[35] P E C T I C E N Z Y M E ASSAYS 329

[35] A s s a y M e t h o d s f o r P e c t i c E n z y m e s
By A L A N COLLMER, JEFFREY L. RIED, and M A R K S. M O U N T

Pectic enzymes are produced in large quantities by many plant-asso-

ciated microorganisms. 1,2 Pectic polymers (chains of predominantly 1,4-
1inked a-D-galacturonic acid and methoxylated derivatives) are major con-
stituents of the middle lamellae and primary cell walls of higher plants. 3
Because of the structural importance and enzymatic vulnerability of these
polymers, pectic enzymes can cause plant tissue maceration, cell lysis, and
modification of cell wall structure, allowing other depolymerases to act on
their respective substrates. 3-5
As typified by the well-characterized extracellular enzyme complexes of
the plant pathogens Erwinia chrysanthemi and Erwinia carotovora, pecto-
lytic microorganisms produce a battery of pectic enzymes differing in
substrate preference, reaction mechanism, and action pattern. 6,7 Pectate
lyases,9 (EC 4.2.2.2), pectin lyase t°,lt (EC 4.2.2.10), and exopolygalactur-
onate lyase 12 (EC 4.2.2.9) cleave by t-elimination and generate products
with a 4,5-unsaturated residue at the nonreducing end. Polygalacturon-
ase 13 (EC 3.2.1.15) and exo-poly-a-l)-galacturonosidase 14,15 (3.2.1.82)
cleave by hydrolysis. The lyases have pH optima of ca. 8.5 and (except for
pectin lyase) require divalent cations. The hydrolases have pH optima at
pH 6.0 or lower and do not require divalent cations. Pectate lyase, pectin
lyase, and polygalacturonase cleave internal glycosidic bonds and generate
a series of oligomeric products. Exopolygalacturonate lyase and exo-poly-

1 D. F. Bateman and R. L. MiUar, Annu. Rev. PhytopathoL 4, 119 (1966).

2 F. M. Rombouts and W. Pilnik, Econ. Microbiol. 5, 227 (1980).
3 M. McNeil, A. G. Darvill, S. C. Fry, and P. Albersheim, Annu. Rev. Biochem. 53, 625
(1984).
4 D. F. Bateman and H. G. Basham, Encycl. Plant Physiol., New Ser. 4, 316 (1976).
5 A. Collmer and N. T. Keen, Annu. Rev. Phythopathol. 24, 383 (1986).
6 j. p. Stack, M. S. Mount, P. M. Berman, and J. P. Hubbard, Phytopathology 70, 267
(1980).
7 j. L. Ried and A. Collmer, Appl. Environ. Microbiol. 52, 305 (1986).
s A. Garibaldi and D. F. Bateman, Physiol. Plant Pathol. 1, 25 (1971).
9 F. Moran, S. Nasun0, and M. P. Starr, Arch. Biochem. Biophys. 123, 298 (1968).
to S. Kamimiya, T. Nishiya, K. Izaki, and H. Takahashi, Agric. Biol. Chem. 38, 1071 (1974).
~' S. Tsuyumu, T. Funakubo, and A. K. Chatterjee, Physiol. Plant Pathol. 27, 119 (1985).
,2 C. Hatanaka and J. Ozawa, Agric. Biol. Chem. 37, 593 (1973).
,3 S. Nasuno and M. P. Starr, J. Biol. Chem. 241, 5298 (1966).
,4 A. Collmer, C. H. Whalen, S. V. Beer, and D. F. Bateman, J. Bacteriol. 149, 626 (1982).
t5 C. Hatanaka and J. Ozawa, Agric. Biol. Chem. 33, 116 (1969).

Copyright© 1988by AcademicPress,Inc.

