[18] BROMELAIN ENZYMES 273

detected only in the latex of F. carica and was absent in the leaves, stem,
and fruit portions of the plant.
Studies of the ficin content of different species of the genus F/cus
have revealed significant differences in proteolytic activity.~°,~1 Of 46
species of Ficus examined, only 13 exhibited appreciable proteolytic
activity; the latex of F. stenocarpa had the highest specific activity,
followed closely by the latices of F. carica and F. glabrata. ~1 A total of
as many as 26 chromatographically distinct active components were
detected in 6 species of Ficus.
Variations in activity have also been observed in different varieties of
the same species of Ficus. Thus, in an examination of 25 varieties of F.
carica, Whitaker 32 observed a 2-fold variation in the casein-digesting
activity of the latex. The number and relative amounts of the active
components of F. carica and F. glabrata differed among varieties within
the same speciesJ ~,"~ For example, in the latices of 9 varieties of F. carica,
a total of at least 16 chromatographically distinct components were
detected, the Kadota variety alone showing 10 distinct active components.
The complexity of this problem is further aggravated by the fact that
there are more than 1800 named species of Ficus, a number of subspecies,
and at least 700 varieties of F. carica aloneJ ° The importance of defining
the source of material and the preparation and properties of the purified
components cannot be emphasized too strongly.

~B. H. Robbins and P. D. Lamson, I. Biol. Chem. 106, 725 (1934).

4,D. C. Williams, V. C. Sgarbieri, and J. R. Whitaker, Plant Physiol. 43, 1083 (1968).

[18] B r o m e l a i n E n z y m e s
B y TAKASHI MURACHI

Nomenclature
Bromelain enzymes are found in tissues of plant family Bromeliaceae
of which pineapple, Ananas comosus (L), is the best known. The pro-
teolytic enzyme found in the juice of pineapple stem is called stem
bromelain and the enzyme in the fruit was first described under the
name of bromelin I and is now called fruit bromelain. 2 Systemic number
EC 3.4.3.24 is given to bromelain.

1R. H. Chittenden, Trans. Connecticut Acad. Sci. 8, 281 (1892).

2R. M. Heinicke and W. A. Gortner, Econ. Botany 11, 225 (1957).
274 ~I]~ CYSTEINE PROTEASES [lS]

Stem Bromelain

A s s a y Method
Principle. Like most proteolytic enzymes, stem bromelain catalyzes
the hydrolysis of protein substrates as well as synthetic substrates.
Casein is most commonly used as a protein substrate and the assay is
made by measuring increase in the amount of trichloroacetic acid-
soluble peptides as a function of time. Various modifications of the
Kunitz method ~,4 are used. The esterase activity of stem bromelain may
be assayed by following the hydrolysis of a-N-benzoyl-L-arginine ethyl
ester (BAEE) in a pH-stat2 Ammonia liberated is determined by a
combination of the microdiffusion technique and the indophenol method
with sodium nitroprusside as catalyst2

Assay ]or Caseinolytic Activity"

Reagents
Casein, 1.2 g/dl. Dissolve 3 g of Hammarsten grade casein in 250
ml of 0.03 M potassium phosphate buffer, pH 7.5, by heating in a
boiling water bath for 15 minutes. The final pH is 7.2.
L-Cysteine, 0.15 M, freshly prepared.
Trichloroacetic acid solution. Dissolve 9 g of trichloroacetic acid,
15 g of sodium acetate, and add 19.5 ml of glacial acetic acid in
water to make a final volume of 500 ml.
Enzyme. The concentration of the enzyme solution should be more
than 20 #g/ml.
Procedure. Place 5.0 ml of casein solution and 0.2 ml of cysteine in a
1.8 X 18 cm test tube and add water and enzyme to give a final volume
of 6.0 ml. After incubation at 35 ° for 10 minutes, add 5.0 ml of trichloro-
acetic acid solution rapidly, shake the mixture well and then allow it to
stand for 30 minutes at 35 ° . Remove the coagulated protein either by
filtration or by centrifugation. Read the absorbance of the filtrate or
the supernatant solution at 275 nm against the blank. Inclusion of
cysteine in the blank run is necessary, since cysteine causes a significant
increase in absorbance at 275 nm of the casein filtrate. The optimal range
for the enzyme is 40 to 80 #g enzyme protein per tube.
Definition of a Unit and Specific Activity. A proteinase unit is defined
a M. Kunitz, J. Gen. Physiol. 39, 291 (1947).
~B. Hagihara, H. Matsubara, M. Nakai, and K. Okunuki, J. Biochem. (Tokyo)
45, 185 (1958).
~T. Inagami and T. Murachi, Biochemistry 2, 1439 (1963).
*A. L. Chaney and E. P. Marbach, Clin. Chem. 8, 130 (1962).
[18] BROMELAIN ENZYMES 275

