslinking m a y occur and affect the susceptibility of
pectins to enzymic attack. One purpose of the present study was to assess the enzymic susceptibility of irradiated pectins. A second objective of these experiments derives from the generally accepted view that enzymes in vivo are well protected and not readily affected by irradiation. Yet, as noted by OKADA et al.,02) radiobiological investigations of enzyme reactions have, with but few exceptions, concentrated on the radiosensitivity of the enzyme and not the radiation effect on substrate. It was thus of radiobiological interest to investigate the effects of radiation on pectin, a substrate for three different pectic enzymes (PME, PG and PTE) each with distinct modes of attack. MATERIALS A N D IVJ[ETHODS
Substrate preparation and irradiation An unbuffered 1 per cent solution was prepared from citrus pectin (Sunkist Growers, Inc., Lot. No. 444) and distilled water. Anaerobic conditions were achieved by bubbling water-pumped N~ through the
* Research supported in part by the U.S. Atomic Energy Commission, Contract AT(11-1)-34, Project 112, Report No. UCD 34P112-39. t Present address, Vacu-Dry Company, Emeryville, California. + Recipient of an I.A.E.A. Fellowship. Present address, The Philippines Women's University, ManiUa. II Abbreviations: PME, pectin methylesterase; PG, pectin polygalacturonase; PTE, pectin transeliminase. 383
384
R.J. ROMANI, L. P. SOMOGYI a n d J U A N I T A MANALO
pectin solution at a rapid rate for 4 hr. Deaerated pectin solution was placed in screw cap vials, these were placed in plastic bags, and the bags in turn in a sealed jar. All manipulationswere made under positive N2 pressure and each container was flushed with N 2 before being sealed. Irradiations were done at ambient temperatures (25-30°C) in a cobalt-60 irradiator of a fixed configuration and delivering 280 krad per hr as determined by ferrous sulfate dosimetry. (xs)
Enzymes and enzyme assays A 0.25 per cent solution of Pectinol 100 D (Rohm & Haas, Philadelphia, Pennsylvania) was initially used as the source of PG. Since Pectinol is known also to contain PTE activity, additional assays were made with a purified PG (0.05 PC units/mg)(*~ obtained through the courtesy of Dr. E. F. Jansen (USDA Laboratory, Albany, California). No PTE activity was detected in the purified PG. PG activity was assayed by two methods. In a technique described by HOBSON,(5) 5-ml samples of the irradiated pectin solutions were brought to pH 5 with 0-1 ml of 1 Mpotassium hydrogen phthalate buffer and held at 30°C for a few minutes prior to the addition of 1 ml of appropriately diluted enzyme. The reducing groups exposed by the enzymatic activity were quantitatively estimated at zero time and at subsequent 15-min intervals. Enzymatically produced decreases in viscosity were also used to assess PG activity. Five ml of the I per cent pectin solution and 0.I ml of 1 M buffer, either potassium hydrogen phthalate or citrate-phosphate buffer, pH 5, were placed in No. 300 Ostwald-CannorFensld vlscometers incubated at 30°C. An appropriate amount of enzyme was then added and the viscossity measured at 10-rain intervals. To obtain PME mature, pitted cherries were homogenized with an equal volume of 0.5 Msodium acetate (pH 8) and held overnight at 0°C with gentle stirring. The homogenate was then centrifuged at 18,000 × g for 10 rain and crude PME obtained from the clear supernatant fraction by precipitation with ammonium sulfate. The fraction precipitating between 50 and 70 per cent (NH4)2SO 4 was utilized, as this portion contained the bulk of the PME activity. Enzymic activity was measured with the rapid, semi-micro method described by SoMoo'~ and RoM.~rt(t~) based on the change in pH as a result of PME activity. Extra precautions were required in adjusting pH when heavily irradiated pectin solutions were used as substrate. These solutions were unstable as indicated by a gradual change in pH even in the absence of enzyme. The standard titration procedure of Rouse and ATKmS(16) was also used. PTE was partially purified from Pectinol 100 D by