METHODS IN ENZYMOLOGY, VOL. 161 All rightsof reproduction in any form reserved.
330 PECTIN [35]

a-D-galacturonosidase attack chain termini, release only digalacturonates,

and reduce the viscosity of solutions containing pectic polymers more
slowly than do the endo-attacking enzymes. Pectin lyase attacks pectin
(polymethoxygalacturonide); the other enzymes attack pectate (polygalac-
turonate). Assays for pectinesterase (EC 3.1.1.11), which deesterifies pectin
to pectate and methanol, are comprehensively discussed by Rexovfi-Ben-
kovfi and Markovi~. 16
The characterization and purification of a pectic enzyme is often com-
plicated by the presence of other pectic enzymes produced by the source
microorganism. Three assay procedures that address this problem will be
described; pectic enzymes from E r w i n i a spp. will be used as examples. The
assays are readily adapted to the analysis of pectic enzyme complexes of
other organisms and can be used with crude preparations. Results from
plant tissue extracts should be interpreted cautiously, however, because of
the prevalence of pectic enzyme inhibitors. 5 The activity stains in the first
procedure enable rapid approximation of the number and type of pectic
enzymes in the sample and can guide purification strategies, particularly
when used in conjunction with titration curves. 17 The second and third
procedures permit quantitative assay of hydrolytic and fl-eliminative pectic
enzymes, respectively. The action patterns of purified pectic enzymes are
determined by viscometric 18 and reaction product analyses; oligogalactur-
onides are resolved by paper chromatography19 or thin-layer chromatogra-
phy 2° and detected with bromphenol blue 21 or thiobarbituric acid spray
reagent 22 (which specifically detects 4,5-unsaturated oligogalacturonides).

Activity Stain for R a p i d Characterization of Pectic E n z y m e s

R e s o l v e d b y Electrophoretic P r o c e d u r e s
Principle. Pectic enzymes diffuse from electrophoresis gels into ul-
trathin polygalacturonate-agarose overlays. After a period of incubation,
the overlays are placed in a solution of cetyltrimethylammonium bromide
or ruthenium red, which precipitates and stains the undegraded substrate,
leaving a clear zone in the overlay that corresponds to the location of the
enzymes in the electrophoresis ge1.17'23Pectic hydrolases and lyases present
~6L. Rexovfi-Benkovaand O. Markovi~,Adv. Carbohydr. Chem. Biochem. 33, 323 (1976).
~7y. Bertheau, E. Madgidi-Hervan, A. Kotoujansky, C. Nguyen-The, T. Andro, and A.
Coleno, Anal. Biochem. 139, 383 (1984).
~sD. F. Bateman, Phytopathology 53, 1178(1963).
~9C. W. Nagel and M. M. Anderson, Arch. Biochem. Biophys. 112, 322 (1965).
2oR. T. Zink and A. K. Chatterjee,Appl. Environ. Microbiol. 49, 714 (1985).
21A. L. Demain and H. J, Phaff,Arch. Biochem. Biophys. 51, 114 (1954).
22L. Warren, Nature (London) 186, 237 (1960).
23j. L. Ried and A. Collmer, Appl. Environ. Microbiol. 50, 615 (1985).
[35] PECTIC ENZYME ASSAYS 331

in the same gel are differentially assayed by manipulating reaction condi-

tions in the overlays.
Reagents
GelBond support film for agarose, 110 × 205 m m (FMC Bioproducts,
Rockland, ME)
Glass plates, two, 110 X 205 m m
Teflon spacers, 0.35 × 5 m m
Agarose (Bethesda Research Laboratories, RockviUe, MD; electropho-
resis grade)
Polygalacturonic acid (Pfaltz and Bauer, Inc., Waterbury, CT; product
P21750)
Sodium hydroxide, 1 N
Pectate lyase substrate: 0.1 M tris(hydroxymethyl)aminomethane
(Tris)-HCl buffer, pH 8.5, 1.5 m M calcium chloride, 0.1% (w/v)
polygalacturonic acid
Exo-poly-a-D-galacturonosidase substrate: 0.1 M potassium phos-
phate buffer, pH 6.0, 10 m M ethylenediaminetetraacetic acid
(EDTA), 0.1% (w/v) polygalacturonic acid
Polygalacturonase substrate: 0.1 M sodium acetate buffer, pH 5.3,
10 m M EDTA, 0.1% (w/v) polygalacturonic acid
Ruthenium red (Sigma Chemical Co., St. Louis, MO), 0.05% (w/v) in
water
Cetyltrimethylammonium bromide (Sigma Chemical Co., St. Louis,
MO), 1.0% (w/v) in water

