Additional PharmBioSci3 Notes

PRECIPITATION OF PROTEINS AT ISOELECTRIC POINT

A Protein solubility
The solubility of proteins in aqueous buffers depends on the distribution of
hydrophilic and hydrophobic amino acid residues on the proteins surface. Proteins
that have high hydrophobic amino acid content on the surface have low solubility in
an aqueous solvent. Charged and polar surface residues interact with ionic groups in
the solvent and increase solubility. Knowledge of amino acid composition of a protein
will aid in determining an ideal precipitation solvent and method.
B Isoelectric point precipitation
Isoelectric point (pI) is the pH-value of a solution at which the total net charge
of a protein equals zero. At a solution pH that is above the pI the surface of the
protein is predominantly negatively charged and therefore like-charged molecules will
exhibit repulsive forces. Likewise the surface of the protein is predominantly
positively charged at a solution pH that is below the pI, and repulsion between
proteins occurs. However, at the pI the negative and positive charges are eliminated,
repulsive electrostatic forces are reduced and the dispersive forces predominate. The
dispersive forces will cause aggregation and precipitation. The pI of most proteins
ranges between the pH 4 to 6.
When microorganisms grow in milk, they often produce acids and lower the pH
of the milk. The phenomenon of precipitation or coagulation of milk protein (casein)
at low pH as milk becomes spoiled is one of the common examples of protein
isolation due to changes in the pH.
C Isoelectric Precipitation of Casein
Casein is a phosphoprotein, which has phosphate groups attached to some of
the amino acid side chains. These are attached mainly to the hydroxyl groups of the
serine and threonine. Actually, casein is a mixture of at least three similar proteins,
which differ primarily in molecular weight and amount of phosphorus they contain
(number of phosphate groups):
Casein

MW
Kd

27.3
24.1
8.0

Phosphate
groups/mole
cule
9
4-5
2

Casein exists in milk as the calcium salt, calcium caseinate. This salt has a
complex structure. It is composed of , , and caseins which form a micelle, or a

Additional PharmBioSci3 Notes

solubilized unit. Neither the nor the casein is soluble in milk, singly or in
combination. If casein is added to either one, or to a combination of the two,
however, the result is a casein complex that is soluble owing to the formation of the
micelle. A structure proposed for the casein micelle is shown in the following figure.
The casein is thought to stabilize the micelle. Since both and casein are
phosphoproteins, they are precipitated by calcium ions

The casein protein, however, has fewer phosphate groups and a high content
of carbohydrate bound to it. It is also thought to have all its serine and threonine
residues (which have hydroxyl groups), as well as its bound carbohydrates, on only
one side of its outer surfaces. This portion of its outer surface is easily solubilized in
water since these polar groups are present. Calcium caseinate has its isoelectric
(neutrality) point at pH 4.6. Therefore, it is insoluble in solutions of pH less than 4.6.
The pH of milk is about 6.6; therefore casein has a negative charge at this pH and is
solubilized as a salt. If acid is added to milk, the negative charges on the outer
surface of the micelle are neutralized (the phosphate groups are protonated) and the
neutral protein precipitates:
The calcium ions remain in solution. When milk sours, lactic acid is produced
by bacterial action (see below), and the consequent lowering of the pH causes the
same clotting reaction.
The casein in milk can also be clotted by the action of an enzyme called
rennin. Rennin is found in the fourth stomach of young calves. However, both the
nature of the clot and the mechanism of clotting differ when rennin is used. The clot
formed using rennin, calcium paracaseinate, contains calcium.

Rennin is a hydrolytic enzyme (peptidase) and acts specifically to cleave

peptide bonds between phenylalanine and methionine residues. It attacks the
casein, breaking the peptide chain so as to release a small segment of it. This
destroys the water-solubilizing surface of the casein, which protects the inner and
caseins and causes the entire micelle to precipitate as calcium paracaseinate. Milk
can be decalcified by treatment with oxalate ion, which forms an insoluble calcium
salt. If the calcium
ions are
removed
from
milk,
a clot
will: not be formed when the
Standard
Buffers
(Take
note
of these)
milk is treated with rennin. The clot, or curd, formed by the action of rennin is sold
commercially as cottage cheese. The liquid remaining is called the whey. The curd
can also be used in producing various types of cheese. It is washed, pressed to
remove any excess whey, and chopped. After this treatment, it is melted, hardened
and ground. The ground curd is then salted, pressed into molds, and set aside to age.

