CAGAYAN STATE UNIVERSITY

COLLEGE OF MEDICINE AND SURGERY

Carig Campus, Tuguegarao City

EXPERIMENT 2
AMINO ACIDS AND PROTEINS

MD-1B GROUP 2
Adam Gray Ayak
Sherwin Caytap
Wilmer Edchamag
Pauline Pulido
Mary Joy Osingat
Kryme Rosito
Hilary Anne Ventura

FLORA P. DAYAG
PROFESSOR, BIOCHEMISTRY
INTRODUCTION

Proteins are the most abundant class of organic compounds in the healthy,
lean human body, constituting more than half of its cellular dry weight. Proteins are
polymers of amino acids and have molecular weights ranging from approximately
10,000 to more than one million. Biochemical functions of proteins include catalysis,
transport, contraction, protection, structure, and metabolic regulation.

Amino acids are the monomeric units, or building blocks, of proteins joined by
a specific type of covalent linkage. The properties of proteins depend on the
characteristic sequence of component amino acids, each of which has distinctive side
chains.

Amino acid polymerization requires elimination of a water molecule as the

carboxyl group of one amino acid reacts with the amino group of another amino acid
to form a covalent amide bond. The repetition of this process with many amino acids
yields a polymer, known as a polypeptide. The amide bonds linking amino acids to
each other are known as peptide bonds. Each amino acid unit within the polypeptide
is referred to as a residue. The sequence of amino acids in a protein is dictated by
the sequence of nucleotides in a segment of the DNA in the chromosomes, and the
uniqueness of each living organism is due to its endowment of specific proteins.

OBJECTIVES

At the end of the experiment, the group should be able to:

 Discuss chemical test to rule out and/or identify amino acids and proteins.
 Learn the principle behind spectrophotometry and apply it in determining
absorbance of sample in comparison to the standard.
 Review the function of amino acids and proteins, including albumin and
globulin, to the body.
 Learn importance of obtaining A/G ratio, as well as, of Total proteins.
 Determine the amount of albumin and total proteins found in blood serum
collected using spectrophotometry.
 Identify diseases that are affected by amino acids and proteins found in the
body.
MATERIALS

LABORATORY
BLOOD COLLECTION REAGENTS
APPARATUSES
Syringe Test tubes
Evacuated Tube Test tube brush ALBUMIN: BROMCRESOL GREEN (Acetate
buffer 100 mmol/L, bromcresol green 0.27
Micropore Test tube racks mmol/L, detergent, pH 4.1.)
Cotton balls Pipettes
Torniquet Pipette tips
PROTEIN: BUIRET REAGENT (Copper (II)
Alcohol Spectrophotometer acetate 6 mmol/L, potassium iodide 12
Centrifuge mmol/L, sodium hydroxide 1.15mol/L,
Beaker detergent.)

SPECTROPHOTOMETRY
It involves the measurement of the light transmitted by a solution to
determine the concentration of the light-absorbing substances in the
solution.

 Single beam spectrophotometer

Simplest type
designed to make one
measurement at a time at
one specified wavelength.
The maximum absorption
of the analyte must be
known in advance when
using this.

 Double beam spectrophotometer

This instrument splits the monochromatic light into two components-
one beam passes through the sample while the other through the reference
solution or blank. The absorbance of the sample can be recorded directly as
electrical output of the sample beam. Types:
1. Double-beam in space – uses two photodetectors, for the sample
beam and the reference beam
2. Double-beam in time – uses one photodetector and alternately
passes the monochromatic light through the sample cuvet and then
reference cuvet using a chopper or rotating sector mirror.
PARTS:
1. Light source
a. Continuum source - Emits radiation that changes in intensity. Tungsten,
deuterium and xenon lamps
b. Line source - Emits limited radiation and wavelength. Mercury and vapor
lamps

2. Entrance slit – It minimizes unwanted or stray light and prevents the

entrance of scattered light into the monochromator system.
 Stray light – refers to any wavelength outside the band transmitted by
the monochromator.

3. Monochromator – It isolates specific or individual wavelength of light.

a. Prisms – wedge-shaped pieces of glass, quartz or sodium chloride.
b. Diffraction gratings – Most commonly used; with better resolution than
prism.
c. Filters – simple, least expensive, not precise but useful.
d. Holographic gratings

4. Exit slit – It controls the width of

light beam (bandpass).