Procedure. The substrate solutions are prepared in advance and stored

frozen. To prevent formation of an insoluble calcium-polygalacturonate
complex during preparation of the pectate lyase substrate, 0.2 g of polyga-
lacturonic acid is first dissolved in 100 ml of 0.2 M Tris-HC1, pH 8.7, and
then 100 ml of 3.0 m M calcium chloride is added. Each of the substrate
solutions must be titrated back to the desired pH with 1 N sodium hydrox-
ide after addition of polygalacturonic acid. Agarose (0.25 g) is then added
to 25 ml of the appropriate substrate solution and heated in a boiling water
bath until it dissolves. The hot solution is then immediately pipetted into
the gel mold with a preheated pipet. The gel mold is assembled by the
following steps. Several milliliters of water are spread on a 110 X 205 m m
glass plate. A sheet of GelBond support film with the hydrophilic side up is
lowered onto the plate. Paper towels are placed on the support film and
excess water is pressed out from beneath it with a rubber roller. A 0.35 )<
5 × 205 m m spacer is placed along one edge of the plate, and then three
0.35 × 5 X 105 m m spacers are placed perpendicularly at the middle and
outer edges. A second glass plate of the same size is laid on the spacers,
332 PECTIN [35]

offset so that 5 m m of the support film is exposed at the open end of the
mold. The plates are damped together and heated to 50 ° in an oven. The
hot polygalacturonate-agarose solution (a little less than 5 ml) is then
carefully delivered to each offset opening in the mold, allowing none to
flow between the support film and the bottom plate. After 30 min at room
temperature, the plates are separated and the support film is cut in half
with scissors along the gap left by the middle spacer. The two overlays can
be used immediately or covered with Saran Wrap (Dow Chemical Corp.,
Indianapolis, IN) and stored at 4 ° in a plastic box containing a wet paper
towel for at least 3 weeks. Assays are initiated by placing the overlay on the
surface of an electrophoresis gel (typically an ultrathin layer isoelectric
focusing gel cast on support film of the same dimensions). Bubbles are
pressed out with a glass rod. After a period of incubation at 30 ° varying
from a few minutes to several hours, depending on the desired sensitivity,
the overlays are placed in solutions of either ruthenium red or cetyltri-
methylammonium bromide for at least 20 rain. Overlays stained with
ruthenium red are then rinsed with water and can be photographed
through a green filter (Wratten No. 58) or dried and stored between plastic
sheets. Overlays precipitated with cetyltrimethylammonium bromide have
a white background and must be stored in the staining solution. The
staining reagents can be reused for several gels. Because the toxicity of
these reagents is unknown, both types of gels are handled with gloves.
Comments. The technique is sufficiently sensitive to detect an isoelec-
trically focused band containing 6.4 × l0 -6 U of pectate lyase activity.23
The sensitivity and high resolution results from the assay mechanism of
detecting zones of enzymatic degradation in a background of limited and
relatively slowly diffusing substrate. The same electrophoresis gel can be
assayed sequentially with substrate overlays buffered to detect pectate
lyases and pectate hydrolases. Overlays buffered for exo-poly-a-D-galactur-
onosidase or polygalacturonase detection can be interchanged without
substantial loss in sensitivity. Pectic enzymes with an exo-cleaving action
pattern will produce partially cleared bands in the overlay. In principle,
pectin lyases should be detectable with overlays containing pectin. When
the pI of the enzyme is substantially different than the pH optimum, the
sensitivity of the assay is enhanced by incubating the electrophoresis gel in
the appropriate buffer before applying the overlay (5 min is optimum for
0.35-mm 5% polyacrylamide isoelectric focusing gels). Pectic enzymes in
0.75-mm 10% polyacrylamide, sodium dodecyl sulfate gels can be rena-
tured by incubating the gels for 2 hr with shaking in three 100-ml changes
of the appropriate buffer (10 m M ) before assaying with substrate overlays.
Not all enzymes renature equally well, and some are inactivated by treat-
ment with dithiothreitol. 23
[35] PECTIC ENZYME ASSAYS 333

