COMPLETE REPORT

BIOCHEMISTRY

Name : Hapida azhari idang

ID : 1413441003

Class/ Group : ICP A of Chemistry

Assitant : Ikhsar

Responcibility : Hardin S.Si., S.Pd., M.Pd

CHEMISTRY LABORATORY
FACULTY OF MATHEMATIC AND SCIENCE
STATE UNIVERSITY OF MAKASSAR
 APPROVAL SHEET

Complete report of Biochemistry Experiment with the title Blood,

which made and arranged by:
Name : Hapida Azhari Idang
Class : ICP Chemistry A
ID : 1413441003
Group : IV (four)
After Checked and consulted by Assistant and Coordination Assistant, so
this report is accepted.

th
Makassar, November 2016
Assistant Coordinator Assistant

Ida Masita Ikshar

Known By,
Responsibility Lecturer,

Hardin S.Si., S.Pd., M.Pd

ID. 19870807 201504 1 004
 CHAPTER 1
INTRODUCTION

A. Background of experiment
Blood is a liquid that is very important for humans because it serves as a
means of transportation as well as having many other uses for sustaining life.
Without enough blood a person can experience health problems and can even lead
to death.
Blood is a combination of fluids, cells and particles that resemble cells,
which flow in the arteries, capillaries and veins; which deliver oxygen and
nutrients to tissues and carrying carbon dioxide and other waste results.
Blood in the human body contains 55% of blood plasma (blood fluid) and 45% of
cells (blood solid). The amount of blood that is on our body is about
sepertigabelas adult body weight, or about 5 or 6 liters.
These components are in the normal individual can experience fuktuasi
because of several factors that vary include nutritional status. These components
are maintained in a level that indicates a balance between anabolic processes and
normal metabolic processes. Deviations from normal values of the components in
the plasma indicates the status of pain. Some examples of normal organic
components are: bilirubin, urea, creatinine, uric acid, glucose, total cholesterol,
total lipid. While the inorganic components are: chloride, phosphate, calcium,
sodium, magnesium, fe.

B. Formulation of experiment
1. How to determine Fe in hemoglobin?
2. How to test the catalytic power of blood?
3. How to test the components of blood?

C. Objective of experriment
1. To determine Fe in hemoglobin
2. To test the catalytic power of blood
3. To test the components of blood
D. Advantage of experiment
These components are maintained in a level that indicates a balance
between anabolic processes and normal metabolic processes. Deviations from
normal values of the components in the plasma indicates the status of pain.
Without enough blood a person can experience health problems and can even lead
to death.
 CHAPTER II
LITERATURE REVIEW

1. Determination of Fe in Hemoglobin
The iron in blood transports oxygen from the lungs to the tissues by virtue
of each iron atom being linked to a bulky, multi-ringed molecule, giving rise to
heme the deep red, non-protein, ferrous component of hemoglobin which has
the formula C34H32FeN4O4. Each heme is attached to a subunit of the respiratory
pigment, hemoglobin, itself a protein. As there are four of these subunits for every
hemoglobin, each entire hemoglobin molecule comprises the same number of
bound iron atoms, enabling it to carry four oxygen molecules. To be able to bind
the heme ring and still have electrons left over for latching onto oxygen, the iron
atoms must be oxidised to the ferrous state, Fe(II). In other words, the iron
prevalent in our blood is not in the metallic state. Instead, it is iron that has
already been rusted or oxidised prior to encounte ring oxygen (Senan, 2011:35).
A second molecule of cytochrome c introduces a second electron that
flows down the same path, stopping at heme 3, which is reduced to the Fe2+ form.
Recall that the iron in hemoglobin is in the Fe2+ form when it binds oxygen.
Thus, at this stage, cytochrome c oxidase is poised to bind oxygen and does so.
The proximity of CuB in its reduced form to the heme a 3 -oxygen complex
allows the oxygen to be reduced to peroxide (O2 2-), which forms a bridge
between the Fe3+ in heme a 3 and CuB 2+. The addition of a third electron from
cytochrome c as well as a proton results in the cleavage of the O-O bond, yielding
a ferryl group, Fe4+ = O, at heme a 3 and CuB 2+-OH. The addition of the final
electron from cytochrome c and a second proton reduces the ferryl group to Fe3+-
OH. Reaction with two additional protons allows the release of two molecules of
water and resets the enzyme to its initial, fully oxidized form (Berg, 2000:746).
Iron deficiency is the most prevalent nutritional deficiency and the most
common cause of anemia in the United States.1 Iron deficiency anemia is
characterized by a defect in hemoglobin synthesis, resulting in red blood cells that
are abnormally small (microcytic) and contain a decreased amount of hemoglobin
(hypochromic).2 The capacity of the blood to deliver oxygen to body cells and
tissues is thus reduced (Koolman, 2005:30).
The main function of iron is to carry oxygen from the lungs to the tissues
and transports electrons in the process of formation of energy in cells. To carry
oxygen, iron and protein must be joined to form hemoglobin in red blood cells
and myoglobin in the muscle fibers. When combined with proteins in the cell, the
iron form the enzymes involved in the formation of energy in cells. So, most of
iron found in red blood cells, where there is they who make up hemoglobin. This
is the substance that gives red color to the blood.

