BIOCHEMISTRY LABORATORY MANUAL

Prepared by Admer C. Daiblio, v2012

Revised by Mary-Ann A. Landiao, v2016

Experiment 2

Amino Acid and Protein Solubility

Score:

Name: Ana Margarita L. Baytion

Date Performed: June 30,

2016
Instructors Signature:

Groupmates: Marla C. Basa

Joann H. Justiniane

I. Objectives
1. To test the solubility of selected amino acids and proteins in various
solutions
2. Describe the effect of pH on the solubility of amino acid and protein
II. Chemicals
3 M Hydrochloric acid
Lysine
Tyrosine
Casein
III.

3 M Sodium Hydroxide
Glycine
Gelatin
Glutamic acid

Apparatus/Materials/Equipment
Test tubes
Test tube rack
Graduated cylinder
Litmus paper

IV. Diagram of the Procedure

Please write on a short bond paper.
V. Summary of Theory
Amino acids are building blocks of proteins, they are organic compounds that
contains both amino (-NH2) group which is basic and the carboxylic (-COOH) group
which in contrast is acidic in nature, and a replaceable side chain (-R). The side chain
structure determines the class of the amino acid: basic, acidic, neutral or nonpolar.
The physical properties of amino acids and proteins are mainly a result of their
structure, both in solid state and in various solutions.

Amino acids are generally soluble in water and insoluble in non-polar organic
solvents such as hydrocarbons. This again reflects the presence of the zwitterions. In
water, the ionic attractions between the ions in the solid amino acid are replaced by
strong attractions between polar water molecules and the zwitterions. This is much
the same as any other ionic substance dissolving in water. The extent of the solubility
in water varies depending on the size and nature of the "R" group.
No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY LABORATORY MANUAL

Prepared by Admer C. Daiblio, v2012
Revised by Mary-Ann A. Landiao, v2016

Zwitterion molecule is responsible for internal acid-base reaction, it is an

internal transfer of a hydrogen ion from the -COOH group to the -NH 2 group to leave
an ion with both a negative charge and a positive charge.

A zwitterion double ion is a molecule that has a positive charge on one

atom and a negative charge on another atom, but which has no net charge.
Zwitterion structures changes when the pH of a solution containing an amino acid is
changed from neutral either to acidic (low in pH) by adding an acid such as HCl or to
basic (high in pH) by adding a base such as NaOH. These ionic attractions take more
energy to break and so the amino acids have high melting points for the size of the
molecules.
VI. Observations
Table VI.1 Solubility in Water
Substances
Glycine
Glutamic Acid
Lysine
Tyrosine
Gelatin
Casein

Observation
Soluble
Insoluble
Insoluble
Insoluble

Table VI.2 Solubility as a Function of Solution pH

Substances
3M NaOH
3M HCl Solution
(weakly acidic)
Glycine
Soluble
Miscible
Glutamic Acid
Soluble
Miscible
Lysine
Tyrosine
Slightly soluble
Miscible
Gelatin
Casein
Immiscible
Slow formation of
precipitate

3M HCl Solution
(strongly acidic)
Miscible
Miscible
Miscible
Formed precipitate

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY LABORATORY MANUAL

Prepared by Admer C. Daiblio, v2012
Revised by Mary-Ann A. Landiao, v2016

VII.

Analysis

The surface of a protein has a net charge that depends on the number and
identities of the charged amino acids, and on pH. At a specific pH the positive and
negative charges will balance and the net charge will be zero. This pH is called the
isoelectric point, and for most proteins it occurs in the pH range of 5.5 to 8. A protein
has its lowest solubility at its isoelectric point. If there is a charge at the protein
surface, the protein prefers to interact with water, rather than with other protein
molecules. This charge makes it more soluble. Without a net charge, protein-protein
interactions and precipitation are more likely.
The pH of an aqueous solution can affect the solubility of the solute. By
changing the pH of the solution, you can change the charge state of the solute. If the
pH of the solution is such that a particular molecule carries no net electric charge,
the solute often has minimal solubility and precipitates out of the solution. At a pH
below a molecule's pI, that molecule will carry a net positive charge; at a pH above
its pI, the molecule will carry a net negative charge.
VIII. Conclusion
A nonpolar amino acid contains one amino group, one carboxyl group, and a
nonpolar side chain. When incorporated into a protein, such amino acids are
hydrophobic (water-fearing): that is they are insoluble in water. Glycine belongs to
this type of amino acid. But since glycine has multiple sites available for
hydrogen bonding, we would expect it to be soluble in water , it can fit
into hydrophilic or hydrophobic environments, due to its minimal side chain of only
one hydrogen atom therefore it is soluble in water. (pI= 5.97)

