50

BIOCHEMICAL EDUCATION

AN EXPERIMENT IN ENZYME
CHARACTERIZATION:
BANANA POLYPHENOLOXIDASE

For the past several years we have been presenting a lecturelaboratory course in modern methods of biochemical analysis for
entering graduate students and advanced undergraduates in
nutrition and food science. The course has included an exercise
on the characterization of banana polyphenoloxidase (PPO).
Bananas are readily available the year round in most parts of
the world, and the properties of banana PPO make possible an
unusually wide range of experiments with a single enzyme.
Polyphenoloxidases (o-diphenol:oxygen oxidoreductase, EC
1.10.3.1) are responsible for the enzymatic browning which
occurs in many fresh fruits and vegetables when they are
damaged.t, z
Banana PPO catalyzes the oxidation of various orthodiphenols
to the corresponding quinones. These highly reactive quinones
react nonenzymatically to form ultimately the melanin pigments.
Figure 1 shows these reactions with dopamine, the primary
substrate for enzymatic browning in bananas. 3 The melanins
that occur in foods are formed by polymerization of the oquinones and/or by condensation of the quinones with amino
acids, peptides, or proteins. 2

EXPERIMENTAL
Enzyme extraction. A highly active PPO can be readily
extracted from banana pulp with a buffer containing an agent
to protect the enzyme from precipitation by tannins. 3
Perform all operations at 0-4C. Homogenize 4g of ripe
banana pulp in 36ml of 1% nonionic detergent (e.g., Cutscum,
Fisher Scientific Co., Pittsburgh, Pa., U.S.A.) in 0.02 M
phosphate buffer (pH 7.0). Homogenization is readily
accomplished with a glass tissue grinder (e.g., Corning Glass
No.7726, Coming, N.Y., U.S.A.), or even with a mortar and
pestle. Centrifuge the mixture for 1Smin. at 20,000xg; the
supernatant fluid is utilized for characterization studies. Store at
4C to retain at least 70% of PPO activity for 1 month.
Enzyme assay. Assay banana PPO by measuring the initial
rate of production of the red 2,3-dihydroindole-5,6-quinone from
dopamine (Fig. 1) at 25C. The reaction mixture contains 1.Sml
of 10~M dopamine hydrochloride (Sigma Chemical Co.,St.
Louis, Mo., U.S.A.; prepared daily) as substrat, 0.02-0.1ml of
enzyme, and 0.1M phosphate buffer (pH 7.0), for a final
volume of 3ml. Measure the increase in absorbance (A) at
470nm of the well-mixed solution for 2-3min. A recording
spectrophotometer is convenient, but the same results can be
obtained by measuring A at 15-20 sec intervals in a simple
colorimeter. Dilute the enzyme (usually about 1:50) so that the
initial linear increase in A is 0.02-0.1 per min. Check for
autoxidation of substrate by measuring for any increase in A in
the absence of PPO.
One unit of PPO activity is defined as that amount of enzyme
which catalyzes the transformation of 1/~mole substrate/min
under the conditions of the assay. If specific activity (units/rag
protein) is required, measure protein (Lowry et al.,4 as described
by Layne s) versus a standard curve prepared with purified
bovine serum albumin.

ENZYME CHARACTERIZATION
The following is a selection of projects which have been
successfully completed in our laboratory. It is preferable to carry
out the characterizations on purified enzyme. However,

July 1975 Vol. 3 No. 3

MICHAEL C. ARCHER and

JAMES K. PALMER
D e p a r t m e n t of Nutrition and F o o d Science
M a s s a c h u s e t t s Institute of Technology
Cambridge, Massachusetts 02319, U . S . A .

