Journal of practice biopharmaceutical and pharmacokinetics

STUDY LITERATURE ABOUT PROTEIN BINDING WITH DRUG USING DYNAMIC

DIALISYS METHOD
Adelia Oktarini, Adnan, Al-Aina, Arina Manasikana, Dyah Ayu Setyarini, Fiony Larasati,
Khairunnisa, Putri Asgaf, Riza Indah Sari, Yutry Rahmi
Email : Farmasiunsri2014@gmail.com
Studi Program of Pharmacy Faculty of Mathematic and Science Sriwijaya University
ABSTRACT
A research on protein binding using dynamic dialysis method. Dynamic dialysis method is based on the
rate of loss of the drug from the dialysis cell is proportional to the concentration of unbound drug. The
drugs used are tetracycline, and blood serum, blood plasma and egg membrane as a binding protein.
Tetracycline to form a bond with plasma proteins in varying amounts this because tetracycline is able to
bind to the protein as much as 65%. The study of protein binding is done by using a UV-VIS
spectrophotometer to determine tetracycline drug release based on various time intervals. Their binding
proteins in blood plasma with tetracycline drugs can be characterized by an increase in the value of the
absorbance at UV-VIS spectrophotometer. Obtained values spread or distributed the amount of drugs
and drug release percent are more in blood plasma and serum and than the group As the amount is
more than the group Bs. At the normality test above 0.05 normality values obtained from all aspects.
So we can conclude significant data means normally distributed.
Keywords: study of protein binding, dynamic dialysis method, tetracycline

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Journal of practice biopharmaceutical and pharmacokinetics

INTRODUCTION

is also too dependent on the pH of plasma and

A critical factor in the distribution of

aged., For example on barbiturates acidosis,

drugs is binding proteins (protein binding),

protein binding in newborns is lower than in

especially plasma proteins, tissue proteins and

adults because of differences in sensitivity

red

(Anief,

blood

cells.

Corresponding

chemical

2000).................................................

structure of proteins, the protein binding can be

Tetracycline compounds derived from

through an ionic bond, hydrogen bridge bonds

Streptomyces rimosus. Alkaline, yellow, and

and

hydrophobic

taste bitter and very low solubility in water.

interactions. This explains why the protein can

These compounds can be administered orally or

bind multiple compounds. But the bond to the

parenterally. Distribution in the body fairly

protein is typically not a typical bond. In human

quickly and the drug can reach almost all

serum albumin can be proven there are two

tissues and body fluids. Excretion through the

distinct

urinary and biliary system (Lachman, 1989).

bond dipole-dipole and

binding

sites

(Syukri,

2002)..................................................

Giving

intravenous

tetracycline

Some compounds are bound selectively on

preferred over intramuscular because the drug

only one of the two binding sites, for example

levels are higher and longer maintained in

antikoagulansia I dikumarol at the binding site,

serum. Drugs that enter the body either orally

the benzodiazepine binding site II and there are

want any parenteral going through a series of

some substances that can be attached to either

biotransformation process, enter the blood

bond. Protein binding is alternating. Ties that

circulation and binds to receptors in the tissue

do not commute (covalent) eg sitostatika

and eventually be eliminated. In the blood drug

reaction that mengalkilasi protein, does not

will be bound by plasma proteins (protein-

belong to the protein binding (Agoes, 2008).

bound drug) or in the form of free compound

The greater the affinity constant of the

(drug

material is concerned, the proteins, the stronger

free)

(Chein,

1987).

Protein binding affect the intensity of

the binding protein. As far as the affinity

work,

length

of

employment

and

the

constants of various proteins, for example to

elimination of ingredients as follows: part of

plasma proteins and protein networks are

drug bound to plasma proteins can not diffuse

different, then the equilibrium distribution of

and generally do not undergo biotransformation

Judah are affected and the equilibrium will shift

and elimination. Means the drug in free form

to proteins with greater affinity. Protein binding

that can achieve the goals that can be

Pharmacy Faculty of Mathematic and Science Sriwijaya University

Journal of practice biopharmaceutical and pharmacokinetics

efficacious. The bound form also serves as a

of human blood plasma, human blood serum 1

backup. If there are several drugs in the blood,

mL, and eggshell membrane.

the possibility of competition for places is

bound to affect the intensity and duration of
work, especially if bound> 80%. It should be
remembered also that the drug also can expel
the body's own substances, such as bilirubin or

Procedures

glucocorticoids from its bond to plasma

1. Preparation of Standard Stock Solutions

proteins and lead to parts that are not bound to

be increased (Williams and Barry, 2004).