as the amount of enzyme that gives an increase in absorbance at 275

nm equivalent to 1 t~g of tyrosine per minute at pH 7.2 and at 35°. 4
Specific activity is expressed as units of enzyme per milligram of protein.

Assay/or Esterolytic Activity 5

Reagents
a-N-Benzoyl-n-arginine ethyl ester (BAEE), 0.1 M, adjusted to
pH 6.O
L-Cysteine, 0.05M, freshly prepared
KCI, 1.0 M
NaOH, 0.1 N
Enzyme. The concentration of the enzyme solution should be more
than 1 mg/ml.
Procedure. The reaction is carried out at 25 °. A Radiometer model
SBR2/SBU1/TTT1 Autotitrator (pH-stat) is used. A syringe-type
buret of 1-ml capacity is filled with 0.1 N. NaOH. Place 5.0 ml of BAEE,
1.0 ml of L-cysteine, and 1.0 ml of KC1 in a reaction vessel of 30-ml
capacity. Adjust the pH to 6.0, and initiate the reaction by adding water
and enzyme to give a final volume of 10.0 ml. Follow the hydrolysis of
the ester substrate by recording the uptake of alkali for a period of l0
to 20 minutes during which the rate of the hydrolysis is practically
constant.
Definition o] a Unit and Specific Activity. An esterase unit is defined
as the amount of enzyme that catalyzes the hydrolysis of 1 micromole
of BAEE per minute at pH 6.0 and 25 °. Specific activity is expressed
as units of enzyme per milligram of protein.

Assay ]or Amidase Activity 5

Reagents
a-N-Benzoyl-L-arginine amide (BAA), 0.05 M, adjusted to pH 6.0
L-Cysteine, 0.05 M, freshly prepared
KC1, 0.1 M
Potassium phosphate buffer, 0.15 M, pH 6.0
Enzyme. The concentration of the enzyme solution should be more
than 2 mg/ml.

Procedure. Incubation is carried out at 25 °. Place 0.5 ml of BAA, 0.5

ml of L-cysteine, 0.5 ml of KC1, and 1.0 ml of buffer in a 1.5 X 15 cm
test tube. Add water and enzyme to give a final volume of 5.0 ml. With-
draw 0.5-ml aliquots from the reaction mixture at 10-minute intervals
276 THE CYSTEINE PROTEASES [18]

and determine the amount of ammonia liberated in each aliquot. The rate
of the reaction is practically constant for the initial 40 minutes.
Definition of a Unit and Specific Activity. An amidase unit is defined
as the amount of enzyme that catalyzes the hydrolysis of 1 micromole
of BAA per minute at pH 6.0 and at 25 °. Specific activity is expressed
as units of enzyme per milligram of protein.

Other Methods of Assay

Hemoglobin may be used as a substrate for proteinase activity. 7
Ninhydrin reaction can be used for determining ammonia liberated from
BAA. s a-N-Benzoyl-DL-arginine p-nitroanilide is an alternative amide
substrate. 9 2-Mercaptoethanol at a final concentration of 0.005M can
be used for L-cysteine as an activator2 Bromelain can be assayed also
by measuring its milk-clotting activity. 1°

