SOLUBILITY OF PROTEINS

Briones Domínguez Luis Beltrán 16270446

German Castle Jennifer Jocelyn 16270451
Ramírez Hernández Alondra Maribi 16270502
Kevin Antonio Ruiz Matus 16270506
INTRODUCTION

The functional properties of proteins are often affected by the

solubility of the protein, especially in the case of swelling, foaming,
emulsification and gelation. Insoluble proteins have a very limited use in
foods. The solubility of a protein is the thermodynamic manifestation of
balance between protein-protein interactions and solvent-protein interactions, which in turn
depend on the hydrophobicity and ionic nature of them. The interactions
hydrophobic interactions promote protein-protein interactions that impact a
decrease in solubility, while ionic interactions promote the
protein-water relationship that causes an increase in solubility.

Proteins are soluble in water when they adopt a globular conformation. The
solubility is due to the free (-R) radicals of amino acids which, when ionized,
they establish weak links (hydrogen bonds) with water molecules. Thus,
when a protein is solubilized, it becomes coated with a layer of molecules of
water (solvation layer) that prevents it from binding to other proteins which
it would cause its precipitation (insolubilization). This property is what makes it possible
the hydration of the tissues of living beings. The solubility data is very
useful for determining the optimal conditions for extraction and purification of
proteins, from their natural sources and to separate protein fractions. The
The degree of insolubility of a protein is a practical measure of the
denaturation + aggregation of the protein, a protein that is presented
denatured, partially aggregated is often ineffective in the processes of
gelification, emulsification or foam formation. Protein solubility at pH
neutral or at pI is often the first of the functional properties that is
measures at each of the stages of preparation and processing of an ingredient
protein.
It has been proposed by Bigelow that the solubility of the protein will be greater the more
the average hydrophobicity is lower and the charge frequency is higher. Although, this
correlation is empirical, it occurs in most proteins and the model
it can be improved if only hydrophobicity and hydrophilicity are taken into account
superficial and not all of the protein, since only the surface that is on
contact with water is what exerts the functional properties.
Classification of proteins based on their solubility;
Albumins: they are those that soluble in water at pH 6.6 (serum albumin,
ovalbumin, and alpha-lactalbumin.
Globulins: they are soluble in dilute saline solutions at pH 7.0 (glycinin,
phaseolin and b-lactoglobulin.
Glutelins: they are soluble in acidic solutions (pH 2) and alkaline solutions (pH 12)
(wheat gluten)
Prolamins: they are soluble in 70% ethanol (zein, corn gluten, and gliadins)
of wheat).

The solubility of proteins depends on numerous factors, among which

they are:
Solubility and pH

At pH values above and below the isoelectric point, proteins have

a net positive or negative charge, respectively. At the isoelectric point the
proteins have a minimal solubility, which, when graphed, allows observation
U-shaped curves. Most proteins are stable for a
a certain pH zone and often denature when subjected to
very high or very low pH values. For pH values close to the point
isoelectric, the protein molecules that show a minimum of interactions
with water and its net charges, are weak enough to allow for
to bring the polypeptide chains closer. Sometimes these aggregates are formed,
what can lead to a precipitation.
Solubility and organic solvents

The addition of organic solvents, such as ethanol or acetone, increases the forces
intra- and intermolecular electrostatics, both repulsive and attractive as
explains above in the denaturation section. The electrostatic interactions
intramolecular repulsions cause the protein molecule to unfold,
state in which the formation of hydrogen bonds is promoted
intermolecular interactions between the peptides now exposed; additionally, they are promoted.
intermolecular electrostatic interactions that are, on the contrary, attractive
between groups with opposite charges. These polar intermolecular interactions
They favor the precipitation of the protein in organic solvents or reduce the
solubility in an aqueous medium.
Solubility and temperature

At a constant pH and ionic strength, the solubility of many proteins generally

it increases by subjecting it to a temperature between 0 and 40ºC. They are presented
exceptions when proteins are highly hydrophobic, such as b-casein and
some proteins from cereals, which have negative relationships with
temperatures above 40ºC. The increase in thermal energy causes
unfolding of proteins (denaturation), as the non-polar groups
they are exposed, aggregation and precipitation of the protein occurs, and therefore the
solubility decreases.