purification doubles the laboratory time required, and the

results with crude PPO extract do not differ significantly from
those obtained with purified PPO.
Kinetics of thermal inactivation. Knowledge in this area has
practical importance because discoloration of processed fruits
and vegetables is often prevented by thermal inactivation of
PPO.
Incubate enzyme solutions at temperatures of 40-95C for up
to 1Stain; then assay activity at 25C. The data on the timetemperature relationship for thermal inactivation illustrates the
concept of high-temperature, short-time processing, which
inactivates enzymes while avoiding significant "cooking" of the
food. Temperature optimum data can also be determined if
thermostatted cell holders are available.
p H Optimum. Banana PPO has a fairly broad pH profile with
an optimum of 7.0 when catalyzing the oxidation of dopamine. 3
Determine the activity of PPO over the pH range 3.0-8.0,
substituting citrate for phosphate below pH 6.0. It is especially
important above pH 7.0 to correct for autoxidation of substrate.
Km for oxygen using oxygen electrode. PPO can be assayed
by measuring the O2 consumed during polyphenol oxidation
(Fig. 1). This assay can be accomplished via Warburg
manometry, hut the Clark oxygen electrodes is quicker and more
convenient. A simple and inexpensive battery-driven electrode
(Rank Bros., Bettisham, Cambs, England) has been utilized
successfully in our laboratory. The Rank electrode conveniently
incorporates an incubation vessel; the electrode itself is in the
base of the vessel. Oxygen electrodes can also be homemade
relatively easily. 7
Calibrate the electrode to read 100% on a 1-2mV recorder in
stirred buffer saturated with air. Establish the 0% line on the
recorder by adding a few crystals of sodium dithionite to the
buffer to deplete the solution of oxygen. Full-scale deflection
then represents 260/~M oxygen. After thoroughly rinsing the cell
and the electrode, add 3ml 0.1M phosphate buffer (pH 7.0)
and 1 ml 10~ M dopamine to the incubation vessel of the Rank
electrode. Then add sufficient PPO to give a rapid rate of
oxygen uptake, and follow the reaction until the oxygen
conctration approaches zero. The entire reaction should be
completed in 10min or less to minimize interference by product
inhibition (see below). Calculate the rate at various oxygen
concentrations from tangents drawn to the progress curve.
(Bendall and Gregorys have illustrated this method for tea leaf
catechol oxidase.) Determine Km from the double reciprocal
plot of Lineweaver and Burk.S A typical value for Km for
oxygen is 7xl0aM.
Substrate studies. A variety of diphenols and monophenols
can be used to demonstrate the substrate specificity of banana
PPO. Because of the variety of products formed, PPO activity
must be monitored with the oxygen electrode or with a coupled
ascorbic acid assay. In the latter assay, the initial quinone
product reacts with ascorbic acid to form dehydroascorbic acid
and regenerates the original phenol.
For the coupled assay, measure the rate of ascorbic acid
oxidation by measuring the decrease in absorhance at 265nm, in
a reaction mixture containing 0.0.5M phosphate buffer (pH 7.0),
l(Y~ M EDTA, 4.2x 10"SM ascorbic acid, substrate, and enzyme
in a final volume of 3ml (EDTA protects ascorbic acid from
oxidation by trace metal impurities).
Determine Michaelis constants (Kin) for a series of diphenols

BIOCHEMICAL EDUCATION

July 1975 Vol. 3 No. 3

51

from measurements of initial rates at substrate concentrations in

the range 10~ to 10tM. Compounds suitable for study (in
addition to dopamine) are norepinephrine, 3,4-dihydroxyphenylalanine (DOPA), catechol, and chlorogenic acid. a Differences
between the D, L, and DL forms of norepinephrine and DOPA
may also be demonstrated.3
Because DOPA, norepinephrine, and dopamine are all
oxidized to dopachrome (Fig. 1), the K m for these three
substrates can be compared by utilizing the standard assay.
Gregory and Bendall t list a number of other potential substrates
and show typical kinetic data for tea PPO.
Inhibitor studies. Interest in chemical inhibitors of
polyphenoloxidases results mostly from the practical need to
prevent enzymatic browning reactions in fruits and vegetables.hZ
For the inhibitor experiments, assay the banana PPO as
described under Enzyme assay, but add potential inhibitors to
the reaction mixture immediately prior to addition of the
substrate.
Banana PPO, like other phenolases,n is a copper
metallocnzyme and can be inhibited by chelating agents. 12 A
progressive increase in inhibition occurs with increasing
concentrations of phenylthiourea and
cuprizone (Biscyclohexanone oxaldihydrazone) in the range 10-3 to 10SM.
Surprisingly, ethylenediaminetetracetic acid (EDTA), an avid
copper chelater, inhibits this enzyme only at higher
concentrations (approximately 10"2M). This result may indicate
steric exclusion of EDTA from the copper binding site. Some
chelating agents cause complete inhibition for a time, followed
by a rate of oxidation somewhat lower than the uninhibited rate.
Examples in this class are sodium diethyldithiocarbamate and
potassium ethylxanthate (10"3 to 1OSM), and sodium mercaptobenzothiazole~z (10 s to 5x 1OSM). The Oz electrode can be used
to show that there is no oxygen uptake during the lag period,
which indicates a direct inhibition of PPO during the lag.
Banana PPO, like many other enzymes, can be inhibited by
structural analogs of its substrates. 13 For example, 4chlororesorcinol inhibits dopamine oxidation about 60% at
1OSM and about 15% at 10"SM.
Reducing agents appear to inhibit PPO,but they actually
prevent the subsequent nonenzymatic reactions, so that no
colored products are formed. "Reducing agents" is a misnomer
in some cases because several different modes of action have
been described.T M Ascorbic acid in the range los to 10"sM does
act primarily by reducing the quinone back to the original
phenol. Cysteine in the range 10"a to los M probably forms an
addition product with quinones, which is not further oxidized or

