Inhibition Study of EDTA and PCMB on Purified Bromelain Activity

from Pineapple Core [Ananas comosus (L.) Merr.] Using Ion

Exchange Chromatography Column and Antiplatelet Activity Test

Nita Magfirah Ilyas

, Siswati Setiasih

and Sumi Hudiyono

Department of Chemistry, FMIPA Universitas Indonesia, Kampus UI Depok, Depok 16424, Indonesia

Keywords: Pineapple core, bromelain, purification, chromatography, kinetic study, antiplatelet.

Abstract: The aim of this research was to isolate and purify bromelain from pineapple core (Ananas comosus) through

fractionation using ammonium sulfate followed by dialysis and ion exchange chromatography.

Fractionation of bromelain with ammonium sulfate produces highest specific activity on fraction 2 (20-

50%) of 260.042 U/mg with purity level 2.548 fold compared to crude extract. Further purification by ion

exchange chromatography DEAE-sepharose and CM-Sephadex C-50 showed an increase in specific

activity, sequentially 500 U/mg with purity level 4.901 fold compared to crude extract and 729.167 U/mg

with purity level 7.150 fold compared to crude extract. The determination of kinetics parameter of purified

bromelain using LM plot gives Km and Vmax value for casein and azocasein substrate of 0.94% (w/v); 0.07

U/min and 0.87% (w/v); 0.05 U/min respectively. Bromelain fraction was inhibited competitively with

EDTA and mix-inhibition type was observed in the presence of PCMB. The addition of EDTA and PCMB

at a concentration of 0.5 ppm can decrease the activity of the enzyme up to 70%. In vitro study of

antiplatelet agent activity using human Platelet Rich Plasma (PRP) revealed that the purified bromelain

show activity as an antiplatelet agent with percentage of aggregation 20.892% and percentage of inhibition

77.994%.

1 INTRODUCTION

The fifth largest pineapple producer in the world is

Indonesia (Rugayah, 2012). Bromelain is a group of

cysteine proteases found in all plant tissues of

pineapple [Ananas comosus (L.) Merr.] (Babagana,

2016). This enzyme break down a protein by

breaking a peptide bond and produce a more simple

peptide. Bromelain can be found in all parts of

pineapple plants. Stem bromelain (EC 3.4.22.32) is a

glycoprotein with isoelectric pH 9.5 and Fruit

Bromelain (E.C. 3.4.22.33) lacks carbohydrate

moiety with isoelectric pH 4.6 (Kaur, 2015).

This enzyme is used in many therapeutic

applications and was known has antithrombic effect

as antiplatelet agent that useful in cardiovascular

diseases treatment (Bhattacharya, 2008). The

benefits of bromelain is quite extensive in the field

of pharmacology and the food industry, stimulate

many researchers to learn more. Bromelain research

in pineapple has been started for a long time until

now. Various methods of isolation and purification

have been done to get the best bromelain activity

from pineapple (Neta, et al, 2012).

Various heavy metals such as mercury, cobalt,

and zinc can inhibit bromelain enzyme. In 2012,

Marshall and Golden characterized bromelain from

Morinda citrifolia (Noni) and observed that

inhibition of bromelain by HgCl2 is the non-

competitive inhibition type. (Kaur, 2015). Protease

group is inhibited by PCMB (p-

Chloromercuriobenzoate), PMSF

(Phenylmethylsulphonyl fluoride), EDTA

(Ethylenediaminetetraacetic acid), benzamidine, and

pepstatin A (Walsh, 2002). Several factors that can

influence enzyme kinetics are the enzymes source

and conditions such as pH or temperature

environment (Kaur, 2015).

In this research the crude bromelain was

extracted from pineapple core and purified through

several stages. We also study the kinetics behavior

in the presence of inhibitor and antiplatelet activity

test by in vitro method.

Ilyas, N., Setiasih, S. and Hudiyono, S.

Inhibition Study of EDTA and PCMB on Puriﬁed Bromelain Activity from Pineapple Core [Ananas comosus (L.) Merr.] Using Ion Exchange Chromatography Column and Antiplatelet Activity

Test.