The action of sales

The addition of neutral salts can increase solubility, as a consequence of

called the salting effect, typical of poorly soluble substances. The ions of the
neutral solutions, with molarities between 0.5 and 1 M, can increase the
solubility of proteins. Ions react with the charges of the proteins and
they reduce the electrostatic attraction between opposite charges of groups
next. On the other hand, the solvation due to these ions allows for an increase in the
solvation of proteins and therefore their solubility. If the concentration of the
neutral sales are greater than 1 M, the solubility of proteins decreases and can
to lead to a precipitation.
Solubility and ionic strength
In a low ionic strength (< 0.5), the ions neutralize the charge on the surface of
the protein. Solubility decreases for proteins that contain a high
incidence of non-polar zones and it increases for proteins that are not. The first
The behavior is typical of soy proteins,86 and the second presents the b-
lactoglobulin. The decrease in solubility is caused by the increase in
hydrophobic interactions, as the greater solubility is due to a decrease in
the ionic activity of the molecule.

Identify the factors that affect the solubility of proteins by modifying the
conditions of the environment in which they are found.
 METHODOLOGY

Break a fresh egg in half and separate the white from the yolk carefully.
Be careful not to mix them. Then a portion of the egg white will be diluted with two.
portions of distilled water and will be mixed gently. The prepared solution is
it will be used in the rest of the experiments. The first experiment will analyze the
Effect of solvent change in this will place 15 ml of the protein solution.
In a beaker, acetone will be allowed to drip slowly and will be stirred.
gently until observing some change, the experiment will be repeated, but now
using ethanol. The second experiment will analyze the effect of the change of
temperature, it will bring water to a boil in a water bath and in a beaker, one
you will place 15 ml of the protein solution, then the glass is immersed in the water
boiling for 2 minutes, then take out the glass and observe what will happen with
the egg, the experiment will be repeated using ice water. The third experiment
It will be the effect of pH change in this, two beakers will be used.
250ml where you will place 50 ml of fresh milk for each one and the numbers will be marked.
1 and 2 to distinguish them. A few drops of vegetable dye are added to both glasses.
and it will be stirred. In glass 1, it will be gradually added with the help of a dropper.
amount of white vinegar and it will be mixed gently. In glass 2, the will be added.
the same amount of lemon juice will be mixed, and it will be left to rest for about 20 minutes
to then observe what will happen. Subsequently, it will be filtered with the help of a
strainer and filter paper separately for each of the cups. The filtered liquid is
you will place it in a beaker, and then observe the filter paper and the liquid
obtained. In the fourth experiment, the effect of the change will be analyzed regarding the
salt concentration, in this 15ml of the protein solution will be placed in a
Beaker and with a spatula, sodium chloride will be added gradually.
to then mix them, he will continue adding sodium chloride until observing some
change, repeat the experiment with ammonium sulfate. In the experiment
The effect of the presence of detergents will be analyzed, 15ml of the
protein solution in a beaker, add a little to it with a pipette
little detergent and mix it, continue adding until observing some
change.
 RESULTS AND DISCUSSION
Effect of solvent change

Solution of
proteína

Fig.1 Protein solution (egg white mixed with distilled water).

The prepared solution was used to conduct the following experiments.

Ethanol

Fig.2 Protein solution with Fig.3 Solution of

ethanol protein with acetone
In the solution, it can be observed In the solution, one can
that the coloration changed from observe that he/she maintained the
transparent to a solution same coloration y
papaya y the solubility consistency that it had the
decreases as this precipitates. egg white.
This as a result of adding
droplets of ethanol into the solution.
Effect of temperature change

Fig. 4 Protein solution in

Fig.5 Protein solution in
cold water
hot water
In the solution, one can observe
that the coloring did not change and In the solution, it can be observed
maintained the consistency that the coloring changed
partially soluble. forming a coagulation of
white color (milky solution)
que disminuye la solubilidad.
Effect of pH change

Vinegar
Lemon

Fig.6 Protein solution with

lemon
Fig.7 Protein solution with
In the solution, it can be observed vinegar
that the milk mixed with drops
of dye when adding drops of In the solution, it can be observed
lemon this precipitates in the part the presence of precipitate due to
from below, the protein of milk the denaturation of the protein
(casein) denatures and from milk (casein) by action of
form large lumps and Very acidic pH of the reagent.
small ones that remain attached to
the walls of the glass.
 Lemon Vinegar

Fig. 8 solution of milk with dye and drops of lemon and filtered liquid vinegar
In the filtered solutions, it can be observed that the obtained liquid contained
Lemon the granules remained on the filter paper and a pure liquid with coloration was obtained.
The drop in the vinegar liquid change, the color was a bit more intense.