Standard stock solution prepared from

tetracycline 100 mg dissolved in 100 mL of
distilled water, then take 10 ml and dilute to
100

mL.

RESEARCH METHODS
2. Preparation of Solutions

Time and place

Pipette 0.2 standard stock solutions; 0.4;

This research was conducted at the

and

0.6; 0.8; 1 and 1.5 mL in 10 mL volumetric

on

flask and adjust the volume to obtain a

September 7th 2016 and September 9th 2016 at

concentration range of 2-15 mg / mL.

Laboratory

of

Biofarmaceutics

Pharmacokinetics
Sriwijaya

University

1.00-5.00 p.m.
3. Measurement Absorbance
Standard

Tools and materials

Equipment used includes UV-Vis
spectrophotometer

(Shimadzu

magnetic

dynamic

solution

absorbance

measurement made at a wavelength of 360 nm

using

UV-Vis

spectrophotometer

and

EB,

Japan),

dialysis

setup,

measuring the absorbance of water as a blank.

analytical weigher (Shimadzu EB, Japan),

Create plot a graph of absorbance against

spatulas, measuring cups, beaker glass, flask,

concentration and tetukan slop and intercept.

stirrer,

pipette, yarn, open cylinders glass, centrifuge,

4. Preparation of Eggshell Membrane

(Shimadzu EB, Japan) and cuvette.

Materials used include tetracycline (PT.

Eggshell membrane can be obtained by

Brataco Chemika Bogor), HCl 5 N, (PT.

soaking the raw chicken eggs in a solution of

Brataco Chemika Bogor), distilled water, 1 mL

HCl 5 N, let eggshell submerged until softened

then separate the eggshell membrane from the

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Journal of practice biopharmaceutical and pharmacokinetics

shell with a hole in the top of the egg and

distilled

water

until

clean.

remove its contents. Membranes have been

separated from their shells are washed with

5. Study of Protein Association Against

Tetracycline

Observations

eggshell

pipette 1 mL of sample and replace with 1 mL

membrane fastened at one end of the glass

of distilled water at time intervals of 5, 10, 15,

cylinder open as protein compartment. Use

30,

beaker glass containing 20 mL of water as non-

spectrophotometer

protein compartment. Place the drug (1 mg /

experiment using 1 mL of human blood plasma

mL) of 2 mL into a tube and dip into a beaker

and drug solution (2 mg / 1 mL) and the

glass, keep the drug solution precisely where

percentage of drug released with the same time

there

the

period. Repeat the experiment using 1 mL

compartment and set it upright. Stir using a

serum of human blood and drug solution (2

magnetic sitirrer on non protein compartment

mg / 1 mL) and specify the percentage of drug

and keep the temperature at 35C. Measure the

released. Create a chart plots the percent

absorbance of the solution of tetracycline with

cumulative

is

were

water

made

on

the

using

outside

of

60,

90

minutes
(

360

using
nm).

UV-Vis
Repeat

DATA ANALYSIS
1. Standard Absorbances
Concentratio
ns
0,2
0,4
0,6
0,8
1
1,5

Absorbances
A
B
0,15
0,161
0,162
0,172
0,238
0,298

0,151
0,162
0,167
0,175
0,207
0,343

So we get the regression equation as follows:

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Journal of practice biopharmaceutical and pharmacokinetics

Curve A

Curve B

0.4
absorbance

0.4

0.2f(x) = 0.12x + 0.11

R = 0.89

absorbancei

f(x) = 0.14x + 0.09

0.2
R = 0.84
0

0 0.5 1 1.5 2
concentration

0 0.5 1 1.5 2
concentration

In this study, carried out the preparation of a

value of the regression equation Y = 0,1423x +

standard stock solution to first use tetracycline

0,941 and R = 0,8353. From both the data

drugs with different concentrations.Based on

obtained a calibration curve obtained curve is

the results of the above data, the data obtained

not linear because the R value <0,999. From

absorbance getting up each time interval.So that

data above, it can be seen that the calibration

a group of data obtained with the value of

curve is more linear in group A than group B.

regression equation Y = 0,1185x + 0,1072 with

This is because the value of R in group A is

a value of R = 0,8904, while group B with the

higher than group B.........................................