column chromatography using the methods of ALBERSHEIM and KILUAS.(Z) PTE activity was measured as described by the same authors, and based on the increase in absorbancy at 235 mtt with time. Every attempt was made to measure the initial rates of the enzymic reactions. However, the accuracy of the methods, especially the viscometric procedure, was greatly circumscribed when applied to heavily irradiated pectin solutions whose viscosity was already markedly reduced by irradiation. Methoxyl content was determined by titration as described by GEE et al.(3)
RESULTS AND DISCUSSION Activities of the three enzymes, PG, P M E a n d P T E , o n pectin substrate, are indicated in Figs. 1-3. Above 10 krad increasing doses of ionizing
IOC
PG
°
~ 8c
~ 6c >- 4¢
\o
w 201
io
ioo
i,ooo
io,ooo
DOSE (KrQd)
FIo. I. Changes in pectin polygalacturonase (PG) activity as a function of radiation dosage of the pectin substrate. Aerobic ( @ - - g ) and anoxic ( O - - O ) irradiation. Enzyme activity based on viscometric effects. r a d i a t i o n decreased the suitability of the irradiated pectin as substrate for P G a n d P T E . Conversely, P M E activity increased with i r r a d i a t i o n of the substrate u p to ca. 250 krad. Beyond a 750-1000 krad threshold drastic alterations of the pectin m a r k e d l y r e d u c e d its suitability as a substrate for all three enzymes. Exposure u n d e r anoxic conditions t e n d e d to only slightly d i m i n i s h the r a d i a t i o n effects. Estimations of methoxyl content, averaging 7.7 per cent for the u n i r r a d i a t e d pectin, p r o v i d e d a d d i t i o n a l evidence for r a d i a t i o n i n d u c e d changes. T h e greatest change occurred at the 1000, 5000 a n d 10,000 krad doses w h e n the
IRRADIATED PECTIN AS A SUBSTRATE FOR PECTIC ENZYMES 140 o
~-~,oo
o , ~
>so
,
PME
~
4O
\
2o o
\ ,b
,~o DOSE(Krad)
' 1,000
' I0,000
FIO. 2. Changes in pectin methylesterase (PME) activity as a function of radiation dosage of the pectin substrate. Aerobic ( @ - - @ ) and anoxic ( © - - © ) irradiation.
"~ so
metric assay, and appropriate corrections were made for the non-enzymic effects, a slight increase in P M E activity with low-dose irradiation of the substrate and a rapid decline as a result of doses above 1000-5000 krad was again observed (Table I). Since both PG and P T E cause a loss in viscosity, similarities in their decline in activity on irradiated substrate (Figs. 1 and 2) could result from P T E contaminant in preparations of crude PG. However, use of purified PG, known to be free of P T E activity, yielded results (Table 2) that corroborate those obtained with crude Table 1. Titrimetric measure of pectin methylesterase (P M E ) activity with irradiated pectin as substrate Dose, krad 0 250 500 750
PTE
1000
5000
~ 6o
385
10,000
PME activity* meq - O H per % of control 10 min I0 12.5 9 11.5
100 125 90 115
10
100
0-22 0.01
2.2 0.1
*Determined by the titration method as described by Rouse and ATKINS. (18)
w 2O
0
l~)
I(~0
' 1,000
i 10,00(2
DOSE (Krod)
Fzo. 3. Changes in pectin transeliminase (PTE) activity as a function of radiation dosage of the pectin substrate. Aerobic ( O - - O ) and anoxic ( O - - O ) irradiation. methoxyl content increased to 8.9, 9.6 and 10.8 per cent respectively. Autocatalytic changes also occurred during refrigerated storage of pectin solutions and, as already noted by GLEGG and KERT~SZ,(* this instability appears to be accentuated in the irradiated solutions. Substrate instability resulting in the non-enzymic release of acidic groups was particularly troublesome in the sensitive semimicro P M E assay. However, when the experiments were repeated using the titra-
Table 2. Changes in pectin polygalacturonase (PC) activity with increasing radiation dosage of the pectin substrate Dose, krad 0 5 10 25 50 100 250 500 1000 5000
% PG activity 100 96 88 82 77 78 71 51 23
7
Range (4 assays)
82-104 76-104 66- 99 62- 96 66- 89 64- 78 I a 69 6 - 40 0- 20
*PG purified according tOJANSENand McDo~,q~L (6) and assayed according to HOBSON.(5)
386
R.J. ROMANI, L. P. SOMOGYI and JUANITA MANALO