Lemon Vinegar
Fig. 9 Filters from the milk solution with dye
The solution that contained lemon drops was a bit difficult to filter since it contained
grains that can be observed that got trapped in the filter paper, in
the filtering of the solution changed, and filtering with vinegar drops was easier now.
that this one didn't have granules, it was a complete liquid.
Effect of change in salt concentration

Fig.10 Protein solution in Fig. 11 Protein solution in

sodium chloride ammonium sulfate
In the solution, one can observe In the solution, it can be observed
that the coloring did not change and
that the coloring does not change but
he/she/it maintained
the consistency what precipitates ammonium sulfate,
partially soluble and the salt positive for denaturation
precipitate for sales.

Effect of the presence of detergents

Detergent

Fig.12 Protein solution with detergent

In the solution, more foam can be observed caused by the detergent apart from what
It contained the solution and the coagulation of the sample.
 DISCUSSION

The solubility of proteins is determined by three main factors: (1) their

degree of hydration, (2) its density and charge distribution along the
chain, and (3) the presence of non-protein compounds such as phosphates, carbohydrates
the lipids that may have a stabilizing effect. From the point of view
thermodynamic, solubilization corresponds to a simultaneous dissociation of the
molecules of the solvent and protein molecules, prior to a dispersion
of these last ones in the solvent, with a maximum contact interfacial surface
between the protein and the solvent.
Effect of solvent change
In figures 2 and 3, the obtained results are due to the fact that in the case of the
aggregation of less polar solvents than water such as ethanol or acetone
they generate a decrease in polarity which creates a decrease in the degree of
hydration of the surface ionic groups of the protein molecule which
finally precipitation, as can be evidenced in the
results obtained. In the case of albumin, it was observed how ethanol
achieves denaturation, coagulation of the egg white while the
I dissolve the acetone. The addition of an organic solvent like ethanol produces
aggregates of protein molecules that tend to precipitate, this is due to the
the solvent has a dielectric constant lower than that of water, which causes
an increase in the attractive forces between opposite charges and a decrease
in the ionization degree of proteins, and consequently a decrease
in the solubility of this.
Effect of temperature change
Figures 4 and 5 present the solution with cold water and with hot water.
solution with hot water after having brought the egg white to a water bath
a significant change in the physical state of the sample was evidenced, moving from
liquid to solid product of cooking the heat on the substance. The chains of
proteins found in egg white are coiled adopting a
spherical shape. They are called globular proteins. When frying or in this case subjecting
The heat causes the egg albumin solution, the heat makes the protein chains
they unfold and form links that connect some chains with others. This change
of structure, gives the egg white the consistency and color that is observed in a
cooked egg, changing from a light and gelatinous color to a white flake,
At high temperatures, the protein favors hydrogen bonds between proteins.
and between these and the water molecules, which changes the conformation and affects
stability since amino acids ionize with difficulty and therefore can
news association and precipitation. In the cold water, no change is observed.
abrupt. It has been proven that each of the protein fractions of the egg white
egg shows a certain susceptibility to pH and treatments
thermal; as acidity increases, ovotransferrin, ovomacroglobulin,
ovomucoid and globulins become more unstable at high temperatures.
During the experimentation on precipitation reactions due to actions of
white clots formed by acids.
Effect of pH change
In figures 6 and 7, the results obtained are similar to what occurs with
the egg, the acid present in vinegar (acetic acid), in lemon (citric acid),
is capable of producing the denaturation of the protein called casein that
there is in the milk. Casein precipitates due to the action of acids. This can occur
through the formation of lactic acid by bactericidal action on lactose or
by adding an acid like in our case where we added lemon until
reaching the isoelectric point of milk, precipitation can occur due to
enzymatic action of chymotrypsin. When we add an acid to casein
(vinegar) its denaturation occurs, leading to a chemical reaction that
it alters its structure, causing casein to stop being soluble in water and precipitate.
Vinegar (acetic acid) contains free hydrogen atoms. The molecules of
The casein in milk combines with the loose hydrogen atoms it contains.
the acid and a chemical reaction occurs. The molecules of casein contained
in the milk they have a negative charge, while the loose hydrogen atoms
what is in the acid has a positive charge. Opposite charges attract, so
that the casein molecules and the loose hydrogen atoms cluster together and
form visible clots.
Effect of the change in the concentration of salts