2. Absorbance tetracycline
Times
5
10
15
30
60
90

Absorbances
A
0,368
0,362
0,318
0,253
0,211
0,202

B
0,378
0,375
0,327
0,223
0,205
0,201

So we get the regression equation as follows:

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Journal of practice biopharmaceutical and pharmacokinetics

curve b

curve A

0.4

0.4
f(x) = - 0x + 0.36
0.2 R = 0.84
absorbnce

absorbance

f(x) = - 0x + 0.36
0.2 R = 0.74
0

50

100

time

50

100

time

Based on the results of the above data, the data

concentration of the drug from the drug

obtained absorbance decreases each time

absorbance data results with the protein. From

interval A group of data obtained with

data above, it can be seen that the calibration

persaaman regression Y = -0,002x + 0,3565

curve is more linear in group A than group B.

with a value of R = 0,8399. While data group B

This is because the value of R in group A is

absorbance value obtained regression equation

higher than group B.

Y = -0,0022x + 0,361 with a value of R =

0.7409. So that the calibration curve obtained is
not linear because the R value <0.999. From the
data obtained can be used to find the
3. Absorbances of tetracycline data + plasma blood
Times
5
10
15
30
60
90

Absorbances
A
B
0,211
0,201
0,279
0,273
0,298
0,32
0,349
0,405
0,399
0,423
0,447
0,441

So we get the regression equation as follows:

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Journal of practice biopharmaceutical and pharmacokinetics

curve A

curve B

0.6

0.6

0.4

f(x) = 0x + 0.25
R = 0.9

absorbance 0.2

0.4

f(x) = 0x + 0.26
R = 0.72

absorbace 0.2

0
0

50

100

50

time

100

time

Based on the results of the above data, the data

obtained absorbance increasing each time
interval. From a group of data values obtained
regression equation Y = -0,002x + 0,245 with a
value of R = 0,895 and than from group B
obtained regeresi value equation Y = 0,0024 +
0,2591 with a value of R = 0.7216.Although the

absorbance increases at each time interval but a

calibration curve obtained is not linear it is
because value R of the two curves <0.999.
From data above, it can be seen that the
calibration curve is more linear in group A than
group B. This is because the value of R in
group A is higher than group B.

4. Absorbances of tetracycline data + serum

Times

Absorbances
A
B
0,246
0,237
0,297
0,281
0,354
0,339
0,386
0,415
0,428
0,43
0,463
0,456

5
10
15
30
60
90

So we get the regression equation as follows:

curve A

curve B

0.6

0.5

0.4

f(x) = 0x + 0.29
R = 0.83

absorbance 0.2
0

f(x) = 0x + 0.28
R = 0.76

absorbance
0

50
time

100

50

100

time

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Journal of practice biopharmaceutical and pharmacokinetics

Based on the results of the above data, the data

the absorbance increases, but the calibration

obtained absorbance increasing each time

curve obtained is not linear it is because the R

interval. From data of group A values obtained

value <0,999. From data above, it can be seen

regression equation Y = -0,002x + 0.2581 with

that the calibration curve is more linear in

a value of R = 0,8284. Data from group B

group A than group B. This is because the value

obtained regeresi value equation y = 0,0023 +

of R in group A is higher than group B.

0,2794 with a value of R = 0,7558. Although

5. Percent Cumulative Drug
a.Tetracycline Drug
Group A

Amount of

Cumulative

Diffused

Percent

Drug (mg)

(%)

5
10
15
30
60
90

0,1097
0,1071
0,0887
0,0613
0,0437
0,0399

10,97
21,68
30,55
36,68
41,04
45,03

Group B

5
10
15
30
60
90

Amount of Diffused

Cumulative Percent

Drug (mg)

(%)

0,1
0,0989
0,082
0,0454
0,0391
0,0377

10
19,89
28,09
32,63
36,54
40,31

Experimental test of the amount of drug that

0,0399 mg. So we get the cumulative percent of

spreads

release.