preparations of the enzyme (Fig. 1). The range in values from quadruplicate assays (Table 2) does illustrate the variability that derives from use of an amorphous substrate rendered unstable by ionizing radiation. The three enzymes used in this study represent three different modes of action: (1) hydrolysis of the C e methylester linkage and de-esterification of the pectin galacturonic acid units by PME, (2) splitting of the 1-4 glycosidic bond by PG, and (3) hydrolysis of the glycosidic bond via a transelimination reaction by PTE. Difference in the response of the enzymes to irradiation of a common substrate provides clues to the nature of the radiation change and to the mode of enzymic attack. Both PG and P T E have specific steric requirementsIl,n) for substrate-enzyme association. Thus even a modest change in tertiary structure of the pectin molecule might affect its suitability as a substrate for these enzymes. MASSEY and FAUST,Cn) reviewing the results of various model experiments, suggested that although random scission of the methylester group m a y result from irradiation, it is apparently minor compared to changes in the degree of depolymerization. Structural changes are also to be expected from radiation scission of the relatively weakC~) interpectin bonds resulting in the exposure of methylester groups to enzymic attack. Such an event would be consistent with the observed increase in P M E activity and concomitant decrease in PG and lYrE activity as the irradiation dose to the substrate is increased to ca. 250 krad. If, as postulated, by JANS~.N and McDoNNEL,(8) in situ de-esterification by P M E must precede glycosidic hydrolysis by PG, irradiation induced de-esterification in the 100-500 krad range could trigger a general enzymic breakdown of the pectins. Citrus pectin is, at best, an impure substrate of poorly defined polymer structure. In this context, the results obtained must be considered preliminary. However, the findings do indicate that the effect of radiation on pectin as a substrate m a y be quite significant and should be taken into account when assessing certain post-irradiation changes in plant tissues. Moreover, the different responses of each pectic enzyme to irradiated pectin encourages a more detailed radiobio-
chemical study with purified enzymes and well defined 'model' substrates that permit kinetic evaluations and further insights into the pectic enzyme-substrate interaction. Acknowledgement--We thank Mrs. BERRITOVEREI~for
very capable technical assistance. REFERENC, ES 1. ALBERSHEIM P. and KXLLL~S URSULA (1962) Studies relating to the purification and properties of pectin transeliminase. Arch. Biochem. Biophys. 97, 107-115. 2. DENNISONR. A., AHMEDE. M. and MARTINF. G. (1967) Pectinesterase activity in irradiated 'Valencia' oranges. Proc. Am. Soc. Hort. Sci. 91j 163-168 3. GEE M., McCOMB E. H. and McCREADY R. M. (1958) A method for the characterization ofpectin substances in some fruit and sugar mares, ft. Food Sd. 23, 72-75. 4. GLEGOR. E. and K~RTESZZ. I. (I 956) 3alter-effect in the degradation of cellulose and pectin by gamma rays. Science124, 893-894. 5. HOBSONG. E. (1962) Determination ofpolygalacturonase in fruits. Nature 195, 804. 6. J~sEN E. F. and McDom~mLL. R. (1945) Influence of methoxyl content of pectic substances on the action of polygalacturonase. Arch. Biochem. 8, 97-112. 7. JOSLYN M. A. (1962) The chemistry of protopectin. Advan. FoodRes. 11, 1-107. 8. KERTESZ Z. I., GLEGG R. E., BOYLE G. F., PARSONSG. F. and MASSEYL. M. (1964) Effect of ionizing radiation on plant tissues. III. Softening and changes in pectins and cellulose of apples, carrots and beets.ft. FoodSci. 29, 40-48. 9. KXRT~SZZ. I., MOROANB. H., TLvr'rLEL. W. and LAVlU M. (1956) Effects of ionizing radiation on pectin. Radiation Res. 5, 372-381. 10. MfiARDImF.J. and NEI-mMIASJ.V. (1956) Effects of gamma radiation on the pectic constituents of fruit and vegetables. Food Technol. 10, 599-60 I. I1. MASSEYL. M.,JR. and FAUSTM. (1969) Irradiation effects on polysaccharides, pp. 269-297. In H. W. SCHULTZ (ed.), Symposium on foods: carbohydrates and their roles. AVI Publishing Co., Westport Co., Westport, Conn. 12. Og~a~A S., KRAtmZ R. and GASS~mRE. (1960) Radiation induced susceptibility of substrates to enzymic degradation. Radiation Res. 13, 607-6 12. 13. REEVE R. M. (1959) Histological and histochemical changes in developing and ripening peaches. II. Cell walls and pectins. Am. a7. Botany 46, 241-248.