Purification Procedure 1~
Starting Material. CommerciaI bromelain may be obtained from the
Dole Company, Honolulu, Hawaii. The product is acetone-dried powder
of the juice of pineapple stem. 2
Step 1. Extraction. Ten grams of commercial bromelain is suspended
in 100 ml of 0.05 M potassium phosphate buffer, pH 6.1, at room tem-
perature for 30 minutes. The suspension is centrifuged at 5000 rpm for
20 minutes and the precipitate is discarded.
Step ~. Treatment with Anion-Exchange Resin. This and all of the
following steps of the procedure are carried out at cold room temperature.
The supernatant fluid (fraction 1) is applied to a 4.5 X 19 cm column of
Duolite A-2 (coarse), which has been equilibrated overnight with 0.05 M
potassium phosphate buffer, pH 6.1. The initial 100 ml of the effluent is
discarded and the following 400-ml effluent (fraction 2) is collected by
a continuous flow of the same buffer. A part of the colored material is
removed by this treatment.
Step 3. Treatment with Cation-Exchange Resin. Fraction 2 is applied
to a 4.5 X 31.5 cm column of Amberlite CG-50, Type 1 (100-200 mesh),
which has been equilibrated with 0.2 M potassium phosphate buffer,
pH 6.1. The colunm is washed with 200 ml of the same buffer and then
with 5 liters of 0.05 M potassium phosphate buffer, pH 6.1, overnight.

~T. Murachi and H. Neur~th, J. Biol. Chem. 235, 99 (1964).

~S. Moore and W. H. Stein, J. Biol. Chem. 211, 907 (1954).
S. Ota, S. Moore, and W. H. Stein, Biochemistry 3, 180 (1964).
1oM. E1-Gharbawi and J. R. Whitaker, Biochemistry 2, 476 (1963).
11T. Murachi, M. Yasui. and Y. Yasuda. Biochemistry 3, 48 (1964).
[18] BROMELAIN ENZYMES 277

The effluent is discarded. The enzyme adsorbed is eluted by washing the

column with 800 ml of 0.2 M potassium monohydrogen phosphate con-
taining 1 M KC1. The initial 250 ml of the effluent is discarded and the
following 250-ml eluate (fraction 3) is collected.
Step ~. Gel Filtration. To fraction 3 is added 100 g of ammonium
sulfate. After 1 hour the precipitate formed is collected by centrifugation
at 8000 rpm for 20 minutes and is dissolved in 50 ml of 0.05 M sodium
acetate buffer, pH 5.2 (fraction 4). Fraction 4 is applied to a 4.5 X 31.5
cm column of Sephadex G-100 (140-400 mesh) which has been washed
with 0.05 M sodium acetate buffer, pH 5.2. The column is washed with
the same buffer and the initial 320-ml effluent is discarded. The following
150 ml effluent (fraction 5) is collected.
Step 6. Ammonium Sul]ate Fractionation. To fraction 5 is added
38.5 g of ammonium sulfate to make 0.42 saturation. After 1 hour the
moderately turbid mixture is centrifuged and to the supernatant fluid is
added 8.8 g of ammonium sulfate to bring the suspension to 0.50
saturation. Bromelain thus precipitated is collected by centrifugation at
8000 rpm for 20 minutes, dissolved in 30 ml of water, and dialyzed
against four changes of 5 liters of water for 48 hours (fraction 6).
The whole procedure of purification up to fraction 6 can be completed
within 5 days, including 2 days for final dialysis. Table I summarizes
the purification procedure. The gel filtration is essential to remove the
contaminant carbohydrates present in fraction 4. Care should be given
not to collect earlier fractions of the effluent which contain carbohydrates
of larger molecular size. 11 The purified preparations are kept frozen and
retain full activity for over a year.

Alternate Purification Procedure 9

Two hundred milligrams of commercial bromelain preparation is dis-
solved in 4.2 ml of cold 0.02 M sodium citrate buffer, pH 5.5. After in-
soluble material has been removed by centrifugation at 30,000 g for 15
minutes, the supernatant solution (4.0 ml) is applied to a 2 X 75 cm
column of Sephadex G-75 (medium particle size) which has been
equilibrated at 25 ° with 0.02 M sodium citrate buffer, pH 5.5, saturated
with phenylmercuric acetate (less than 10-4M). The column is washed
with the same buffer at a flow rate of 10 ml per hour at room temperature.
Several effluent fractions with high proteolytic activity are pooled and
added directly to a 2 X 35 cm column of sulfoethyl-Sephadex (C-25, fine
mesh size, 2.0 meq/g) equilibrated with 0.3M sodium citrate buffer,
pH 6.0, containing 5 X 10-4 M phenylmercuric acetate. The column is
washed with the same buffer at room temperature and the fractions that
have high proteolytic activity are pooled. The main component isolated
2~8 THE CYSTEINE PROTEASES [18]

~ddddd

dddddd

~z dddddd

r8

o
[18] BROMELAIN ENZYMES 279

accounts for 42% of the total protein and 60% of the caseinolytic
activity of the original crude stem bromelain.