In figures 10 and 11, the solution of sodium chloride and ammonium sulfate results
The results obtained are due to the fact that the solubility of the protein is sensitive to the concentration.
of salt, the concentration of salt is expressed in terms of ionic strength. When mixing
the egg white sample with Ammonium Sulfate, the latter being a salt
neutralized the interactions (hydrogen bonds) of the tertiary structure of the
protein, thus causing its unfolding, which precipitated to
consequence. In a solution with ammonium sulfate, precipitation due to variation of
ionic strength is due to neutral salts exerting pronounced effects on
the solubility of globular proteins. At low concentration, salts
they increase the solubility of many proteins, a phenomenon known as
salinization by salting. The salts of divalent ions such as MgCl2 and
(NH4)2SO4 is much more effective in the solubilization of proteins than the
monovalent ion salts. The ability of neutral salts to influence the
the solubility of proteins is dependent on their ionic strength which constitutes a
measure of both the concentration and the number of existing electric charges
in the cations and anions provided by the salt. Which in the case of sulfate
of ammonium would be the presence of a monovalent cation and a divalent anion, and the
the maximum concentration that the solvent used can solvate, that is, a
high concentration. As the ionic strength increases, the solubility of a
protein begins to decrease. At a sufficiently high ionic strength,
some proteins can be almost completely precipitated from their solution,
effect called salting out.

Effect of the presence of detergents

In Figure 12, the obtained results were due to the fact that detergents are
similar to soaps because they have a soluble ionic end in their molecule
in water and another nonpolar end that displaces oils. Detergents have
the advantage, over soaps, of forming soluble calcium and magnesium sulfates
in water, so they do not form clots when used with hard water. It is produced the
solubilization not only of the polar "residue" (interphase level) but also of
that situated in the middle of the detergent micelles. Heat facilitates solubility.
of the detergents, reducing on the other hand the viscosity of the greasy residue,
making it more easily dispersible in that way. For not coagulating the albumin
and thanks to the low surface tension, those substances penetrate deeply into
all the concavities, the canaliculi, and the inflections of the duct
root moistening the organic remains and microorganisms inside it.
 CONCLUSION

In the experiments conducted, the solubility of the protein was analyzed.

from egg white and milk, the factors that are related to their increase
or decrease are the following; the change of pH, the temperature, the concentration
of sales, of solvent and presence of detergents each of these plays a role
important in the solubility of proteins, as we add lemon
the milk is starting to form a kind of lumps which is indicative of
that the isolectric point of the milk is being reached, which is known as
commonly known as 'sour milk'. An increase in temperature causes the energy
kinetics of the molecules, which generates a disorganization of the envelope
aqueous phase of the proteins, this disorganization ultimately generates the
denaturation of proteins. On the other hand, at high temperatures it
destroys weak interactions and disrupts the protein structure, of
the way the hydrophobic interior interacts with the aqueous medium and the production of the
aggregation and precipitation of the denatured protein. In the reactions where
the precipitation obtained was due to a change in the physical state of the protein,
while in the coagulations a change in the physical state has occurred and
in the chemical structure that's why it is irreversible. At the physicochemical level, proteins
react differently depending on the factors that affect
solubility, the chemical reactions and interactions that take place are
Ionic interactions (Increase protein-water interactions, Increase
solubility), Hydrophobic interactions (Decrease solubility, Increase
protein-protein interactions, It was found that pH, temperature, and salts
they influence the process of aggregation and coagulation of proteins. In
We observed that white-colored clots formed during the precipitation of albumin.
The theory tells us that it should precipitate, it may take longer for these.
clots form a white precipitate.
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