DOI: 10.5220/0009843200002406

In Proceedings of BROMO Conference (BROMO 2018) - Symposium on Natural Product and Biodiversity, page 1

ISBN: 978-989-758-347-6

2 EXPERIMENTAL

2.1 Enzyme Extract from Pineapple

Core

The pineapples that were used in this research

were obtained from Palembang, Kramat Jati Market.

Crude enzyme was prepared by making pineapple

core puree using 0.2 M phosphate buffer, pH 7.0 at

4°C and then filtered. The filtrate had been

centrifuged at 6000 rpm for 20 minutes at 4°C. The

supernatant obtained from each sample was crude

bromelain extract. The crude bromelain was stored

in a refrigerator to be used as a source of enzyme.

2.2 Fractionation of Enzyme with

Ammonium Sulfate

Fractionation of crude bromelain was done by

using ammonium sulfate at different range of

concentration (0-20%, 20-50%, 50-80%). About 150

ml of crude enzyme extract was put in a beaker glass

which was placed in a salt ice bath. The crude

enzyme was slowly added by ammonium sulfate (0-

20%) with constant stirring using a magnetic stirrer.

After the addition of salt was completed, the stirring

process had been continued for 20 minutes. The

solution was then allowed to settle overnight in the

refrigerator. The solution was centrifuged (6000

rpm) for 20 minutes at 4

C and the precipitate was

dissolved in 0.2 M phosphate buffer, pH 7.0.

2.3 Dialysis

The enzyme solution was put into a cellophane bag.

The cellophane bag was then immersed in a solution

of 0.05 M phosphate buffer, pH 7.0. The dialysis

was taken place at 4°C using ice salt bath by

constant stirring. The buffer was changed for every 2

hours.

2.4 Protein and Enzyme Activity Assay

Lowry method is used to determine the protein

concentration. The enzymatic activity assay was

perfomed by Kunitz method using casein as

substrate at 37°C for 30 minutes. The enzyme was

inactivated by adding 3 ml of 5% TCA. The solution

then had been incubated in ice water bath for 30

minutes.

2.5 Kinetics Studies

2.5.1 Determination of Kinetics Parameter

Kinetic parameters (Km: Michaelis-Menten constant

and Vmax: maximum velocity) were determined

from Lineweaver Burk Plot between the enzyme

activity at optimum pH and temperature.

2.5.2 The Effect of EDTA and PCMB on

Bromelain Activity

The absence and presence of EDTA (0,1; 0,3 and 0,5

mM) and PCMB (0,1; 0,3 and 0,5 mM) were used to

assay the bromelain activity. Km and Vmax were

calculated using Lineweaver Burk plot.

2.6 In vitro Antiplatelet Activity Test

The antiplatelet activity test of the enzyme

bromelain fraction was carried by Born method. The

absorbance of the sample solution was measured

before and after the addition of aggregators.

Aggregators that used was ADP (Adenosine

Diphosphate 5'). Samples that tested were crude

enzymes and enzyme fractions obtained from each

stage of purification with the highest specific

activity. Aspirin was used as a positive control and

distilled water is used as a negative control.

In the sample tubes containing enzyme sample

(70 ml), was added PRP (Platelet Rich Plasma) (560

ml) and saline water and then shaken with a vortex

(low speed). The solution was incubated for 2 min at

37 °C. The absorbance of the solution was measured

by a spectrometer at a wavelength of 600 nm. Once

measured, the solution was added by 70 ml of ADP

and incubated for 10 min at 37 °C, subsequently re-

measured the absorbance. For the control solution is

done the same way, but the sample solution is

replaced with distilled water (negative control) and

asetosal 1 mg / ml (positive control). Calculation of

antiplatelet action can be seen from the percentage

inhibition of platelet aggregation and presentation of

aggregation that occurs. Calculation of percentage

inhibition were also performed to determine how

large the inhibition of platelet aggregation in each

solution (Moriyama, 2009).