Properties
Unless otherwise noted, description of properties will be made below
for the step 6 preparation obtained by the procedure of Murachi et al. ~
Stability. The enzyme retains full activity against casein when kept
at 5 ° for 24 hours over a range of p H from 4 to 10. 5 T h e enzyme is stable
in 25% ( v / v ) methanol at 25 ° for 20 minutes, while it loses 33%
caseinolytic activity in 20% ( v / v ) ethanol at 37 ° for 20 minutes. ~
A 50% loss of the activity is caused by heating the enzyme solution at
55 ° for 20 minutes at p H 6.10. ~° Lyophilization causes 27% decrease in
the activity. ~
Purity. The purified enzyme is homogeneous by ultracentrifugal
sedimentation, free-boundary electrophoresis, and diffusion analyses. ~
C h r o m a t o g r a p h y on Amberlite CG-50, CM-Sephadex, D E A E - S e p h a d e x ,
or SE-Sephadex yields a single s y m m e t r i c a l p e a k 2 ,~ The homogeneity
of the enzyme is verified by disc electrophoresis on acrylamide
gel. ~ The chromatographically purified enzyme, which migrates as a
single band upon electrophoresis on cellulose acetate, 9 contains small
but significant amounts of extraneous end groups in addition to the

TABLE II
PHYSICAL PROPERTIES OF STEM BROMELAIN

Sedimentation constant, s 20,w

o 2.73 S
Diffusion constant, D~0.w 7.77 × 10-7 cm2 sec-1
Partial specific volume, V 0.743 ml/g
Intrinsic viscosity, [~] 0.039 dl/g
Frictional ratio, fifo 1.26
Isoelectric point, pI 9.55
Absorbancy, ~ %c m at 280 nm
~A1 20.1 ~
ORD parameters, ~ 241 nm
-ao 190°
-b0 78
Molar ellipticity, I0]~ -4200 b
Molecular weight 33,200, c 32,100, ~ 33,500,
a Revised value [T. Murachi, T. Inagami, and M. Yasui, Biochemistry4, 2815 (1965)].
bT. Sakai, K. Ikeda, K. Hamaguchi, and T. Murachi, Biochemistry 9, 1939 (1970).
By sedimentation-diffusion.
From sedimentation constant and intrinsic viscosity,
By Archibald method.

T. Murachi, unpublished observations.

"L. P. Chao and I. E. Liener, Biochem. Biophys. Res. Commun. 27, 100 (1967).
280 THE CYSTEINE PROTEASES [18]

terminal valine2 In view of this and other facts, particularly the observa-
tions made by Whitaker and associates I°,14 that stem bromelain can be
fractionationated further into 5 proteolytically active components, each
having a different amino acid composition, strict homogeneity of the
"step 6 preparation" can hardly be claimed at the present moment.

TABLE III
AMINO ACID COMPOSITION OF STEM AND FRUIT BROMELAINS

Fruit bromelain b
Stem bromelain a
Green Ripe
Amino acid 1 2 3 4 fruit fruit

Lysine 20 23 12 20.2 7.8 8.3

Histidine 1 2 1 1.34 1.4 1.3
Arginine 10 12 6 10.2 8.6 9.1
Aspartic acid 27 29 16 28.9 29.8 29.8
Threonine 12 14 8 12.4 13.5 13.8
Serine 24 28 16 24.9 32.2 32.0
Glutamic acid 20 23 12 23.0 23.2 23.4
Proline 13 14 8 13.1 11.6 12.0
Glycine 29 35 19 30.5 32.6 32.2
Alanine 30 35 20 32.4 23.8 24.4
Half-cystine 11 10 5 7.4 10.0 10.0
Valine 19 22 12 20.7 19.8 20.1
Methionine 4 5 2 5.0 6.0 5.8
Isoleucine 20 21 12 18.4 16.4 16.2
Leucine 9 10 5 9.0 10.0 10.0
Tyrosine 19 21 11 18.2 22.4 22.2
Phenylalanine 9 9 5 8.0 7.6 8.0
Tryptophan 8 8 5 -- 5.6 --
Total (285) (321) (179)
Ammonia (amide) 25 42 19 -- 43.0 43.4
Glucosamine 2c 6 4 -- <0.2 < 0.2
Carbohydrate (%) 2.1 1.46 2.0 -- 3.2 3.3