polymerized. Sodium disulfite, commonly used in the food

industry to inhibit enzymatic browning, reacts in the range 10"3
to 1OSM to deplete the assay mixture of oxygen. With
"reducing agents", regardless of mechanism, once all the
inhibitor has reacted, formation of colored products will begin,
but often at a rate lower than the original uninhibited rate.
More rigorous experiments on inhibition kinetics and
mechanisms may be carried out, if desired, as outlined by
Gregory and Bendall 1 for tea PPO and by Pierpoint14 for tobacco
PPO.
Product inhibition. Banana PPO, like other phenolases, is
irreversibly inhibited by the product of enzyme action, probably
by an actual reaction of the o-quinone products at the active
site. ts
Measure the time required for a significant decrease (10-20%)
in the rate of dopamine oxidation and for the reaction rate to
approach zero (about 10 and 30 min, respectively). These
experiments emphasize the desirability of measuring initial rates
in enzyme assays. The extent of substrate oxidation at any point
can also be calculated from the absorbance of the assay mixture
and the extinction coefficient of the red product (~ = 2.515; Fig.
1), or from the Oz uptake. Normally, the reaction is essentially
complete when only about 5% of the substrate has been
oxidized. Add more substrate or PPO to the reaction mixture
after the reaction has ceased, to see if either addition reinitiates
the reaction.
Enzyme purification. A 10- to 20-fold purification of the
enzyme can be readily accomplished with a minimum of
equipment, as detailed by Palmer)
First concentrate the PPO in the detergent extract and free it
of most of the detergent by acetone precipitation at about -10C.
Follow this procedure bY chromatography on DEAE-ceUulose
using a step gradient. Elute the inert protein with 0.04M
phosphate buffer (pH 8.0), and then elute the PPO with 0.08M
phosphate at the same pH. Because the PPO peak emerges
immediately after increasing the ionic strength of the buffer, an
automatic fraction collector is not essential.
Polyacrylamide gel electrophoresis (PAGE). This important
technique in protein chemistry can be illustrated with protein
fractions obtained during purification of PPO.
Use operating conditions exactly as defined by Gabriel is for
an anionic enzyme sample (system I, 7.5% acrylamide). In
addition to staining protein bands with Coomassie Blue, PPO
activity can be localized by soaking gels in 10"=M dopamine
solution for about 5 min and then allowing them to stand in air.
PPO activity is shown by development of red-brown bands.

H|

.o

N
DOPAMINE

PPO 4.

"

",

= "To.

H
2,3- OlHY O ROINDOL(
-5,6 QUINONE (RED)
MAX.- 3 0 0 = ~ , L 0 6 ( , 3 . N
470mp, LOG E,5.40

DOPAMINE
QUINONE

SLOW

FAST
4
*~ 0 8

eMELANIN " 7
H
I N O O L [ - 5 , 6 QUI NONE
(PURPLE)
MAX- 540rap

HO
H
6,6 01HY DROXYIN DOLE

Fig. 1. Proposed mechanism for the oxidation of dopamine by banana PPO.

52

BIOCHEMICAL EDUCATION

July 1975 Vol. 3 No. 3

DISCUSSION
Each characterization exercise normally requires about two
laboratory periods of 3hr. each. Two students collaborate on a
selected project for which we supply appropriate equipment,
procedure outlines, and one or two reprints as models. We
encourage the students not simply to reproduce data in the
reprints, but to attempt some additional experiments. For
example, an ammonium sulfate-dialysis step has sometimes been
interposed between acetone precipitation and chromatography in
purifying the enzyme, ascorbic acid has been found to enhance
inhibition by mercaptobenzothiazole, etc. This "research"
approach engenders considerable interest. After completion of
the exercises, the class meets in a seminar and each group
summarizes its procedures, results, and conclusions.

6 Strickland, E. H., Ziegler, F. D., and Anthony, A., Nature

191:969 (1961).
r Foster, J. M., Bioscience 19:541 (1969).
B Bendall, D. S. and Gregory, R. P. F., in Enzyme Chemistry of
Phenolic Compounds (Pridham, J. B., ed.), Pergamon Press,
1963, p. 7.
9 Lineweaver, H. and Burk, D., J. Am. Chem. Soc. 5 6 : 6 5 8
(1934).
a0 Gregory, R. P. F. and Bendall, D. S., Biochem. J. 101:569
(1966).
n

Kertesz, D. and Zito, R., in Oxygenases (Hayaishi, O., ed.),

Academic Press, 1962, p. 134.

a2 Palmer, J. K. and Roberts, R. B., Science 157:200 (1967).