Additional PharmBioSci3 Notes

EXPERIMENT 5: PROTEIN HYDROLYSIS AND CHROMATOGRAPHIC ANALYSIS OF AA

Proteins must be broken down into amino acids and peptides by hydrolysis, utilizing
any of a number of methods, in order to provide nutrients in forms that cells may easily
utilize. Protein hydrolysates, also called peptones, are the result of the hydrolysis process
on protein material. For over a century, peptones have been used as the main nutritive
components in culture media. Peptones supply the carbon, nitrogen, minerals and growth
factors needed to support microbial and mammalian cell metabolic processes, thereby
promoting cell proliferation and production. Additionally, peptones have been used as
serum replacements and media supplements in mammalian cell culture media.
Commonly used protein sources, as starting material, include meat, casein and whey
(milk proteins), gelatin, soybean. Enzyme sources include animal organs (pancreatin and
pepsin), papaya (papain), fig (ficin), pineapple (bromelain) and microbes.
Acid Hydrolysis
Acid hydrolysis is a fairly harsh process, usually carried out at high temperature. This
process attacks all peptide bonds in the protein substrate, destroying some of the individual
amino acids. For example, tryptophan is usually totally lost in an acid hydrolysis. Cystine,
serine and threonine are partially broken down and asparagine and glutamine are
converted to their acidic forms. Vitamins are mostly destroyed by acid hydrolysis. Salt may
be formed during neutralization of an acid hydrolysis, resulting in a product with high salt
content. *(In the experiment, 6N Sulfuric Acid was used)
Alkaline Hydrolysis
Same mechanism with Acid hydrolysis but it employs metal hydroxides (e.g., barium
hydroxide in the experiment). It also destroys some individual amino acids and it is done at
a high temperature.
Enzymatic Hydrolysis
Proteolytic enzymes hydrolyze proteins more gently than acids, do not require high
temperature and usually target specific peptide bonds. The material that results from a
proteolytic digestion is a mixture of amino acids and polypeptides of varying lengths. For
example, the enzyme pepsin will cut an amino acid chain where there is a phenylalanine or
leucine bond. Papain will cut the chain adjacent to arginine, lysine and phenylalanine.
Pancreatin shows activity at arginine, lysine, tyrosine, tryptophan, phenylalanine and
leucine bonds.
Chromatographic analysis
Amino acid

Rf value

Additional PharmBioSci3 Notes

alanine

0.38

arginine

0.20

asparagine
aspartic acid
cysteine

0.5
0.24
0.4

glutamine

0.13

glutamic acid

0.30

glycine

0.26

histidine

0.11

isoleucine

0.72

leucine

0.73

lysine

0.14

methionine

0.55

phenylalanine

0.68

proline
not a true amino acid - shows up as yellow

0.43

serine

0.27

threonine

0.35

tryptophan

0.66

tyrosine

0.45

valine

0.61

Source: http://www.biotopics.co.uk/as/amino_acid_chromatography.html

Additional PharmBioSci3 Notes

Solvent used in the experiment: ethanol-water-ammonia (80+10+10 ratio)

Nindhydrin spray reagent for visualization of the AA spots (since they are
invisible)

Rf =

distance travelled by the spot

distance travelled by the solvent front

EXPT 6: IONIC PROPERTIES OF AA

Amino acids are organic compounds containing both an amino group (- NH3) and a
carboxyl group (- COOH).
Each of these amino acids has a side chain (R group) with distinctive chemical
properties.
Amino acids are grouped into five main classes based on the properties of their R
group: nonpolar AA, Polar Neutral AA, Polar Basic AA and Polar Acidic AA.
When amino acid is dissolved in water, it exists in solution as the dipolar ion, or
zwitterion.
In physiological systems where the pH is near neutrality, the amino group of an
amino acid will be protonated and the carboxylic acid group will be deprotonated.
This is called the zwitterion form.

Additional PharmBioSci3 Notes

A zwitterion can act as either an acid (proton donor) or a base (proton acceptor).

Substances having this dual nature are amphoteric. So, amino acids in aqueous
solution exist predominantly in isoelectric form.
Characteristic pH at which the net electric charge is zero is called the isoelectric point
(pI)
An amino acid has a net negative charge at any pH above its pI, and has a net
positive charge at any pH below its pI.
Each amino acid has its own pI value.
The ionizable groups of amino acids act as weak acids or bases, giving off or taking
on protons when the pH is altered.
Common amino acids are weak polyprotic acids (an acid that supplies two or more
protons during an acid-base rxn)
A simple amino acid (that does not have an acid or base group in the "R" group) is a
diprotic acid in its fully protonated form; it can donate two protons during its
complete titration with a base.
Titration curves are produced by monitoring the pH of given volume of a sample
solution after successive addition of acid or alkali. Titration curves are usually plots of
pH against the volume of titrant added or more correctly against the number of
equivalents added per mole of the sample.
Upon titration of amino acid with acid, it acts as a base, and upon titration with base,
it acts as an acid.
In the experiment, the amino acid represents either the conjugate base A- or the
conjugate acid HA from in the Henderson-Hasselbalch equation, depending on the
type of titration.