2. Test of Catalytic Power of Blood

In all living cells, DNA serves to store genetic information. Specific
segments of DNA (genes) are transcribed as needed into RNAs, which either
carry out structural or catalytic tasks themselves or provide the basis for
synthesizing proteins. In the latter case, the DNA codes for the primary structure
of proteins. The language used in this process has four letters (A, G, C, and T).
All of the words (codons) contain three letters (triplets), and each triplet
stands for one of the 20 proteinogenic amino acids (Koolman, 2005:84).
Metabolic flow is based on anabolic and catabolic reactions. Metabolism
begins with the ingestion of food that is foreign to the organism (containing a
varying amount of smaller and larger compounds), which is broken down in the
digestive tract to smaller molecules by hydrolysis. An anabolic phase occurs when
the smaller molecules are taken up into the organisms bloodstream and become
part of the organism. Catabolic reactions constantly break down the organism
again for its functional needs. We will demonstrate the connections of processes
in organs and organisms as well as the interconnectedness of organisms within the
whole of living nature (Tellingen, 2001:10).
Enzymes accelerate reactions by factors of as much as a million or more.
Indeed, most reactions in biological systems do not take place at perceptible rates
in the absence of enzymes. Even a reaction as simple as the hydration of carbon
dioxide is catalyzed by an enzyme namely, carbonic anhydrase. The transfer of
CO2 from the tissues into the blood and then to the alveolar air would be less
complete in the absence of this enzyme. In fact, carbonic anhydrase is one of the
fastest enzymes known. Each enzyme molecule can hydrate 106 molecules of
CO2 per second.This catalyzed reaction is 107 times as fast as the uncatalyzed
one. We will consider the mechanism of carbonic anhydrase catalysis in Chapter
9. Enzymes are highly specific both in the reactions that they catalyze and in their
choice of reactants, which are called substrates. An enzyme usually catalyzes a
single chemical reaction or a set of closely related reactions. Side reactions
leading to the wasteful formation of by-products are rare in enzyme-catalyzed
reactions, in contrast with uncatalyzed ones (Berg, 2000:303).
The catalytic power in the blood that is enzym catalase which can
accelerate the decomposition of H2O2.

3. Test Component of Blood

Carbohydrates, proteins, and lipids are primary nutritional ingredients for
humans. The breakdown of nutrients (carbohydrates, proteins and lipids) in the
intestines results in small compounds (metabolites) that can pass through the wall
of the intestines into the blood. Complex carbohydrates and proteins are polymers
that break up into a large number of smaller similar compounds, the monomers.
Lipids do not have monomer and polymer forms. All complex compounds in an
organism are produced within the organism itself (Tellingen, 2001:26).
Polymeric carbohydratesabove all starch, as well as some disaccharides
are important (but not essential) components of food. In the gut, they are broken
down into monosaccharides and resorbed in this form. The form in which
carbohydrates are distributed by the blood of vertebrates is glucose (blood
sugar). This is taken up by the cells and either broken down to obtain energy
(glycolysis) or converted into other metabolites. Several organs (particularly the
liver and muscles) store glycogen as a polymeric reserve carbohydrate The
glycogen molecules are covalently bound to a protein, glycogenin.
Polysaccharides are used by many organisms (Koolman, 2005:34).
 Not all vitamins function as coenzymes. The fat-soluble vitamins, which
are designated by the letters A, D, E, and K, have a diverse array of functions.
Vitamin K, which is required for normal blood clotting (K from the German
koagulation), participates in the carboxylation of glutamate residues to -
carboxyglutamate, which makes modified glutamic acid a much stronger chelator
of Ca2+. Vitamin A (retinol) is the precursor of retinal, the light-sensitive group
in rhodopsin and other visual pigments. A deficiency of this vitamin leads to night
blindness. In addition, young animals require vitamin A for growth. Retinoic acid,
which contains a terminal carboxylate in place of the alcohol terminus of retinol,
serves as a signal molecule and activates the transcription of specific genes that
mediate growth and development. A metabolite of vitamin D is a hormone that
regulates the metabolism of calcium and phosphorus. A deficiency in vitamin D
impairs bone formation in growing animals. Infertility in rats is a consequence of
vitamin E (-tocopherol) deficiency. This vitamin reacts with and neutralizes
reactive oxygen species such as hydroxyl, radicals before they can oxidize
unsaturated membrane lipids, damaging cell structures (Berg, 2000:304).
Components of human blood is composed of red blood cells
(erythrocytes), white blood cells (WBCs), pieces of blood (platelets), and blood
plasma.
 CHAPTER III
METHOLOGY EXPERIMENT