5. Cuvet – holds the solution whose

concentration is to be measured.
a. Alumina silica glass
b. Quartz/plastic
c. Borosilicate glass
d. Soft glass

6. Photodetector – detects and

converts transmitted light into
photoelectric energy.
 a. Photocell – simplest, with a wide bandpass
b. Phototube – Contains cathode and anode enclosed in a glass case. It has
photosensitive material that gives off electron when light energy strikes it.
c. Photomultiplier tube – Most commonly used detector which measures the
visible and the UV region. It has excellent sensitivity. Should never be
exposed to room light because it will burn out.
d. Photodiode – Not as sensitive as PMT but with excellent linearity.

7. Read-out device – It displays output of the detection system. Examples are

galvanometer, ammeter and Light Emitting Diode (LED) display.

ABSORBANCE
The amount of light absorbed. It is proportional to the inverse logarithm of
transmittance. It is mathematically derived from %T.

A = abc = 2 – log %T

Where: A = Absorbance
a = molar absorptivity
b = length of light through the solution
c = concentration of absorbing molecule

Unknown solution: Au x Cs
As

% TRANSMITTANCE
The ratio of the radiant energy transmitted divided by the radiant energy
incident on the sample.

%T = It x 100
I0

Where: It = transmitted light thru the sample

I0 = intensity of light striking the sample

 In actual practice, the light transmittance by a blank is substituted for

I0

PROCEDURE

A. ALBUMIN
1. Pipette into labelled test tubes:

BLANK STANDARD SAMPLE

ALBUMIN (S) -- 10µL --
SAMPLE -- -- 10µL
REAGENT 1.0 mL 1.0mL 1.0mL
 2. Mix thoroughly and let stand the tubes for 1 minute at room temperature.
3. Read the absorbance (A) of the Standard and the Sample at 630 nm against
the Blank. The color is stable for 30 minutes.

B. PROTEIN (TOTAL)

1. Pipette into labelled test tubes:

BLANK STANDARD SAMPLE
DISTILLED WATER 20µL -- --
PROTEIN (S) -- 20µL --
SAMPLE -- -- 20µL
REAGENT 1.0 mL 1.0mL 1.0mL

2. Mix thoroughly and let stand the tubes for 10 minutes at room temperature.
3. Read the absorbance (A) of the Standard and the Sample at 545 nm against
the Blank. The color is stable for at least 2 hours.

RESULTS AND DISCUSSION

A. ALBUMIN

ABSORBANCE ABSORBANCE CONCENTRATION CONCENTRATION

(SAMPLE/UNKNOWN) (STANDARD) OF STANDARD OF UNKNOWN
Group 1 0.299 27.89 g/L
Group 2 0.502 46.82 g/L
Group 3 0.218 0.550 51.3 g/L 20.33 g/L
Group 4 0.509 47.48 g/L
Group 5 0.434 40.48 g/L
Group 6 0.549 51.21 g/L

REFERENCE VALUES:
Newborn 2 to 4 28-44 g/L
days
4 days to 14 years 38-54 g/L
Adult 35-50 g/L
>60 years 34-48 g/L

COMPUTATION

Conc (sample) = Absorbance of Sample x Conc of Standard

Absorbance of Standard

= 0.502 x 51.3 g/L

0.550

= 46.82 g/L
 B. PROTEIN

ABSORBANCE ABSORBANCE CONCENTRATION CONCENTRATION

(SAMPLE/UNKNOWN) (STANDARD) OF STANDARD OF UNKNOWN
Group 1 0.391 66.82 g/L
Group 2 0.305 52.13 g/L
Group 3 0.345 0.385 65.8 g/L 58.96 g/L
Group 4 0.322 55.03 g/L
Group 5 0.374 63.92 g/L
Group 6 0.175 29.90 g/L
REFERENCE RANGE:
Ambulatory 64-83 g/L
Recumbent 60-70 g/L

COMPUTATION

Conc (sample) = Absorbance of Sample x Conc of Standard

Absorbance of Standard

= 0.305 x 65.8 g/L

0.385
= 52.13 g/L

POST LABORATORY QUESTIONS

1. Will amino acids give a positive Biuret test? Why?

No, amino acids do not give a positive biuret test. Biuret test is most widely
used method for the determination of total protein. In this reaction, cupric ion
complex reacts with the groups involved in the peptide bond. In addition to the
NHCO group that occurs in the peptide bond, Cu2+ will react with any compound
that has two or more of the following groups: NHCH2 and NHCS.