EXPT 7: ENZYMES AND THEIR ACTIVITIES

Enzymes are proteins that act as catalysts for biological reactions. Enzymes, like all
catalysts, speed up reactions without being used up themselves. They do this by lowering
the activation energy of a reaction. All biochemical reactions are catalyzed by enzymes.
Since enzymes are proteins, they can be denatured in a variety of ways, so they are most
active under mild conditions. Most enzymes have optimum activity at a neutral pH and at
body temperature.

Additional PharmBioSci3 Notes

Enzymes are also very specific they only act on one substrate or one class of related
substrate molecules. The reason for this is that the active site of the enzyme is
complementary to the shape and polarity of the substrate. Typically, only one kind of
substrate will fit into the active site.
Effect of Enzyme Concentration
During catalysis, the first step is the substrate (S) binding to the enzyme (E), giving an
enzyme-substrate complex (ES). This is an equilibrium reaction, and will be favored by a
high concentration of enzyme and/or substrate. After the substrate is bound, the reaction
takes place, and then the product is released.

E + S ES E + P

Effect of Temperature
All reactions are faster at a higher temperature. However, enzyme-catalyzed reactions
become slower or stop if the temperature becomes too high, because enzymes become
denatured at high temperatures. Therefore, enzymes have an optimum temperature that
corresponds to maximum activity. (At higher or lower temperatures, the activity of the
enzyme is lower.) The optimum temperature is usually around body temperature (37C).
Effect of pH
Each enzyme has an optimum pH. Above or below an enzymes optimum pH, its activity is
lower. The optimum pH of a particular enzyme corresponds to the pH of its natural
environment. For many enzymes, this corresponds to pH values of around 7. For pepsin,
which is active in the stomach, the optimum pH is 2 (the pH of the stomach).
Trypsin, which is active in the small intestine, has an optimum pH of 8 that matches the pH
of the small intestine.
Effect of Inhibitors
Inhibitors are substances that slow down or stop enzymes. Competitive inhibitors are
molecules that are very similar to the substrate, so they can bind to the enzyme but cannot
react. They compete with the substrate for the active site of the enzyme. Noncompetitive
inhibitors are molecules that are not similar to the substrate and therefore do not bind to
the active site. They do, however, bind to a different location on the enzyme and change
the shape of the active site so that the substrate can no longer bind. Irreversible inhibitors
form covalent bonds to the enzyme and therefore cannot be removed.
Amylase. This enzyme is responsible for hydrolyzing starch. In the presence of amylase, a
sample of starch will be
hydrolyzed to shorter polysaccharides, dextrins, maltose, and glucose. The extent of the
hydrolysis depends on how long it is allowed to react if the starch is hydrolyzed
completely, the resulting product is glucose.
Catalase is an enzyme that is present in many living cells to help convert hydrogen
peroxide (H2O2) to water and oxygen gas (H2O + O2). Hydrogen peroxide is a product of
lipid metabolism. It is a strong oxidizer, causing free radical formation which can be very
toxic and damaging to cells. It is important for cells to have enzymes such as catalase to
protect themselves against free radical formation by breaking hydrogen peroxide down into
safe and useful substances. As the catalase reaction occurs in a test tube, effervescence
(bubbling) develops that is easily observable as oxygen gas is formed. The general equation
for this reaction is:

2 H2O2 + catalase 2 H2O + O2 + catalase

A) Extraction and Activity of Amylase

Additional PharmBioSci3 Notes

(+) with iodine - bluish black (due to presence of starch) but disappears
eventually due to action of amylase
(+) with Fehlings R brick red (Cu2O) ppt due to presence of glucose
(reducing sugar) as a result of hydrolysis of starch
Preparation of Fehling's reagent:
Fehlings reagent is prepared by mixing exactly equal volumes of
Fehlings A and Fehlings B solution in a 1:1 ratio immediately before use
(usually 1 mL of each).
Fehlings A solution is an aqueous solution of copper sulfate
pentahydrate (CuSO45H2O) with few drops of concentrated sulfuric
acid.
Fehlings B solution is an aqueous solution of potassium sodium tartrate
and sodium hydroxide