drug release by 45,03%. At trial and found the

Cumulative percent drug release determines

number of drugs that spread in minutes to 5, 10,

how much of the drug is released into the

15, 30, 60, and 90 of 0,1 mg ; 0,0989 mg ;

membrane. The amount of drug that is spread is

0,0082 mg ; 0,0454 mg ; 0,0391 mg ; 0,0437

proportional to the cumulative percent. The

and 0,0399 mg so we get the cumulative

higher the amount of drug that spreads the

percent of drug release by 40,31%. The value of

higher the value percent cumulative. For A trial

the cumulative percent in group A is higher than

found the number of drug spread in minutes to

group B. This indicates that the drugs which are

5, 10, 15, 30, 60, and 90 of 0,1097 mg ; 0,1071

bounded in group A is lower than in group B.

percent

cumulative

drug

mg ; 0,0887 mg ; 0,0613 mg ; 0,0437 and

b.Drug tetracycline + blood plasma

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Group A

Amount of

Cumulative

Diffused

Percent

Drug (mg)

(%)

5
10
15
30
60
90

0,0437
0,0723
0,0803
0,1017
0,1227
0,1429

4,37
11,59
19,62
29,79
42,06
56,35

Group B

5
10
15
30
60
90

Amount of Diffused

Cumulative Percent

Drug (mg)

(%)

0,0151
0,0258
0,0318
0,0438
0,0463
0,0488

1,51
4,09
7,27
11,65
16,28
21,16

Experimental test of the amount of drug that

At trial and found the number of drugs that

spreads

release.

spread in minutes to 5, 10, 15, 30, 60, and 90 of

Cumulative percent drug release determines

0,0151 mg ; 0,0258 mg ; 0,0318 mg ; 0,0438

how much of the drug is released into the

mg ; 0,0463 and 0,0488 mg.So we get the

membrane. The amount of drug that is spread is

cumulative

proportional to the cumulative percent. The

21,16%.The value of the cumulative percent at

greater the amount of drug that spreads the

A is higher than group B. This indicates that the

greater the value per cent cumulative. For A

drug is bound to an A to be lower than in the

trial found the number of drug spread in

experiments B. From experimental results the

minutes to 5, 10, 15, 30, 60, and 90 of 0,0437

amount of drug that spreads percent cumulative

mg ; 0,0723 mg ; 0,0803 mg ; 0,1017 mg ;

drug release. Cumulative percent drug release

0,1227 and 0,1429 mg. So we get the

determines how much of the drug is released

cumulative percent of drug release by 56,35%.

into the

membrane. The amount of drug that is spread is

and found the number of drugs that spread in

proportional to the cumulative percent.The

minutes to 5, 10, 15, 30, 60, and 90 of 0,0504

higher amount of drug that spreads the higher

mg ; 0,0658 mg ; 0,0863 mg ; 0,1130 mg ;

the value per cent cumulative. For group A

0,1183 and 0,1275 mg. So we get the

found the number of drug spread in minutes to

cumulative percent of drug release by 56,13%.

5, 10, 15, 30, 60, and 90 of 0,0584 mg ; 0,0798

The value of the cumulative percent at group A

mg ; 0,1083 mg ; 0,1172 mg ; 0,1349 and

is higher than group B. This indicates that the

0,1496 mg. So we get the cumulative percent of

drugs which are bounded in group A is lower

drug release in the serum of 64,37%. At trial

lower than group B.

percent

cumulative

drug

percent

of

drug

release

by

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Journal of practice biopharmaceutical and pharmacokinetics

It can be concluded amount of drug spread and

because the plasma contains more protein than

the cumulative percent of drug release occurs

in serum so that more drug in plasma is bound

more frequently less serum and plasma.This is

to proteins.

6. Normality Value Analysis and Correlation Using SPSS

a. 2 ml Tetracycline
Tests of Normality
Kolmogorov-Smirnova
group
amount_of_drug_diffused

cumulative_percent

Statistic

df

Shapiro-Wilk

Sig.

Statistic

df

Sig.

blue a

.191

.200*

.847

.148

blue b

.312

.069

.767

.059

blue a

.172

.200*

.950

.738

blue b

.289

.129

.885

.291

b. 1 ml Tetracycline + 1ml Serum

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Journal of practice biopharmaceutical and pharmacokinetics

Tests of Normality
Kolmogorov-Smirnova
group
amount_of_drug_diffused

cumulative_percent

Statistic

df

Shapiro-Wilk

Sig.