IRRADIATED PECTIN AS A SUBSTRATE F O R PECTIC ENZYMES 14. ROMANIR . J . (1967) Radiobiological parameters in the irradiation of fruits and vegetables, pp. 57-103. In, Advances in food research, Vol. 15. Academic Press, N. Y. 15. Ro~mui R.J., ROBINSONB.J., R . ~ H. L., MAXlE E. C. and SOMMERN. F. (1963) Fruit irradiationphysical methods. Radiation Botany 3, 345-350. 16. Rouse A. H. and ATKXNSC. D. (1955) Pectinesterase and pectin in commercial citrus juices as determined by methods used at the Citrus
387
Experiment Station. Bull. Fla Agric. Exp. Stn. 570, 6-8. 17. SoMooYI L. P. and ROMANIR. J. (1964) A simplified technique for the determination of pectin methylesterase activity. Anal. Biochem. 8, 498-501. 18. SOMOOYIL. P. and ROMAN1R. J. (1964) Irradiation-induced textural change in fruits and its relation to pectin metabolism, or. Food Sci. 29, 366-371.
I R R A D I A T E D P E C T I N AS A S U B S T R A T E F O R P E C T I C ENZYMES* a . j. ROMANZ, L. P. SOMOGYI I" and JUANITA M.ANALO~ Department of Pomology, University of California, Davis, California, U.S.A.
(Received 24 May 1971) ROM.~'¢~R.J., SOMOO~aL. P. and I~IA_,NALOJUANITO.Irradiatedpectin as a substratefor pectic enzymes. tL~aTXON BOTANY11, 383--387, 1971.--Gamma irradiation of a 1 per cent pectin solution with doses up to 250 krad increased the susceptibility of the pectin to pectin methylesterase and decreased its susceptibility to pectin-polygalacturonase and pectin-transeliminase. Major physicochemical changes occurred in the 500--1000 krad dose range as revealed by a rapid decrease in susceptibility to all three enzymes. Irradiation under anoxic conditions did not significantly alter the radiation effect. Uniquely altered interactions of the enzymes with irradiated substrate offer a diagnostic tool for the study of both the nature of radiation damage and the mode of enzymic attack. INTRODUCTION
IRRADIATION of pectin results in significant degradation and loss of viscosity.(9, as) Since natural softening of fruit is also accompanied by degradative changes in pectic constituents,0S) softening of irradiated fruit is often attributed to a radiation scission of the pectins. However, analyses of pectins extracted from irradiated fruit have led to conflicting opinions as to whether observed radiation damage to the pectins is adequate(S, 1°) or inadequate(is) to account for the gross tissue changes. Moreover, an assessment of causal factors must take into account the dynamic, physiological responses of the tissues themselves to irradiation stress. Indeed, evidence has been presented that PMEll activity in cherriesCa s) and in expressed juice from irradiated oranges(2) actually increased after doses in the order of 300 krad. In a review of this general subject ROMAmI14) called attention to the possibility that both radiation scission and crosslinking m a y occur and affect the susceptibility of
pectins to enzymic attack. One purpose of the present study was to assess the enzymic susceptibility of irradiated pectins. A second objective of these experiments derives from the generally accepted view that enzymes in vivo are well protected and not readily affected by irradiation. Yet, as noted by OKADA et al.,02) radiobiological investigations of enzyme reactions have, with but few exceptions, concentrated on the radiosensitivity of the enzyme and not the radiation effect on substrate. It was thus of radiobiological interest to investigate the effects of radiation on pectin, a substrate for three different pectic enzymes (PME, PG and PTE) each with distinct modes of attack. MATERIALS A N D IVJ[ETHODS
Substrate preparation and irradiation An unbuffered 1 per cent solution was prepared from citrus pectin (Sunkist Growers, Inc., Lot. No. 444) and distilled water. Anaerobic conditions were achieved by bubbling water-pumped N~ through the
* Research supported in part by the U.S. Atomic Energy Commission, Contract AT(11-1)-34, Project 112, Report No. UCD 34P112-39. t Present address, Vacu-Dry Company, Emeryville, California. + Recipient of an I.A.E.A. Fellowship. Present address, The Philippines Women's University, ManiUa. II Abbreviations: PME, pectin methylesterase; PG, pectin polygalacturonase; PTE, pectin transeliminase. 383
384
R.J. ROMANI, L. P. SOMOGYI a n d J U A N I T A MANALO