Assay for Polygalacturonase

Principle.The increase in reducing groups resultingfrom release of
oligogalacturonatesfrom polygalacturonateis measured with the copper-
arsenomolybdate reagentsof Nelson,24 as modified by Somogyi. 2s
Reagents
Potassium sodium tartrate
Anhydrous sodium carbonate
Cupric sulfate pentahydrate
Sodium hydrogen carbonate
Anhydrous sodium sulfate
Ammonium molybdate
Sulfuric acid
Disodium hydrogen arsenate heptahydrate
Substrate stock solution: 60 m M sodium acetate buffer, pH 5.3,
0.12 M sodium chloride, 6.0 m M EDTA, 0.24% (w/v) polygalactur-
onic acid
o-Galacturonic acid (Sigma Chemical Co., St. Louis, MO)
Procedure. The copper reagent is prepared by dissolving 12 g of sodium
potassium tartrate and 24 g of anhydrous sodium carbonate in 250 ml of
water. The following reagents are then added in sequence while stirring: (1)
a solution of 4.0 g of cupric sulfate pentahydrate in 100 ml of water; (2)
16 g of sodium hydrogen carbonate; and (3) a solution of 180 g of anhy-
drous sodium sulfate in 500 ml of water, boiled before addition to expel
air. The volume of the final solution is brought to 1 liter with water. A
precipitate that forms during the first week is removed by filtration or
sedimentation and the clear blue supernatant is then used in assays. The
arsenomolybdate reagent is prepared by dissolving 25 g of ammonium
molybdate in 450 ml of water and adding 21 ml of 96% sulfuric acid and a
solution of 3.0 g of disodium hydrogen arsenate heptahydrate in 25 ml of
water. The final solution is incubated at 37 ° for 24 hr before use and is
stored in a brown bottle. The substrate stock solution is prepared by adding
20 ml of 0.6 M sodium chloride with rapid mixing to 80 ml of a solution
containing 75 m M sodium acetate, pH 5.3, 7.5 m M EDTA, and 0.3%
(w/v) polygalacturonic acid (final pH adjusted to 5.3 with 1 N sodium
hydroxide). The substrate stock solution can be preserved with 0.02%
sodium azide and stored at 4 °.
Enzyme assays are initiated by mixing 2.5 ml of substrate stock solu-
tion and 0.5 ml of enzyme sample plus water. The reactions are incubated
24N. Nelson, J. Biol. Chem. 153, 375 (1944).
25 M. Somogyi, J. Biol. Chem. 195, 19 (1952).
334 PECTIN [35]

at 30 °. Samples (0.5 ml) are removed from the reaction mixture at timed
intervals and immediately mixed with 0.5 ml of copper reagent (which
stops the reaction). Sample tubes are subsequently incubated in a vigor-
ously boiling water bath (with a marble over each tube) for 10 min. After
the tubes have cooled to room temperature, 1.0 ml of the arsenomolybdate
reagent is added. After a 15- to 40-min incubation at room temperature,
the assay mixture is centrifuged in a clinical centrifuge to remove precipi-
tated residual substrate, and the absorbance of the supernatant is read at
500 nm in a spectrophotometer. The increase in absorbance over time in
reaction samples, relative to a substrate blank or relative to samples taken
at several intervals, is then calculated. These values are used in reference to
a o-galacturonic acid standard curve to determine the rate of oligogalac-
turonic acid formation. One unit of enzyme forms 1 gmol of oligogalac-
turonate in 1 min under the conditions of the assay. Oligogalacturonates as
large as the pentamer yield the same absorbance at 500 nm as galacturonic
acid in the Somogyi-Nelson assay.26
Comments. Purification of polygalacturonase from E. carotovora has
been described. 13 EDTA is present in the reaction mixture only to inhibit
the activity of contaminating pectate lyases. Citrate buffer should be
avoided because it interferes with the Somogyi-Nelson a s s a y . 27 The E.
chrysanthemi exo-poly-ot-D-galacturonosidase is assayed by a similar pro-
cedure.14 A reducing sugar assay based on the formation of UV-absorbing
complexes with 2-cyanoacetamide has been reported to be simpler and
more sensitive than the Somogyi-Nelson assay for pectic enzymes but has
not yet met wide u s e . 28'29