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

3 RESULTS AND DISCUSSION

3.1 Purification of Bromelain from

Crude Extract by Ammonium

Sulfate Precipitation

Crude bromelain extract from pineapple core has

been purified by fractionation using ammonium

sulfate, followed by dialysis. Crude enzyme

obtained from pineapple core have a specific activity

of 102.018 U/mg protein. The next stage to purify

crude enzyme was the fractionation method using

ammonium sulfate with different of range

concentrations produced enzyme fractions that have

different proteolytic activity and protein content

(Table 1). Fraction 2 of pineapple core has higher

proteolytic activity than fraction 1 and 3.

Table 1: Purification of Crude Enzyme by using Ammonium Sulfate

Fraction

Volume

(mL)

Total

Specific

Activity

(U/mg)

Purity

Level

(Fold)

Activity

(

)

Protein

(

Fraction 1

(

0-20%

)

7.1 122.356 0.818 149.465 1.465

Fraction 2

(20-50%)

11 223.116 0.858 260.042 2.548

Fraction 3

(

50-80%

)

4 32.4 0.532 60.902 0.596

Leftove

186 306.9 8.556 35.869 0.351

Table 2 shows that the specific activity before

and after dialysis. The fraction 2 from the pineapple

core after dialysis has the highest specific activity.

This is because in dialysis some proteins that has

molecular weight less than bromelain can pass

through the cellophane membrane. The specific

activity of bromelain from pineapple core is 381.287

U/mg protein with a purity level of 3.737 fold to the

crude enzyme extract.

Table 2: The Result of Dialysis on Bromelain Enzyme

Fraction

Volume

(mL)

Total

Specific

Activity

(U/mg)

Purity

Level

(Fold)

Activity

(U)

Protein

(mg)

Fraction 2

Before Dial

sis

8 223.116 0.858 260.042 2.548

Fraction 2

After Dial

sis

11 208.450 0.546 381.287 3.737

3.2 Bromelain Fractionation with Ion

Exchange Chromatography

Columns

Ion exchange chromatography column is an

advanced stage to purified bromelain. In this

research, the reversible interaction between charged

molecules and a resin which has the opposite charge

of the protein molecules will be affected by the

eluent through the column. This method is called

stepwise gradient elution (gradient method and

elution rise). If the ion charge of protein molecules

was same with resin’s ion charge, then the molecules

weren’t bound and eluted out first. The molecules

that have different ion charge interacted with the

resin in different strengths, then it required eluent

that has a gradually concentration to release protein

molecules bound from the resin. The resin that used

as the stationary phase in the column is a DEAE

(diethylaminoethyl) Sepharose (anion exchange

resin). Here are the values of proteolytic activity

against fraction number can be mapped in the form

of chromatograms and had 5 peak proteins (Figure

1).

Inhibition Study of EDTA and PCMB on Puriﬁed Bromelain Activity from Pineapple Core [Ananas comosus (L.) Merr.] Using Ion

Exchange Chromatography Column and Antiplatelet Activity Test

0, 25

0, 5

0, 75

0 102030405060708090100110120

Proteolytic

A c tiv ity

(U /m L)

Protein

Absorbance

(2 8 0

nm )

Fraction

Number

Pr otein

(280

nm) Prot eolytic

Activity

(U/mL)

Figure 1: Anion exchange chromatography DEAE – Sepharose Chromatogram. SP: protein absorption peak (280 nm); AP:

proteolytic activity peak.

The value of proteolytic activity, protein content and specific activity at this stage of purification by ion-

exchange chromatography column in Table 3.