A. Apparatus and Chemicals

1. Apparatus
a. Porceline cup 2 pieces
b. Test tube 6 pieces
c. Stirring bar 2 pieces
d. Drop pipette 5 pieces
e. Tripot and asbestos 1 piece
f. Funnel 1 piece
g. Erlenmeyer 250 mL 1 piece
h. Bunsen burner 1 piece
i. Heating mantle 1 piece
j. Measuring glass 50 mL 1 piece
k. Measuring glass 10 mL 1 piece
l. Spatula 1 piece
m. Stopwatch 1 piece
n. Knife 1 piece
o. The test tube shelf 1 piece
p. Rought and smooth cloth @1 piece
2. Chemicals
a. Chickens blood
b. Hydrochloric acid solution (HCl) 0.1 N and 1M
c. Diluted Nitric acid (HNO3)
d. Sodium hydroxide solution (NaOH) 2.5N
e. Sodium hydroxide solution (NaOH) 10 %
f. Filter paper
g. Potassium Thiocyanate (KSCN)
h. Potassium ferrocyanide (K4Fe(CN)6)
i. Hydrogen peroxide (H2O2) 3%
j. Acetic acid (CH3COOH) 2 M
k. Benedict reagent
l. Millon reagent
m. Aquadest (H2O)
n. Silver nitrate AgNO3 0.2M
o. Tissu
p. Sunlight

B. Work Procedures
1. Determining Fe in hemoglobin
a. 10 ml of blood was entered into a porcelain dish
b. Blood was heated until simmering
c. HCl 0,1 N and dilute HNO3 (HNO3 has been heated previously) was added a
little bit
d. The mixture was stirred then filtered
e. The filtrate was divided into two parts
f. [K4Fe (CN)6] was added Filtrate in the first test tube and KSCN was added
another tube with
g. The color was observe change and write the equation.
2. Power Catalytic Blood Tests
a. 2 ml (H2O2) 3% was entered in a test tube and then observed
b. A few drops of blood was added and Observed what happens
1. Test for Blood Components
a. 10 ml blood and 50 ml of distilled water was mixed into a porcelain dish and
b. The mix solution was heated in the bath (spiritus burner
c. Two drops of 2M acetic acid was added and continue heating for 5 minutes
until coagulation occurs and then filtering it.
d. The filtrate was evaporated to half
e. Filtrate was divided into 3 part
f. Reagent Benedict was added into first tube,
g. Reagent Millon was added into second tube
h. AgNO3 was added into third tube
i. Sediment (3b) was taked and put it in a porcelain dish and incandansce. Once
cool, 1 mL of 1M HCl , 1 mL HNO3 and 1mL [K4Fe (CN) 6] 2M was added
 CHAPTER IV
RESULT AND DISCUSSION

A. Observation Result
1. Determination of Fe in Hemoglobin
No. Activity Result
1. Burnt and black color
20 drops of blood >

Several drops oh HCl + burnt blood

2. Black suspention
>

Black suspention + several drops of HNO3

3. Black brownish

> suspension
4. Brownish solution
Black suspension > and

filteresd
5. Green solution
Tube I + K4Fe(CN)2
6. Brown solution
Tube II + KSCN

2. Test of Catlytic power of blood

No. Activity Result
1. Several 10 drops of H2O2 3% Colorless solution
(Colorless)

2. Colorless soluton + Several drops of blood Red solution and

bubble gas

3. Test Component of Blood

No. Activity Result
1. 50 mL of H2O (colorless) + 10 mL of Red solution

blood (red) >
 2. Red solution + 2 drops of CH3COOH Red solution with
(colorless) percipitate

Red solution with percipitate > 5 Red solution with brown
3. percipitate
minute

Red solution with brown
Red solution with brown percipitate >
4. percipitate
until part of before