a Sources of values are as follows. Column 1 : T. Murachi, Biochemistry 3, 932 (1964).

N u m b e r of residues per mole of M W 33,000. Column 2: S. Ota, S. Moore, a n d W. H.
Stein, Biochemistry 3, 180 (1964). N u m b e r of residues per mole of M W 35,730.
Column 3: G. Feinstein and J. R. Whitaker, Biochemistry 3, 1050 (1964). For
component II, t a k e n methionine as two residues per molecule. Column 4: S. S.
Husain and G. Lowe, Biochem. J. 110, 53 (1968). Taken leucine as nine residues
per molecule.
b S. Ota, S. Moore, a n d W. H. Stein, Biochemistry 3, 180 (1964). Mole ratios with
leucine set as 10.
c Revised value [N. Takahashi, Y. Yasuda, M. Kuzuya, and T. Murachi, J. Biochem.
(Tokyo) 66, 659 (1969)].

14G. Feinstein a n d J. R. Whitaker, Biochemistry 3, 1050 (1964).

[18] BROMELAIN ENZYMES 281

Physical Properties. 11 Physical constants of stem bromelain protein are

listed in Table II. The data suggest that the enzyme is a basic protein
with a molecular weight of approximately 33,000. It is more basic and
about 1.5 times larger in size if compared with papain. From values of
-bo and molar ellipticity at 222 nm, the content of a-helix in stem
bromelain can be calculated to be approximately 10%. A time-dependent
and finally irreversible conformational change occurs at pH values higher
than 10.3. ~, ~a
Chemical Properties. In Table III are shown the amino acid composi-
tions of stem bromelain reported by different investigators. ~,~e,l~ The
principal amino terminal residue is valine 9 and the carboxyl terminal is
glycine28 The enzyme is a glycoprotein having one oligosaccharide moiety
per molecule which is covalently linked to the peptide chain. ~9,~9~ The
proposed structure of undecaglycopeptide isolated from the pepsin
digest of stem bromelain is 2o-22~.
L-Fuc D-Xyl
~)
D-Man D-Man-
I (~1 --* 6 or 2)
-D-Man D-G: cNAe
(al --~ 2) (al --* 2 or 6) (a) ~1 --* 3 or 4)
D-G]cNAc
(~1 --* t~NHrN)
Ala-Arg-Val-Pro-Arg-Asn-~ tsn-Glu-Ser-Ser-Met
Stem bromelain has one reactive sulfhydryl group per molecule as
determined by titration with p-ehloromereuribenzoate. This sulfhydryl
group is essential for catalytic activity. ~3 The reported amino acid
sequences of the active site are:
Cys-Gly-Ala-Cys-Trpls
Asn-Gln-Asp-Pro-Cys-Gly-Ala-Cys-Trp
u

UA. Tachibana and T. Murachi, Biochemistry 5, 2756 (1966).

~" T. Murachi and M. Yamazaki, Biochemistry 9, 1935 (1970).
~T. Murachi, Biochemistry 3, 932 (1964).
1'S. S. Husain and G. Lowe, Biochem. J. 110, 53 (1968).
~8S. Ota, Seikagaku 37, 433 (1965) (Abstract, in Japanese).
~T. Murachi, A. Suzuki, and N. Takahashi, Biochemistry 6, 3730 (1967).
~ J. Scocca and Y. C. Lee, J. Biol. Chem. 244, 4852 (1969).
~K. Kito and T. Murachi, J. Chromatog. 44, 205 (1969).
~tN. Takahashi, Y. Yasuda, M. Kuzuya, and T. Murachi, J. Biochem. (Tokyo)
66, 659 (1969).
T. Murachi and N. Takahashi, in "Structure-Function Relationships of Proteolytic
Enzymes" (P. Desnuelle, H. Neurath, and M. Ottesen, eds.), p. 298, Munksgaard,
Copenhagen, 1970.
Y. Yasuda, N. Takahashi, and T. Murazhi, Biochemistry 9, 25 (1970).
~ T. Murachi and M. Yasui, Biochemistry 4, 2275 (1965).
282 ~. CYSTEIN~. rROTF~S~.S [18]

where Cys indicates the reactive cysteinyl residue.