pectin solution at a rapid rate for 4 hr. Deaerated pectin solution was placed in screw cap vials, these were placed in plastic bags, and the bags in turn in a sealed jar. All manipulationswere made under positive N2 pressure and each container was flushed with N 2 before being sealed. Irradiations were done at ambient temperatures (25-30°C) in a cobalt-60 irradiator of a fixed configuration and delivering 280 krad per hr as determined by ferrous sulfate dosimetry. (xs)
Enzymes and enzyme assays A 0.25 per cent solution of Pectinol 100 D (Rohm & Haas, Philadelphia, Pennsylvania) was initially used as the source of PG. Since Pectinol is known also to contain PTE activity, additional assays were made with a purified PG (0.05 PC units/mg)(*~ obtained through the courtesy of Dr. E. F. Jansen (USDA Laboratory, Albany, California). No PTE activity was detected in the purified PG. PG activity was assayed by two methods. In a technique described by HOBSON,(5) 5-ml samples of the irradiated pectin solutions were brought to pH 5 with 0-1 ml of 1 Mpotassium hydrogen phthalate buffer and held at 30°C for a few minutes prior to the addition of 1 ml of appropriately diluted enzyme. The reducing groups exposed by the enzymatic activity were quantitatively estimated at zero time and at subsequent 15-min intervals. Enzymatically produced decreases in viscosity were also used to assess PG activity. Five ml of the I per cent pectin solution and 0.I ml of 1 M buffer, either potassium hydrogen phthalate or citrate-phosphate buffer, pH 5, were placed in No. 300 Ostwald-CannorFensld vlscometers incubated at 30°C. An appropriate amount of enzyme was then added and the viscossity measured at 10-rain intervals. To obtain PME mature, pitted cherries were homogenized with an equal volume of 0.5 Msodium acetate (pH 8) and held overnight at 0°C with gentle stirring. The homogenate was then centrifuged at 18,000 × g for 10 rain and crude PME obtained from the clear supernatant fraction by precipitation with ammonium sulfate. The fraction precipitating between 50 and 70 per cent (NH4)2SO 4 was utilized, as this portion contained the bulk of the PME activity. Enzymic activity was measured with the rapid, semi-micro method described by SoMoo'~ and RoM.~rt(t~) based on the change in pH as a result of PME activity. Extra precautions were required in adjusting pH when heavily irradiated pectin solutions were used as substrate. These solutions were unstable as indicated by a gradual change in pH even in the absence of enzyme. The standard titration procedure of Rouse and ATKmS(16) was also used. PTE was partially purified from Pectinol 100 D by
column chromatography using the methods of ALBERSHEIM and KILUAS.(Z) PTE activity was measured as described by the same authors, and based on the increase in absorbancy at 235 mtt with time. Every attempt was made to measure the initial rates of the enzymic reactions. However, the accuracy of the methods, especially the viscometric procedure, was greatly circumscribed when applied to heavily irradiated pectin solutions whose viscosity was already markedly reduced by irradiation. Methoxyl content was determined by titration as described by GEE et al.(3)
RESULTS AND DISCUSSION Activities of the three enzymes, PG, P M E a n d P T E , o n pectin substrate, are indicated in Figs. 1-3. Above 10 krad increasing doses of ionizing
IOC
PG
°
~ 8c
~ 6c >- 4¢
\o
w 201
io
ioo
i,ooo
io,ooo
DOSE (KrQd)
FIo. I. Changes in pectin polygalacturonase (PG) activity as a function of radiation dosage of the pectin substrate. Aerobic ( @ - - g ) and anoxic ( O - - O ) irradiation. Enzyme activity based on viscometric effects. r a d i a t i o n decreased the suitability of the irradiated pectin as substrate for P G a n d P T E . Conversely, P M E activity increased with i r r a d i a t i o n of the substrate u p to ca. 250 krad. Beyond a 750-1000 krad threshold drastic alterations of the pectin m a r k e d l y r e d u c e d its suitability as a substrate for all three enzymes. Exposure u n d e r anoxic conditions t e n d e d to only slightly d i m i n i s h the r a d i a t i o n effects. Estimations of methoxyl content, averaging 7.7 per cent for the u n i r r a d i a t e d pectin, p r o v i d e d a d d i t i o n a l evidence for r a d i a t i o n i n d u c e d changes. T h e greatest change occurred at the 1000, 5000 a n d 10,000 krad doses w h e n the
IRRADIATED PECTIN AS A SUBSTRATE FOR PECTIC ENZYMES 140 o
~-~,oo
o , ~
>so
,
PME
~
4O
\
2o o
\ ,b
,~o DOSE(Krad)
' 1,000
' I0,000
FIO. 2. Changes in pectin methylesterase (PME) activity as a function of radiation dosage of the pectin substrate. Aerobic ( @ - - @ ) and anoxic ( © - - © ) irradiation.