There are three types of polar molecules. One is a polar neutral molecule, it
contains one amino group, one carboxyl group, and a side chain that is polar but
neutral. These amino acids are more soluble in water than non-polar amino acids; the
R group present can hydrogen bond to water. Tyrosine is an example of this type
since both its side chain of a polar neutral amino acid is neither acidic nor basic. (pI=
5.66)

The second type of polar amino acid is polar acidic amino acid; it contains one
amino group and two carboxyl groups, the second carboxyl group being part of the
side chain. In solution at physiological Ph, the side chain of a polar acidic amino acid
bears a negative charge; the side-chain carboxyl group has lost its acidic hydrogen
atom. Glutamic acid belongs to this type of amino acid. (pI= 3.22)

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

The addition of 3 M NaOH to casein produced sodium caseinate. After

addition of acid in the solution a white colored, wool-like precipitate appeared. (pI=
4.6)

BIOCHEMISTRY LABORATORY MANUAL

Prepared by Admer C. Daiblio, v2012
Revised by Mary-Ann A. Landiao, v2016

Strong solute-solvent attractions equate to greater solubility while weak

solute-solvent attractions equate to lesser solubility. Polar solutes dissolve best in
polar solvents while non-polar solutes tend to dissolve best in non-polar solvents. In
the case of a polar solute and non-polar solvent (or vice versa), it tends to be
insoluble or only soluble to a miniscule degree. A general rule is, "Like dissolves like.
The results of our experiment do not coincide with this rule as there are other factors
to be considered that may influence protein solubility: Ionic strength, ionic
composition, pH, and temperature. Another factor is that the larger the molecules of
the solute are, the larger is their molecular weight and their size. Since proteins are
substances of high molecular weight it is more difficult for solvent molecules to
surround bigger molecules. If other factors are altered, we can conclude that larger
particles are generally less soluble.

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.

BIOCHEMISTRY LABORATORY MANUAL

Prepared by Admer C. Daiblio, v2012
Revised by Mary-Ann A. Landiao, v2016

IX. References
Millio F. & Loffredo W. (n.d). Qualitative testing for Amino Acids and Proteins.
Retrieved
from
https://labopslton.wikispaces.com/file/view/Qualitative+Testing+for+Amino+Acids+
%26+Proteins.pdf

Bryan W. P. (26 June 2010). THE ISOIONIC POINT OF AMINO ACIDS AND PROTEINS.
Retrieved from http://onlinelibrary.wiley.com/doi/10.1016/0307-4412(78)90164-4/pdf

Boundless. The Effect of pH on Solubility. Boundless Chemistry. (26 May. 2016).

Retrieved
from https://www.boundless.com/chemistry/textbooks/boundlesschemistry-textbook/acid-base-equilibria-16/solubility-equilibria-120/the-effect-of-phon-solubility-490-6881/
Ophard C. (2003). Characteristics and Properties of Amino Acids. Retrieved from
http://chemistry.elmhurst.edu/vchembook/561aminostructure.html

Toledo
M.
(n.d).
Protein
Solubility.
Retrieved
http://www.chemistryexplained.com/Pr-Ro/Protein-Solubility.html

from

Nehete J., Bhambar R., Narkhede M., and Gawali S. (7 December 2013). Natural
proteins: Sources, isolation, characterization and applications. Retrieved from
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3841988/

No part of this manual may be reproduced without written permission from the Chemistry Department of
the College of Arts and Sciences, Mindanao University of Science and Technology, Cagayan de Oro City.