REFERENCES
i Eskin, N. A. M., Henderson, H. M., and Townsend, R. J.,
Biochemistry of Foods, Academic Press, 1971, p. 83.
2 Mathew, A. G., and Parpia, H. A. B., Advan. Food Res. 19"
75 (1971).
3 Palmer, J. K., Plant Physiol. 38:508 (1963).
4 Lowry, O. H., Rosebrough, N. J., Farr,
Randall, R. J.,J. Biol. Chem. 193:265 (1951).

5 Layne, E., in Methods in Enzymology, Vol. 3 (Colowick, S. P.

and Kaplan, N. O., eds.), Academic Press, 1957, p. 488.

A.

L.,

and

Membranes and their Cellular Functions

By J. B. F i n e a n , R. C o l e m a n a n d R. M . M i c h e l l . P p 124.
Biackwell
Scientific
Publications,
Oxford.
1974.
P a p e r b a c k , 2.80.
After a long period of relative quiescence, research into the
structure and role of membranes has received fresh impetus as a
result of the development of new methodology and techniques.
Not surprisingly, therefore, most of the old concepts have, at the
very least, been modified or more often abandoned altogether.
An important and rapidly expanding new dimension is being
added to the fields of Biochemistry and Cell Biology. As is
usually the case, however, this new knowledge has undergone a
lag phase before its introduction into the standard text books. It
is to fulfil this need that three members of the Department of
Biochemistry at Birmingham University have compiled a short
text which incorporates most of the current thoughts on
membrane structure and function. The Book appears to be
geared principally for the undergraduate worker and, less
obviously, to non-specialists in the area.
The material is divided into six chapters starting with a
general introduction to the concept of membranes, their
structure, function and diversity and approaches to their study.
Subsequent chapters deal with the part played by the membrane
in various transport phenomena, cell surface interactions and
the control and organisation of certain metabolic processes.
Finally the Authors discuss in more detail recent developments
relating to the organisation and dynamics of this structural
feature of all cells. On the whole, the organisation of the various
sections follows a logical progression except for the final
chapter. Much of the material in this section could be more
profitably included very much earlier in the book. For example,
the idea of fluidity is mentioned long before its discussion in the
last chapter.
The subject matter covered in this book is surprisingly
comprehensive for a volume of this size. Most aspects of
membrane form and function have been mentioned if, at times,
only very briefly. It is not to be expected in such a rapidly
changing field that the information given is the most up to date
but the Authors have certainly dealt with those areas where
something akin to revolutionary change is taken place. In this

13 Kull, F. C., Grimm, M. R., and Mayer, R. L., Proc. Soc.

Exptl. Biol. Med. 86:330 (1954).
14 Pierpoint, W. S., Biochem. J. 98:567 (1966).
15 Brooks, P. W. and Dawson, C. Z., in The Biochemistry of
Copper (Peisach, J., Aisen, P., and Blumberg, W. C., eds.),
Academic Press, 1966, p. 346.
16 Gabriel, O., in Methods in Enzymology, Vol. 22 (Jakoby, W. J.,
ed.), Academic Press, 1971, p. 565.

respect, therefore, readers can be reasonably sure of obtaining a

sound background to the subject. Not so satisfactory, however,
is the content in terms of experimental design and evidence.
This is a regrettable omission perhaps necessitated by their
desire to produce a relatively inexpensive text, but it does
prevent the authors stressing important developments or
proceeding beyond the stage of broad and in some cases
misleading generalisations. The result is a certain uniformity of
emphasis which is not helpful to the uninitiated student. It is
perhaps particularly disappointing that the action of detergents
receives little attention since anyone reading original
publications in the field of membranology is certain to be faced
with the use of these substances.
The format of the book is admirable, the text flows without
interruption and by using displaced subtitling, the reader is
afforded the facility of easy reference. The style is pleasantly
readable and, if at times a little clumsy, nevertheless maintains
the interest of the reader. However, the frequent use of
analogies
to
explain
particular
situations
in
readily
understandable terms is only partially successful. This is always
a tricky technique to introduce since it often leaves the reader
with a mistaken impression. A singularly unfortunate example of
this occurs on the very first page where the membrane of a cell
is likened to a two-dimensional stockade. Research workers in
the field of transport would I suspect be rather perturbed by the
impression that small substances could easily penetrate this
"stockade" but most larger ones succeed only by "jumping",
"climbing", "flying" or using "controlled gates".
One of the most dominating features of this book is the
extensive use of diagrams. Like the written style, this aspect
illustrates both the considerable strengths and flaws of this
publication. By and large the drawings are both clear and
extremely helpful but on occasion some of the cartoons appear
to confuse rather than clarify. This is particularly noticeable
when related to the transport properties of the cell. It is only
with second sight that one can interpret these diagrams - - and
surely although amusing, this defeats the object.
Notwithstanding these grumbles, however, the authors have
succeeded in producing a text which all undergraduates in the
biological sciences would do well to read.
John Findlay