Table 3: Results of Purification Phase by Ion Exchange Column Chromatography with DEAE-Sepharose

Fraction

Volume

(mL)

Total

Specific

Activity

(U/mg)

Purity

Level

(Fold)

Activity

(U)

Protein

(mg)

AP1

(13-20)

40 13.320 0.612 21.764 0.213

AP2

(

36-55

)

100 28.300 0.110 257.272 2.521

AP3

(

59-80

)

110 143 0.286 500 4.901

AP4

(82-89)

40 44 0.272 161.764 1.585

AP5

(

92-115

)

70 56 0.224 250 2.450

Further purification, the bromelain fraction was

purified by ion exchange chromatography CM

(carboxymethyl)-Sephadex C-50 (cation exchange

resin). Here are the values of proteolytic activity

against fraction number can be mapped in the form

of chromatograms and had 4 peak proteins (Figure

2).

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

0,2

0,4

0,6

0,8

1,2

1,4

1,6

1,8

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45

0,5

0 10 20 30 40 50 60 70 80 90 100 110 1 20 130 140

Proteolytic

A c tiv ity

(U /m L)

Protein

Absorbance

(2 80

nm )

Fraction

Number

Protein

280

nm Proteolytic

Activity

(U/mL)

Figure 2: Cation exchange chromatography CM–Sephadex C-50 Chromatogram. SP: protein absorption peak (280 nm); AP:

proteolytic activity peak.

The value of proteolytic activity, protein content and specific activity at this stage of purification by ion-

exchange chromatography column in Table 4.

Table 4: Results of Purification Phase by Ion Exchange Column Chromatography with CM–Sephadex C-50

Fraction

Volume

(mL)

Total

Specific

Activity

(U/mg)

Purity

Level

(Fold)

Activity

(

)

Protein

(

AP1

(16-27)

60 35 0.048 729.167 7.150

AP2

(

32-47

)

80 10.667 0.240 44.444 0.435

AP3

(

53-67

)

75 7.500 0.450 16.67 0.163

AP4

(88-100)

65 1.083 0.104 10.417 0.102

3.3 Kinetics Studies

3.3.1 Determination of Kinetics Parameter

Kinetic parameters (Km and Vmax) were

determined from Lineweaver Burk Plot between the

enzyme activity at optimum pH and temperature in

Figure 3 and 4.

Inhibition Study of EDTA and PCMB on Puriﬁed Bromelain Activity from Pineapple Core [Ananas comosus (L.) Merr.] Using Ion

Exchange Chromatography Column and Antiplatelet Activity Test

Figure 3: Lineweaver Burk Plot for the Hydrolysis of Casein by Purified Bromelain.

Figure 4: Lineweaver Burk Plot for the Hydrolysis of Azocasein by Purified Bromelain.

The value of Kinetics Parameter and Proteolytic Activity Bromelain on Casein and Azocasein in Table 5.

Table 5: Kinetics Parameter and Proteolytic Activity Bromelain on Casein and Azocasein.

Substrate

Proteolytic

Activity

(

U/mL

)

(%)

Vmax

(U/min)

Casein 1.133 0.94 0.07

Azocasein 0.866 0.87 0.05

Based on these data, the value of Km fraction of

bromelain on casein and azocasein is relatively same

and low. A low Km bromelain on casein and

azocasein indicate that the Enzyme-Substrate

complex is good with a high affinity between

bromelain to both substrates. Several factors that can

influence enzyme kinetics are the enzymes source

and conditions such as pH or temperature

environment (Kaur, 2015).

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

3.3.2 The Effect of EDTA and PCMB on

Bromelain Activity

The absence and presence of EDTA (0,1; 0,3 and 0,5

mM) and PCMB (0,1; 0,3 and 0,5 mM) were used to

assay the bromelain activity. Lineweaver Burk plot

in the presence of EDTA and PCMB were used to

calculate Km and Vmax (Figure 5 and 6). Bromelain

fraction was inhibited competitively with EDTA and

mix-inhibition type was observed in the presence of

PCMB.

Figure 5: Lineweaver Burk Plot for the Hydrolysis of Casein in the Presence of EDTA (0,1; 0,3; 0,5 mM).

Figure 6: Lineweaver Burk Plot for the Hydrolysis of Casein in the Presence of PCMB (0,1; 0,3; 0,5 mM).