2. Aside from the Biuret reaction, what other test is frequently used to quantitate
proteins? How does this compare with biuret in terms of sensitivity?
 METHOD PRINCIPLE ADVANTAGES AND DISADVANTAGES

Reference method (assume average nitrogen content

of 16%)
Digestion of protein: Time consuming and too tedious for routine use. Actual
KJELDAHL measurement of nitrogen nitrogen content of serum proteins is 15.1% to 16.8%,
content therefore, error is introduced if a protein standard is
used that differs in composition from the serum
specimen to be analyzed because the percentage of
nitrogen will not be the same.
Rapid and simple: Assume protein solids are present in
same concentration as in calibrating serum.
Measurement of
Error is introduced when the sample is icteric, lipemic, or
REFRACTORY refractive index due to
hemolyzed. The refractive index is also temperature
solutes in serum
dependent, and some refractometers incorporate a
built-in temperature correction.

Protein binds to dye and Research use.

causes a spectral shift in Simple and fast, require caution when applying this test
DYE BINDING
the absorbance maximum to the complex mixture of protein found in serum.
of the dye
Formation of violet- Routine method: Requires at least two peptide bonds
colored chelate between and on alkaline medium.
BIURET Recommended by the International Federation of
Cu2+ ions and peptide
bonds. Clinical Chemistry Expert Level.

3. Explain why in chronic liver disease, for example cirrhosis, serum proteins
are decreased. Give 2 other conditions where is hypoproteinemia.
 In chronic liver disease like cirrhosis, the serum proteins are decreased
because there is a decrease in the synthesis of serum proteins in the
liver in which the liver is the site of all non-immune protein synthesis.
 Conditions like Chronic Kidney Disease and loss of blood in open
wounds, internal bleeding or extensive burns are also examples of
diseases where hypoproteinemia is present.
4. Give two conditions that may exhibit hyperproteinemia.
 Dehydration- when excess water is lost from the vascular system, the
proteins, because of their size, remain within the blood vessels. The
quantity remains unchanged but the concentration is elevated.
 Excessive production of gamma- globulins
5. What is the importance of determining the albumin/ globulin ratio? What is the
normal albumin/ globulin ratio? What is meant by an inverted albumin/globulin
ratio? What is Total Serum Protein?
  The importance of determining the albumin/globulin ratio is useful as
diagnostic information for kidney and liver diseases when there is
reversal or significant change in the ratio.
 Normal A/G ratio is 1.3- 3.1
 Inverted A/G ratio means that the globulin is greater than albumin
which is seen in cirrhosis, multiple myeloma, and Wald Enstrom’s
macroglobulin.
 A total serum protein test measures the total amount of protein in the
blood. It also measures the amounts of two major groups
of proteins in the blood: albumin and globulin.

6. What is the function of (a) Albumin and (b) Globulin in the body?
Serum albumin accounts for 55% of blood proteins, and is a major contributor
to maintaining the osmotic pressure of plasma to assist in the transport of lipids
and steroid hormones. Globulins make up 38% of blood proteins and transport
ions, hormones, and lipids assisting in immune function.

SUMMARY AND CONCLUSION

Proteins are the most abundant and functionally diverse molecules in living
systems. Virtually every life process depends on this class of molecules. For example,
enzymes and polypeptide hormones direct and regulate metabolism in the body,
whereas contractile proteins in muscle permit movement. In bone, the protein
collagen forms a framework for the deposition of calcium phosphate crystals, acting
like the steel cables in reinforced concrete. In the bloodstream, proteins, such as
hemoglobin and plasma albumin, shuttle molecules essential to life, whereas
immune-globulins fight infectious bacteria and viruses. In short, proteins display an
incredible diversity of functions, yet all share the common structural feature of being
linear polymers of amino acids. This chapter describes the properties of amino acids.

APPENDICES
References:

Bhagavan, N.V. & Chung, E. H. (2011). Essentials of Medical Biochemistry With

Clinical Cases. San Diego, CA: Academic Press.

Harvey, R. & Ferrier, D. (2011). Lippincott’s Illustrated Reviews: Biochemistry 5th Ed.
Philadelphia, PA: Wolters Kluwer.

Bishop, M. et al. (2010). Clinical Chemistry: Techniques, Principles, Correlations 6th

Ed. Philadelphia, PA: Wolters Kluwer.