"~ so
metric assay, and appropriate corrections were made for the non-enzymic effects, a slight increase in P M E activity with low-dose irradiation of the substrate and a rapid decline as a result of doses above 1000-5000 krad was again observed (Table I). Since both PG and P T E cause a loss in viscosity, similarities in their decline in activity on irradiated substrate (Figs. 1 and 2) could result from P T E contaminant in preparations of crude PG. However, use of purified PG, known to be free of P T E activity, yielded results (Table 2) that corroborate those obtained with crude Table 1. Titrimetric measure of pectin methylesterase (P M E ) activity with irradiated pectin as substrate Dose, krad 0 250 500 750
PTE
1000
5000
~ 6o
385
10,000
PME activity* meq - O H per % of control 10 min I0 12.5 9 11.5
100 125 90 115
10
100
0-22 0.01
2.2 0.1
*Determined by the titration method as described by Rouse and ATKINS. (18)
w 2O
0
l~)
I(~0
' 1,000
i 10,00(2
DOSE (Krod)
Fzo. 3. Changes in pectin transeliminase (PTE) activity as a function of radiation dosage of the pectin substrate. Aerobic ( O - - O ) and anoxic ( O - - O ) irradiation. methoxyl content increased to 8.9, 9.6 and 10.8 per cent respectively. Autocatalytic changes also occurred during refrigerated storage of pectin solutions and, as already noted by GLEGG and KERT~SZ,(* this instability appears to be accentuated in the irradiated solutions. Substrate instability resulting in the non-enzymic release of acidic groups was particularly troublesome in the sensitive semimicro P M E assay. However, when the experiments were repeated using the titra-
Table 2. Changes in pectin polygalacturonase (PC) activity with increasing radiation dosage of the pectin substrate Dose, krad 0 5 10 25 50 100 250 500 1000 5000
% PG activity 100 96 88 82 77 78 71 51 23
7
Range (4 assays)
82-104 76-104 66- 99 62- 96 66- 89 64- 78 I a 69 6 - 40 0- 20
*PG purified according tOJANSENand McDo~,q~L (6) and assayed according to HOBSON.(5)
386
R.J. ROMANI, L. P. SOMOGYI and JUANITA MANALO
preparations of the enzyme (Fig. 1). The range in values from quadruplicate assays (Table 2) does illustrate the variability that derives from use of an amorphous substrate rendered unstable by ionizing radiation. The three enzymes used in this study represent three different modes of action: (1) hydrolysis of the C e methylester linkage and de-esterification of the pectin galacturonic acid units by PME, (2) splitting of the 1-4 glycosidic bond by PG, and (3) hydrolysis of the glycosidic bond via a transelimination reaction by PTE. Difference in the response of the enzymes to irradiation of a common substrate provides clues to the nature of the radiation change and to the mode of enzymic attack. Both PG and P T E have specific steric requirementsIl,n) for substrate-enzyme association. Thus even a modest change in tertiary structure of the pectin molecule might affect its suitability as a substrate for these enzymes. MASSEY and FAUST,Cn) reviewing the results of various model experiments, suggested that although random scission of the methylester group m a y result from irradiation, it is apparently minor compared to changes in the degree of depolymerization. Structural changes are also to be expected from radiation scission of the relatively weakC~) interpectin bonds resulting in the exposure of methylester groups to enzymic attack. Such an event would be consistent with the observed increase in P M E activity and concomitant decrease in PG and lYrE activity as the irradiation dose to the substrate is increased to ca. 250 krad. If, as postulated, by JANS~.N and McDoNNEL,(8) in situ de-esterification by P M E must precede glycosidic