Inhibition Study of EDTA and PCMB on Puriﬁed Bromelain Activity from Pineapple Core [Ananas comosus (L.) Merr.] Using Ion

Exchange Chromatography Column and Antiplatelet Activity Test

The value of Kinetics Parameter and Inhibition Percentage Bromelain by EDTA and PCMB in Table 6.

Table 6: Kinetics Parameter and Inhibition Percentage Bromelain by EDTA and PCMB

[I]

Inhibitor (I)

EDTA PCMB

(

)

Vmax

(

U/min

)

%Inhibition

(

)

Vmax

(

U/min

)

%Inhibition

0 0.940 0.070 - 0.940 0.070 -

0.1 1.780 0.070 33.824 2.000 0.040 22.059

0.3 2.532 0.075 39.706 2.322 0.030 61.765

0.5 4.676 0.072 55.882 2.900 0.020 70.588

The addition of EDTA and PCMB at a concentration of 0.5 ppm can decrease the activity of the enzyme up

to 70%. Bromelain belongs to the cysteine protease containing thiol groups. PCMB is an organic compound

that reacts through a mercury-sulfur affinity with a sulfhydryl group in peptides, proteins and other molecules.

PCMB interacts and reacts with thiol groups in bromelain and therefore PCMB is an inhibitor that can inhibit

the reaction between bromelain and casein substrate. The interaction between cysteine protease (bromelain) and

PCMB is highly dependent on the reactivity of the thiol (-SH) group.

3.4 Antiplatelet Activity Test In Vitro

Antiplatelet agent activity test (in vitro) was used to

observe the value of the platelet aggregation

percentage and inhibition of platelet aggregation

percentage. Platelet aggregation (thrombosis) is the

ability of platelets to form a clot in the blood. The

percentage of platelet aggregation showed the effect

from a compound on the process of platelet clot

formation, while the percentage of inhibition of

platelet aggregation showed the ability from a

compound to inhibit the aggregation process. If the

inhibition percentage was high, it shows that the

compound was more active as well as the ability of

these compounds as antiplatelet agents. An in vitro

study using Platelet Rich Plasma (PRP) with

turbidimetric method (Sathyapriya, 2012). The

platelet inhibition percentage by bromelain fraction

shown in Figure 7 and 8.

100

Aquadest

Dialysis DEAE-Sephar ose CM-Sephadex

Bromelain

Std Asetosal

94,939

51,772

36,100

20,892

5,031

2,22 7

Aggregation

Enzyme

Fraction

Figure 7: Percentage of platelet inhibition by bromelain fraction (Aggregation Percentage)

BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity

100

Dialysis DEAE-Sepharose CM-Sephadex

Bromelain

Std Asetosal

45,469

61,976

77,994

94,700

97,655

Inhibition

Enzyme

Fraction

Figure 8: Percentage of platelet inhibition by bromelain fraction (Inhibition Percentage)

4 CONCLUSIONS

Bromelain was successfully purified through several

purification methods. The result of bromelain after

purified by ion chromatography exchange CM-

Sephadex C-50 gave the highest specific activity.

Bromelain fraction was inhibited competitively with

EDTA and mix-inhibition type was observed in the

presence of PCMB. The addition of EDTA and

PCMB at a concentration of 0.5 ppm can decrease

the activity of the enzyme up to 70%. All samples

with the highest specific activity of each stage of

purification has the ability as an antiplatelet agent.

The highest value of antiplatelet activity was from

the purest enzyme fraction that purify using ion-

exchange chromatography column with percentage

of aggregation 20.892% and percentage of inhibition

77.994%.

ACKNOWLEDGEMENTS

This research was funded by Penelitian

Unggulan Perguruan Tinggi (PUPT)

Universitas Indonesia 2018.

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Inhibition Study of EDTA and PCMB on Puriﬁed Bromelain Activity from Pineapple Core [Ananas comosus (L.) Merr.] Using Ion

Exchange Chromatography Column and Antiplatelet Activity Test

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BROMO 2018 - Bromo Conference, Symposium on Natural Products and Biodiversity