Red solution with brown

5. Yellow soultion with brown precipitate percipitate

>

6. Yellow solution devided in tube -Orange Brownish

Tube I + Benedict reagent solution

Tube II + Millon reagent -White coagulant

Tube III + AgNO3 + HNO3 -White solution with

preciptate

B. Discussion
1. Penentuan Fe dalam Hemoglobin
Percobaan ini bertujuan untuk mengetahui kadar Fe dalam darah.
Mengambil sampel darah, kemudian dipanaskan hingga membara (hangus).
Tujuan dari perlakuan ini yaitu agar zat- zat lain selain Fe akan menguap. Ada
tidaknya logam Fe perlu adanya penambahan HCl hal ini bertujuan untuk
melarutkan besi. Reaksinya:
Fe + 2 HCl Fe2+ + Cl- + H2
Kemudian penambahan HNO3 encer yang bertujuan mengubah Fe2+
menjadi Fe3+. Reaksinya:
 Fe2+ + HNO3 Fe3+ + NO + 2 H2O

Setelah terbentuk ion Fe2+ dan ion Fe3+, maka campuran darah tersebut
disaring. Hasil filtrat dibagi menjadi dua bagian. Tabung pertama ditambahkan
K4Fe(CN)6 yang berfungsi untuk mengikat ion Fe2+ sesuai dengan persamaan
reaksi:
Fe2+ + [Fe(CN)6] + 4K+ Fe3+ + [Fe(CN)6]4-
4 Fe3+ + [Fe(CN)6]4- Fe4 [Fe(CN)6]3
Warna larutan menjadi hijau hal ini tidak sesuai dengan teoi yang
seharusnya menjadi biru prusi yang menunjukkan adanya Fe2+ dalam darah.
Tabung kedua ditambahkan KSCN yang bertujuan sama dengan K4Fe(CN)6 yaitu
mengikat ion Fe3+. Reaksi yang terjadi:
Fe3+ + SCN- Fe(SCN)3

Warna larutan yang diperoleh adalah larutan cokelat. Hal ini sesuai dengan
teori menunjukan bahwa di dalam darah terdapat ion Fe3+

2. Tes Daya Katalitik Darah

Percobaan ini bertujuan untuk menguji daya katalitik adanya enzim
katalase yang mengurai hidrogen peroksida dengan cepat. Mengambil H2O2 3%
beberapa mili, warna larutan bening dan tak terdapat gelembung. Setelah
ditambahkan beberapa tetes darah, maka warna larutan menjadi merah dan
terbentuk gelembung gas. Hal ini membuktikan bahwa dalam darah terdapat
enzim katalase yang berfungsi mempercepat prosese penguraian H2O2. Reaksinya:

2H2O2 2H2O + O2
Hb + H2O2 HbO2 + H2

3. Tes terhadap Komponen-komponen Darah

Percobaan ini bertujuan un tuk menguji komponen-komponen yang ada
dalam darah. percobaan ini mengambil 50 ml aquadest dan 10 ml sampel darah.
Campuran larutan dipanaskan hingga setengah volumenya. kedua campuran
tersebut dipanaskan dan ditambahkan beberapa tetes asam asetat yang bertujuan
agar terbentuk koagolasi. Kemudian kuagolasi tersebut disaring dan filtrat dibagi
menjadi tiga buah tabung. Tabung pertama ditambahkan pereaksi benedict yang
telah dipanaskam sebelumnya. Tujuan dari pengujian ini yaitu mengetahui adanya
gula pereduksi dalam darah, uji positifnya yaitu terbentuk larutan yang berwarna
merah bata. Reaksinya

Pada tabung II ditambahkan AgNO3 yang bertujuan mengidentifikasi ion

Cl- dalam darah. Uji positifnya yaitu terbentuknya endapan putih. Reaksinya:
AgNO3 + Cl- AgCl + NO3-
Selanjutnya, pada tabung III ditambahkan pereaksi millon yang bertujuan
mengidentifikasi protein dalam darah. Uji positifnya yaitu terbentuk endapan
putih. Reaksinya:
Untuk endapan yang telah ditambahkan dengan HCl yang sebelumnya
telah dipijarkan diuji dengan K4Fe(CN)6 dan terbentuk larutan biru prussi.
 CHAPTER V
CONCLUTION AND SUGGESTION

A. Conclution
1. In the determination of Fe in hemoglobin, in addition K4Fe (CN) 6 prussi the
blue color whereas chocolate menendakan KSCN solution Fe ions present in
the blood
2. The presence of bubbles indicates that the blood may accelerate the process
of decomposition of hydrogen peroxide
3. The components of blood consists of the addition of AgNO3 Cl-

B. Suggestion
It is expected that the next practitioner so that the blood sample used
should be utterly inconceivable coming from healthy chickens that tests carried
out in accordance with the purpose
 BIBLIOGRAPHY

Berg, M. Jeremy, John, L. Tymozco, and Lub ert Stryer. 2000. Biocheistry fifth
edition. New York. W.H. Freeman and Company.