Assay for P e c t a t e Lyase

Principle. The increasing absorbance at 232 nm of 4,5-unsaturated
reaction products is monitored with a recording spectrophotometer.
Reagents
Substrate stock solution: 60 m M Tris-HC1, pH 8.5, 0.6 m M calcium
chloride, 0.24% (w/v) polygalacturonic acid
Procedure. The substrate stock solution is prepared in the manner
described for the pectate lyase substrate in the activity stain procedure. To
initiate the assay, 2.5 ml of the substrate stock and 0.5 ml of enzyme

26 L. Rexov~i-Benkov~, Fur. J. Biochem. 39, 109 (1973).

27 L. G. Paleg, A n a l Chem. 31, 1902 (1959).
28 K. C. Gross, HortScience 17, 933 (1982).
29 S. Honda, Y. Matsuda, M. Takahashi, and K. Kakehl, A n a l Chem. 52, 1079 (1980).
[3 6] PROTOPECTINASE 3 35

sample plus water (both previously equilibrated to 30 °) are rapidly mixed

in a 3-ml cuvette with a 1-cm light path. The subsequent increase in
absorbance at 232 nm is monitored as a function of time with a recording
spectrophotometer. The amounts of enzyme and water that are added
must be empirically adjusted to yield a linear rate of reaction for at least 30
sec. One unit of enzyme forms I pmol of 4,5-unsaturated product in 1 min
under the conditions of the assay. The molar extinction coefficient for the
unsaturated product at 232 nm is 4600 M-1 cm-l.19
Comments. Purification of pectate lyase from E. chrysanthemi and
Escherichia coli clones carrying individual pectate lyase isozyme genes has
been described, s,3°'31 The same assay procedure can be used with exopoly-
galacturonate lyase. 32 The assay for Erwinia chrysanthemi pectin lyase is
altered by the deletion of calcium chloride from the substrate solution and
by the substitution of highly methoxylated Link pectin for polygalacturon-
ate) ~ Tsuyumu et al. ~ describe a recent modification of the method of
Morell et al. 33 for the preparation of this substrate. The molar extinction
coefficient of the unsaturated product is 5550 M -~ c m - ~ : 4'35 The thiobar-
bituric acid procedure of Waravdekar and Saslaw 36 modified by Weissbach
and Hurwitz, 37 provides an alternative procedure for assaying pectic lyases;
4,5-unsaturated products, following treatment with periodic acid, form a
complex with thiobarbituric acid that absorbs maximally at 545-550 nm.

30 C. Schoedel and A. Collmer, J. Bacteriol. 167, 117 (1986).

3, N. T. Keen, D. Dahlbeck, B. Staskawicz, and W. Belser, J. Bacteriol. 159, 825 (1984).
32 j. D. Macmillan and H. J. Phaff, this series, Vol. 8, p. 632.
33 S. Morell, L. Bauer, and K. P. Link, J. Biol. Chem. 105, 1 (1934).
34 p. Albersheim, this series, Vol. 8, p. 628.
35 R. D. Edstrom and H. J. Phaff, J. Biol. Chem. 239, 2403 (1964).
36 V. S. Waravdekar and L. D. Saslaw, Biochim. Biophys. Acta 24, 439 (1957).
37 A. Weissbach and J. Hurwitz, J. Biol. Chem. 234, 705 (1959).

[36] Protopectinase from Yeasts and a Yeastlike Fungus

By TAKUO SAKAI

Protopectin is a water-insoluble pectic substance found in plants. The

decomposition of protopectin was originally attributed to one specific
enzyme, "protopectinase," ~ but further research on pectolytic enzymes

C. S. Brinton, W. H. Dore, H. J. Wichmann, J. J. Willaman, and C. P. Wilson, J. Am.

Chem. Soc. 49, 38 (1927).

Copyright© 1988by AcademicPress,Inc.

METHODS IN ENZYMOLOGY, VOL. 161 All rightsof reproductionin any formreserved.