The reagent 1,3-dibromoacetone cross links a cysteinyl and a histidyl
residue within the same enzyme molecule. The amino acid sequence
around the latter residue is described2~:
His-Ala-Val-Thr-Ala-Ile-Gly-Tyr
The result is interpreted as showing the presence of the imidazole group
of a histidyl residue within 5 A of the reactive sulfhydryl group.
Activators and Inhibitors. The purified enzyme obtained by the
routine procedure 11 shows 60-70% activity as assayed by casein hy-
drolysis in the absence of activatorsY 8 The enzyme can be fully activated
in the presence of 0.005 M cysteine, 2-mercaptoethanol, or dithiothreitol.
K C N is less effective.7 Stem bromelain is reversibly inhibited by inorganic
mercuric ion, 7 organic mercurials, ~'23 and tetrathionate. 22 Irreversible
inhibition occurs when stem bromelain is reacted with N-ethylmaleimide,
N-(4-dimethyl-3,5-dinitrophenyl)maleimide ( D D P M ) , 25 monoiodoacetic
acid, 18 and 1,3-dibromoacetoneY' These reagents alkylate the essential
sulfhydryl group of the enzyme protein. Chloromethyl ketone derivative
of N-tosyl-L-phenylalanine (TPCK) and 1-chloro-3-tosylamido-7-amino-
2-heptanone (TLCK) also alkylate the SH group, resulting in inactiva-
tion of the enzyme. 22'26 The second-order rate constant of the reaction
between the enzyme and T P C K or TLCK is 2.3 or 11.9 1 mole -~ sec-~,
respectively, as determined at pH 7.0 and 30°. 22 Diisopropylphosphoro-
fluoridate (DFP) does not inhibit stem bromelain but it alkylphos-
phorylates the enzyme-protein at pH 8.2 without inhibition of proteinase
activity. 28 The alkylphosphorylation occurs at the phenolic hydroxyl
groups of tyrosyl residues. 27 The diisopropylphosphorylated enzyme
shows an alteration in specificity toward synthetic substratesY 8
Specificity and Kinetic Properties. The specificity can be described
as being broad, since a variety of synthetic substrates are hydrolyzed by
stem bromelain as shown in Table IV. Basic amino acyl residues are
preferred, but the preference is less strict than in the case of papain. The
fact that in bromelain catalysis kcat for BAEE is 140 times as large as
kcat for BAA, is in sharp contrast to the finding that with papain or
ficin kcat values are almost identical for the ester and for the correspond-
ing amide substrates, suggesting some important difference in the

24S. S. Husain and G. Lowe, Chem. Commun. p. 1387 (1968).

25T. Murachi, T. Miyake, and M. Mizuno, Abstr. Intern. Congr. Biochem. 7th
August 1967, Tokyo, p. 765.
a T . Murachi and K. Kato, J. Biochem. (Tokyo) 62, 627 (1967).
27T. Murachi, T. Inagami, and M. Yasui, Biochemistry 4, 2815 (1965).
~8T. Murachi, Abstr. Intern. Congr. Biochem. 6th July 1964, New York, p. 324.
[18] BROMELAIN ENZYMES 283

TABLE IV
SUBSTRATE SPECIFICITY AND KINETIC PARAMETERS OF STEM BROMELAIN

Rate of hydrolysis
of 0.01 M substrate

X lOs Km(app) kc~t

Substrate (sec-1) (%) (M) (sec-1)