Koolman, Jan and Roehm, Klaus-Heinrich. 2005. Color Atlas Biochemistry.

Marburg, Germany: Juergen Wirth.

Senan, C. 2011. Blood. United Kingdom: library publishing.

Tellingen, Christaa van. 2001. Biochemistry. Louis Bolk Instituut: Point Of View
 DOCUMENTATION

1. Determination Fe in Blood

20 drops of blood was Several drops of HCl Tube I: K4Fe(CN)6

heated and HNO3 was added Tube II: KSCN

2. Test of catalytic power of blood

H2O2 was added in the Tube and Several drops of

blood was added
3. Test component of blood

50 mL H2O + 10 mL 2 drops of CH3COOH was The soluion was heated

blood was heated added

The precipitate was The filtrat was devided in The precipitate was
filtered to three tube heated and was added 1
I: Benedict mL of HCl and
II:AgNO3 K4Fe(CN)6
III: Milon
 JOURNAL OF EXPERIMENT

Title of experiment : Blood

Day/Date of experiment : Monday/November 20th 20156
Name : Hapida Azhari Idang
ID : 1413441003
Class/Group : ICP A/ IV (four)
Members : 1. Anugrawati Asri
2. Khairunnisa
3. Yuli Astuti
Assistant : Ikshar

A. OBJECTIVE OF EXPERIMENT
4. To determine Fe in hemoglobin
5. To test the catalytic power of blood
6. To test the components of blood

B. APPARATUS AND CHEMICALS

1. Apparatus
q. Porceline cup 2 pieces
r. Test tube 6 pieces
s. Stirring bar 2 pieces
t. Drop pipette 5 pieces
u. Tripot and asbestos 1 piece
v. Funnel 1 piece
w. Erlenmeyer 250 mL 1 piece
x. Bunsen burner 1 piece
y. Heating mantle 1 piece
z. Measuring glass 50 mL 1 piece
aa. Measuring glass 10 mL 1 piece
bb. Spatula 1 piece
cc. Stopwatch 1 piece
dd. Knife 1 piece
ee. The test tube shelf 1 piece
ff. Rought and smooth cloth @1 piece
2. Chemicals
q. Chickens blood
r. Hydrochloric acid solution (HCl) 0.1 N and 1M
s. Diluted Nitric acid (HNO3)
t. Sodium hydroxide solution (NaOH) 2.5N
u. Sodium hydroxide solution (NaOH) 10 %
v. Filter paper
w. Potassium Thiocyanate (KSCN)
x. Potassium ferrocyanide (K4Fe(CN)6)
y. Hydrogen peroxide (H2O2) 3%
z. Acetic acid (CH3COOH) 2 M
aa. Benedict reagent
bb. Millon reagent
cc. Aquadest (H2O)
dd. Silver nitrate AgNO3 0.2M
ee. Tissu
ff. Sunlight

C. WORK PROCEDURES
1. Determination of Fe in hemoglobin
litle HCl 0.1 N

add HNO3 diluted have

cooled been hoted
10 drops of blood heated
observed the color
change in each tube

filtered stirred
add KSCN add K4Fe(CN)6

2. Catalytic power of blood test

observed the colot before

and after added the blood

add several mL add several

of H2O2 3% drops of blood

3. Components of blood test

67 67
67 67 45 45
45 45 31 11
8 3 8
31 8 3 8 2 9 2 9
67 67
45 45
11
2 9 2 9 1 1
31 11
8 3 8 1 1 0
2 9 2 9 0
1 1
0

2 drops of heated until 5 minutes

10 mL of blood and heated CH3COOH
50 ml of H2O

observed the
45
31
67
11
8
9
3
45
67
8
9
color change in
2 2
1 1
0 each tube
taked the sediment
and heated filtered
benedict Millon AgNO3

observed the
color change
add 5 drops of HCl 1M
and 1 mL of K4Fe(CN)6

Makassar, 19 November 2016

Assistant, Apperantice,

Ikshar Hapida Azhari Idang