hydrolysis by PG, irradiation induced de-esterification in the 100-500 krad range could trigger a general enzymic breakdown of the pectins. Citrus pectin is, at best, an impure substrate of poorly defined polymer structure. In this context, the results obtained must be considered preliminary. However, the findings do indicate that the effect of radiation on pectin as a substrate m a y be quite significant and should be taken into account when assessing certain post-irradiation changes in plant tissues. Moreover, the different responses of each pectic enzyme to irradiated pectin encourages a more detailed radiobio-
chemical study with purified enzymes and well defined 'model' substrates that permit kinetic evaluations and further insights into the pectic enzyme-substrate interaction. Acknowledgement--We thank Mrs. BERRITOVEREI~for
very capable technical assistance. REFERENC, ES 1. ALBERSHEIM P. and KXLLL~S URSULA (1962) Studies relating to the purification and properties of pectin transeliminase. Arch. Biochem. Biophys. 97, 107-115. 2. DENNISONR. A., AHMEDE. M. and MARTINF. G. (1967) Pectinesterase activity in irradiated 'Valencia' oranges. Proc. Am. Soc. Hort. Sci. 91j 163-168 3. GEE M., McCOMB E. H. and McCREADY R. M. (1958) A method for the characterization ofpectin substances in some fruit and sugar mares, ft. Food Sd. 23, 72-75. 4. GLEGOR. E. and K~RTESZZ. I. (I 956) 3alter-effect in the degradation of cellulose and pectin by gamma rays. Science124, 893-894. 5. HOBSONG. E. (1962) Determination ofpolygalacturonase in fruits. Nature 195, 804. 6. J~sEN E. F. and McDom~mLL. R. (1945) Influence of methoxyl content of pectic substances on the action of polygalacturonase. Arch. Biochem. 8, 97-112. 7. JOSLYN M. A. (1962) The chemistry of protopectin. Advan. FoodRes. 11, 1-107. 8. KERTESZ Z. I., GLEGG R. E., BOYLE G. F., PARSONSG. F. and MASSEYL. M. (1964) Effect of ionizing radiation on plant tissues. III. Softening and changes in pectins and cellulose of apples, carrots and beets.ft. FoodSci. 29, 40-48. 9. KXRT~SZZ. I., MOROANB. H., TLvr'rLEL. W. and LAVlU M. (1956) Effects of ionizing radiation on pectin. Radiation Res. 5, 372-381. 10. MfiARDImF.J. and NEI-mMIASJ.V. (1956) Effects of gamma radiation on the pectic constituents of fruit and vegetables. Food Technol. 10, 599-60 I. I1. MASSEYL. M.,JR. and FAUSTM. (1969) Irradiation effects on polysaccharides, pp. 269-297. In H. W. SCHULTZ (ed.), Symposium on foods: carbohydrates and their roles. AVI Publishing Co., Westport Co., Westport, Conn. 12. Og~a~A S., KRAtmZ R. and GASS~mRE. (1960) Radiation induced susceptibility of substrates to enzymic degradation. Radiation Res. 13, 607-6 12. 13. REEVE R. M. (1959) Histological and histochemical changes in developing and ripening peaches. II. Cell walls and pectins. Am. a7. Botany 46, 241-248.
IRRADIATED PECTIN AS A SUBSTRATE F O R PECTIC ENZYMES 14. ROMANIR . J . (1967) Radiobiological parameters in the irradiation of fruits and vegetables, pp. 57-103. In, Advances in food research, Vol. 15. Academic Press, N. Y. 15. Ro~mui R.J., ROBINSONB.J., R . ~ H. L., MAXlE E. C. and SOMMERN. F. (1963) Fruit irradiationphysical methods. Radiation Botany 3, 345-350. 16. Rouse A. H. and ATKXNSC. D. (1955) Pectinesterase and pectin in commercial citrus juices as determined by methods used at the Citrus
387
Experiment Station. Bull. Fla Agric. Exp. Stn. 570, 6-8. 17. SoMooYI L. P. and ROMANIR. J. (1964) A simplified technique for the determination of pectin methylesterase activity. Anal. Biochem. 8, 498-501. 18. SOMOOYIL. P. and ROMAN1R. J. (1964) Irradiation-induced textural change in fruits and its relation to pectin metabolism, or. Food Sci. 29, 366-371.