Universidad Autnoma De Coahuila

Facultad De Ciencias Biolgicas

Practica #8
EXTRACCIN DE LA CASEINA Y DETERMINACIN DEL
PUNTO ISOELECTRICO

Fernndez Michel Silvia Guadalupe Cuauhtemoc Medellin Luna

Matricula 14318264
Entrega 8/05/2017

Torren, Coahuila.
 EXTRACCIN DE LA CASEINA Y DETERMINACIN DEL
PUNTO ISOELECTRICO

SUMMARY.
In this practice we can determined the point isoelectric of the milk and
then we have determined also the absorbancia through a process
chemical.
INTRODUCTION.
The isoelectric point (pI) of a peptide is defined as the pH at which the
peptide has a net charge of zero. At pH values below the pI, peptides
carry a net positive charge; Above the pI, they carry a net negative
charge pI-values of peptides can be utilized as a basis for separation in
solution. If a voltage is applied to a complex peptide mixture in a pH
gradient, the peptide will migrate to the pH at which they are neutrally
charged. The pI of a peptide maybe accurately calculated from its
sequence, and provides information that complements mass
spectrometric methods for protein and peptide identification.
 (Mathematical formula to calculate the Isoelectric
point.)
Protein purification includes a series of processes to isolate a particular
protein from a complex mixture. This technique is critical for
characterizing the function, structure, and interaction of the protein of
interest. An analytical purification generally uses three distinct
properties to separate proteins, including the isoelectric technique.
Protein purification, protein isoelectric pointA protein can be purified
according to its protein isoelectric point by running the said protein
through an ion exchange column or a pH-graded gel. At the isoelectric
point, a protein has no net charge. Above the isoelectric point, a protein
carries a net negative chargebelow it, a net positive charge.
Because a majority of weakly acid remains in nearly every protein, they
are generally negatively charged at neutral pH. The isoelectric point is
significant in protein purification because it represents the pH where
solubility is typically minimal. Here, the protein isoelectric point signifies
where mobility in an electro-focusing system is zeroand, in turn, the
point where the protein will collect.
HYPOTHESIS.
We use chemical methods to get a good amount of caisena.
MATERIALS AND METHODS.
MATERIALS.
10 vasos de precipitado de 25 ml (o frascos Gerber chicos)
2 vasos de precipitado de 50ml
1 vaso de precipitado de 250ml
1 probeta de 50ml
1 matraz aforado de 50 ml
1 pipeta graduada de 5,0 ml
1 pipeta graduada de 1,0 ml
1 pipeta graduada de 10,0 ml
1 Embudo Buchner
1 Matraz Kitazato
1 Manguera
1Termmetro
1 Potencimetro (pH-metro)
2 Papel de filtro

CHEMICAL REAGENTS.
Agua destilada
Acetato sdico 0,1 N
cido actico 0,01 N
cido actico 0,1 N
cido actico 1,0 N
NaOH 1N
ter etlico
Etanol al 70%
Soluciones buffer pH 4,0 y 7,0 para calibrar el potencimetro
Leche entera corriente ( 1 litro)

EQUIPMENT.
Bascula analtica.
METHODS.
Milk
EXPERIMENTAL DEVELOPMENT.
A. Aislamiento de la casena:

Calentar 150 ml de
Aadir 50
agua destilada a 38
mL de Leche
C.

Adicionar Ac.
Acetico 1M hasta Gota a gota con
observar agitacion
precipitado

Sedimentar y filtrar

Lavar el precipitado
con 20 mL de etanol

Secar el precipitado

Colocar el
precipitado pesado Agregar 5 mL ter
en un vaso de etilico
precipitado

Filtrar
 Desechar liquido
B. Preparacin de la solucin de casena:

Colocar 250 g de
caseina

Agregar 20 mL de
agua y 5 Ml de
NaOH 1N

Verter en un matraz Adicionar 5 mL de

aforado de 50 ml Ac. Acetico

Diluir con agua

destilada hasta 50
mL

Desechar liquido
 C. Determinacin del PI de la casena:

RESULTS

TUBO Acetato Ac. Actico Ac. Actico pH resultante pH Real Absorbancia

0.1N 0.1N 0.01 N (aprox.) 640 n.m

1 0.5 9.5 - 3.2 2.56 .056

2 1 9 - 3.6 3.22 .020
3 1.5 8.5 - 3.8 3.35 .030
4 2 8 - 4.0 3.55 .024
5 3 7 - 4.2 4.15 .045
6 4 6 - 4.5 4.30 .048
7 6 4 - 4.7 4.50 .035
8 8 2 - 5.1 5.00 .022
9 6 - 4 5.5 5.30 .013
10 8 - 2 6.1 5.58 .028

This table shows the actual pH results taken from the reagent samples
as per the table and then measured at absorbance at 640 nm.
QUESTIONARY.
1. Representar la absorbancia a 640 nm frente al pH de cada tubo en
el apartado
2. Qu sucedera si se representa la transmitancia?
3. Cul es el punto isoelctrico de la casena?
Estas micelas de casena se estabilizan por iones de calcio e
interacciones hidrofbicas. Otro dato interesante, utilizado para
separar las casenas del resto de las protenas lcteas mediante su
precipitacin, es que su punto isoelctrico (pI) promedio es de 4,6.

4. Por qu las protenas tienen solubilidad mnima en el PI?

Por desnaturalizacin de las protenas a la prdida de las estructuras cualquier

El proceso mediante el cual la protena desnaturalizada recupera su estructura nativa se

llama renaturalizacin Esta propiedad es de gran utilidad durante los procesos
de aislamiento y purificacin de protenas, ya que no todas la protenas reaccionan de
igual forma ante un cambio en el medio donde se encuentra disuelta.

factor que modifique la interaccin de la protena con el disolvente

disminuir su estabilidad en disolucin y provocar la precipitacin. La
precipitacin suele ser consecuencia del fenmeno.
Llamado desnaturalizacin y se dice entonces que la protena. se
encuentra desnaturalizada

DISCUSSIONS.
Casein is a protein found in milk. Casein is a set of proteins. We
obtained the precipitation of the casin from the milk successfully by
using the filtration method.
CONCLUSIONS.
Isolation of casein from milk is possible using the low solubility that this
protein has when it is brought to its isoelectric point, although the yield
is less than the amount of milk used.
BIBLIOGRAPHY.
Food Chemestry 4th revised and extended etidion, Professor Dr. Hans-
Dieter Belitz
Cappllary Electrophoresis Of Proteins, Tim Wehr
A survey of molecular descriptors used in mass spectrometry based
proteomics, Audain E.
The Protein Isoelectric Point database, Bunkute E.