Benzoyl-L-arginine ethyl ester (BAEE) 29.4 100 0.17 0.50

Benzoyl-L-arginine amide (BAA) 3.1 10.5 0.0012 0.0035
Benzoyl-L-arginine methyl ester 91.7 312 0. 032 0.11
Tosyl-~arginine methyl ester 14.8 50.3
Tosyl-~lysine methyl ester 3.96 a 13.4 ~ 0. 0 8 4 , 0.035 ~
L-Lysine ethyl ester 2.81 9.56
L-Histidine ethyl ester <0.1 --
Glycine ethyl ester 5.40 18.4
Benzoylglycine ethyl ester 11.9 40.5 0.21 a 0.36"
Benzoylglycine amide 0.28 0.96
Acetylglycine ethyl ester <0.2 -- 33" 0.55 ~
Benzoyl-DL-alanine ethyl ester 3.96 13.5
L-Leucine ethyl ester 5.48 18.6
L-Phenylalanine ethyl ester 8.60 29.3
L-Tyrosine ethyl ester 6.39 21.7

T. Murachi, unpublished data.

mechanism of catalysis? Stem bromelain rapidly cleaves glucagon at

either ArK (18)-Ala (19) or Ala (19)-Gln (20) bond, while it leaves intact
L y s ( 1 2 ) - T y r ( 1 3 ) and Arg(17)-Arg(18) bonds. 7 The B-chain of oxidized
insulin is a relatively poor substrate for bromelain. ~ Bradykinin is rapidly
and almost exclusively cleaved between Phe(5) and Ser(6).22.29

F r u i t Bromelain

Assay M e t h o d
The assay procedures and reagents are the same as those for stem
bromelain except that a smaller amount of fruit enzyme is needed for
assaying the hydrolyses of casein, a-N-benzoyl-L-arginine amide, and
a-N-benzoyl-DL-arginine p-nitroanilide2 There is no report of the assay
for the esterolytic activity of fruit bromelain.

Purification Procedure 9
Acetone Powder o] Fruit Juice. The fresh fruit, green or ripe, is freed
of leaves and epithelium and the juice is obtained by pressing with a

2~T. Murachi and T. Miyake, Physiol. Chem. Phys. 2, 97 (1970).

284 THE CYSTEINE PROTEASES [18]
hydraulic press. The juice (pH 3.2-3.5) is cooled to 00-4 ° and 1 volume
of cold acetone is added. The precipitate is discarded. The enzyme is
precipitated by the addition of two more volumes of cold acetone and
the precipitate is collected by centrifugation and dried under reduced
pressure. The dried product is ground to a powder in a mortar.
DEAE-CeUulose Chromatography. Two hundred milligrams of the
acetone powder is added to 10 ml of cold 0.02 M sodium citrate buffer,
pH 6.0. After centrifugation at 30,000 g for 15 minutes, 9 ml of the
clear supernatant solution is applied to a 2 X 20 cm column of DEAF,-
cellulose (0.96 meq/g) which has been equilibrated at 25 ° with 0.02 M
sodium acetate buffer, pH 6.0, containing 5 X 10-~ M phenylmercuric
acetate. The buffer change elutes the adsorbed enzyme. Several fractions
that have high proteolytic activity are obtained and are pooled.
Yields of the products are summarized in Table V.

TABLE V
PURIFICATION OF FRUIT BROMELAIN

Step Yield

Acetone powder 3.3-3.7 g per liter of juice

DEAE chromatography
Green fruit 43% of total protein; 88% of activity
Ripe fruit 32% of total protein; 87% of activity

Properties ~
Unlike stem bromelain, the fruit enzyme is an acidic protein. The
enzyme is apparently homogeneous as judged by reehromatography and
electrophoresis on cellulose acetate. The principal NH2-terminal residue
is alanine (0.9 residue per 3 X I0 ~ g protein), but additional NH2-
terminal residues are noted (valine 0.~, serine 0.2, and glycine 0.1). The
amino acid compositions of the enzymes from green and ripe fruits are
shown in Table III. The fruit enzyme also contains carbohydrate that
cannot be removed by the purification procedures used thus far. Fruit
bromelain is much more active against BAA than the stem enzyme. The
enzyme catalyzes synthesis of acylamino acid anilides2 ° Fruit bromelain
is inhibited by mercurials and the activity is restored by eysteine.

wS. Ota, T. Fu, and R. Hirohata, J. Biochem. (Tokyo) 49, 532 (1961).