Statistic

df

Sig.

blue a

.127

.200*

.990

.988

blue b

.238

.200*

.945

.700

blue a

.153

.200*

.968

.876

blue b

.158

.200*

.964

.848

c. 1 mL Tetracycline + 1 mL Plasma

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Journal of practice biopharmaceutical and pharmacokinetics

Tests of Normality
Kolmogorov-Smirnova
Group
amount_of_drug_diffused

cumulative_percent

Statistic

df

Shapiro-Wilk

Sig.

Statistic

df

Sig.

blue a

.156

.200*

.981

.955

blue b

.267

.200*

.904

.398

blue a

.142

.200*

.970

.890

blue b

.192

.200*

.971

.901

conclude significant data means normally

From the observation above 0.05 normality

distributed.

values obtained from all aspect so we can

7. Corelation Analysis

Correlations
cumulative_perc Ammount_of_diff
Group
Group

Pearson Correlation

ent
1

-.111

-.165

.540

.607

12

12

12

Pearson Correlation

-.011

.963**

Sig. (2-tailed)

.730

Sig. (2-tailed)
N
cumulative_percent

used_drug

N
Ammount_of_diffused_dru Pearson Correlation

.000

12

12

12

-.155

.909**

.607

.000

12

12

g
Sig. (2-tailed)
N

12

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**. Correlation is significant at the 0.01 level (2-tailed).

Journal of practice biopharmaceutical and pharmacokinetics

After testing the value of normality, conducted

ammount of diffused drug have correlation.

a correlation analysis between the cumulative

This indicates that there is a correlation

percent and the ammount of diffused drug from

between the the cumulative percent and

significant value, it can be concluded that the

ammount diffused drug have correlation.

correlation between the cumulative percent and

8. T-Test Analysis

One-Sample Test
t-test for equality of means
Df
amount_of_drug_diffused

blue a
blue b

cumulative_percent

blue a
blue b

Sig. (2-tailed
6

.000

.000

.000

.000

Mean difference
1.00375
1.009
50.0100
50.0100

Sig value which is gotten is under 0,05 so it

between ammount of diffused drugs and

can be concluded that there are differences

cumulative percent in group A and group B.

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Journal of practice biopharmaceutical and pharmacokinetics

CONCLUSION

release. From T-Test analysis, we can conclude

A decrease in protein binding may result

that there are differences between ammount of

in increases in the concentration of free drug

diffused drugs and cumulative percent in group

allowing more drug pass through the cell

A and group B.

membrane and large. The cumulative percent of

drug obtained increased each time interval.
From the experimental results and the value of

REFERENCES

the amount of drug diffused and percent of drug

Agoes, Goeswin. 2008,

Pengembangan

release is higher in serum than plasma. This is

Sediaan Farmasi, Institute

because there are more in the plasma protein

Teknologi Bandung, Bandung.

Anief, M. 2000. Ilmu Meracik Obat Teori

binding compared to the plasma so that more

dan Praktek Cetakan ke-9.

medicine is bound in the plasma and the drug

release percent smaller than in serum. SPSS
data obtained from normality values above 0.05

Universitas Gajah Mada,

Yogyakarta.
Chein, Cameiro. 1981. Basic Histology,

from every aspects. So we can conclude

3rd edition. Lange Medical

significant data means normally distributed.

Results of correlation analysis shows
that the correlation between the cumulative
percent, and the number of drugs have spread
correlation. This indicates that the distributed
throughout the network, so more of the drug

Publication, Drawer Los

Altos, California.
Lachman, 1989.
Pengantar

Sediaan Farmasi, Institut Teknologi

Bandung, Bandung...........................
Syukri, 2002, Biofarmasetika, UII-Press,
Yogyakarta.

could be available to interact with the receptor

to produce a pharmacological effect more

Bentuk

William & Barry, 2004.

Biofarmasetika &

powerful. The greater the absorbance, the

Farmakokinetika Terapan, Edisi

binding protein is getting there a correlation

kelima, Airlangga University Press,

between the amount of drug spread of drug

Surabaya.

Pharmacy Faculty of Mathematic and Science Sriwijaya University