3.

AISLAMIENTO Y
CARACTERIZACIN DE ENZIMAS

3.1 Tcnicas de purificacin de enzimas

3.2 Tcnicas analticas para cuantificacin de protenas
3.3 Medicin de actividad enzimtica

Tamao y nmero de cromosomas en diferentes organismos

Organismo

Tamao del
genoma (Mpb)*

Nmero de
cromosomas

Nmero de
genes

Secuencias
codificadoras para
protenas(%)

Virus de la
Hepatitis B

0.5

12

99

Escherichia coli

4.6

4,288

88

Saccharomyces
cervisiae

12

16

6,000

70

Caenorhabditis
elegants

97

19,000

25

Arroz

440

~35,000

~10

Drosophila

180

13,600

13

Pollo

1,200

39

Ratn

3,000

20

Vaca

3,000

30

Humano

3,000

23

~35,000

1-1.5

* Millones de pares de bases

* * Tamao del genoma y nmero de cromosomas referidos a clulas haploides

PARA ENTENDER LA FUNCIN DE UNA ENZIMA

HAY QUE PRUFICARLA

Fuente
El ensayo
Como liberar una la protena de su nicho natural
Solubilidad, Tamao, Carga y Afinidad

PURIFICACIN

CARACTERIZACIN

Electroforesis
Secuencia peptdica
Mapeo Trptico
Intercambio inico
Interaccin hidrofbica
SEC
Fraccionamiento por

diferencia de coeficioente
de difusin

Ultracentrifugacin

analtica
Espectroscopa
Dispersin de luz
dinmica
Anlisis de glicosilacin
Espectrometra de masas
Calorimetra
Determinacin de
estructura

LA FORMA DE ROMPER EL TEJIDO

Fundamento

Esfuerzo de corte producido por

lquidos

Homogeneizador
Potter-Elvehjem

Tejidos blandos

Polytron

Tejidos blandos y
duros

Prensa de French

Bacterias

Esfuerzo de corte producido por slidos Mortero con mazo

Cavitacin gaseosa

Medios qumicos y bioqumicos

Materiales

Vegetales

Bomba de nitrgeno

Clulas

Sonicador

Orgnelos y clulas

Medio hipotnico

Clulas de la sangre

Enzimas (lisozima)

Hidrlisis de la pared
celular bacteriana

El ensayo

Centrifugacin Diferencial. Las clulas se homogenizan y se centrifugan con

cambios en la fuerza centrfuga. El material ms denso se va la fondo del tubo.
Cada una de las fracciones pueden usarse para fines especficos.
http://www.ncbi.nlm.nih.gov/books/NBK22410/figure/A441/

Fraccionamiento: Salting out y dilisis

Dilisis: Las molculas de protenas (crculos rojos) quedan retenidos dentro de la

bolsa de dilisis, en la medida que molculas pequeas (azules) pueden difundir.
http://www.ncbi.nlm.nih.gov/books/NBK22410/#A448

Cromatografa de tamices moleculares

(permeacin en gel, gel filtration)

En la cromatografa de tamices moleculares un mezcla de protenas se

aplica en la parte superior de una columna llena de esferas porosas.
http://www.ncbi.nlm.nih.gov/books/NBK22410/#A448

Cromatografa de intercambio inico

En la cromatografa de intercambio inico se

separan protenas de acuerdo a su carga neta.

Cromatografa de afinidad

Como ejemplo, la Cocanavalina A (amarilla) se une a

un soporte slido que contiene glucosa. La adicin de
una concentracin ms alta de glucosa desprende la
concavalina del soporte.

Electroforesis en Gel de poliacrilamida (PAGE)

Step

Total protein
(mg)

Total activity
(Unidades=mi
cromoles/min)

Specific
activity,
(U/mg)

Yield (%)

Purification
level

Homogenization

15,000

150,000

10

100

Salt fractionation

4,600

138,000

30

92

Ion-exchange
chromatography

1,278

115,500

90

77

Molecular
exclusion
chromatography

68.8

75,000

1,100

50

110

Affinity
chromatography

1.75

52,500

30,000

35

3,000

La actividad enzimtica es una medida de la cantidad de enzima activa

presente y del nivel de actividad de la misma. La actividad expresa la
cantidad de sustrato convertido por unidad de tiempo, teniendo
condiciones.
1 UI = 1 mol sustrato convertido x min-1

electroforesis
Elecroforesis de zona
Isoelectroenfoque
PAGE-SDS

PAGE-SDS

Isoelectroenfoque

2D

HPLC

m is the mass of the particle

is the partial specific volume
is the density of the medium
f is the frictional coefficient (a measure of the
shape of the particle).
The (1 - ) term is the buoyant force exerted by
liquid medium

Centrifugacin por Zona. Formacin de un gradiente,

Colocacin de la muestra, Centrifugacin y Coleccin de
fracciones.
http://www.ncbi.nlm.nih.gov/books/NBK22410/figure/A479/

matrix-assisted laser desorption-ionization (MALDI) and electrospray spectrometry

MALDI-TOF Mass Spectrometry http://www.youtube.com/watch?v=OKxRx0ctrl0&feature=related

(1) The protein sample, embedded in an appropriate matrix, is ionized by the application of a laser beam.
(2) An electrical field accelerates the ions formed through the flight tube toward the detector. (3) The
lightest ions arrive first. (4) The ionizing laser pulse also triggers a clock that measures the time of flight
(TOF) for the ions. [After J. T. Watson, Introduction to Mass Spectrometry, 3d ed. (Lippincott-Raven,
1997), p. 279.]

http://www.ncbi.nlm.nih.gov/books/NBK56011/

MALDI-TOF Mass Spectrum of Insulin and lactoglobulin

A mixture of 5 pmol each of insulin (I) and -lactoglobulin (L) was ionized by MALDI, which produces
predominately singly charged molecular ions from peptides and proteins (I + H+ for insulin and L + H+ for
lactoglobulin). However, molecules with multiple charges as well as small quantities of a singly charged dimer of
insulin, (2 I + H)+, also are produced. [After J. T. Watson, Introduction to Mass Spectrometry, 3d ed. (LippincottRaven, 1997), p. 282.]

Technique

Process

Application

2-DE (2-dimensional gel electrophoresis)

Soluble protein separation in gel by isoelectric

focusing (IEF) followed by separation based on
molecular weight (SDS-PAGE)

Fractionation of proteins, through highly

simplified protein spots

1-DE (1-dimensional gel electrophoresis)

Soluble protein separation in gel by isoelectric

Low-resolution separation of proteins.
focusing (IEF) or molecular weight (SDS-PAGE) Generally used prior to subsequent
separation steps

DIGE (differential gel electrophoresis)

Fluorescent dye labeled soluble proteins

separated by isoelectric point followed by
separation based on molecular weight

Quantitative 2-D fractionation used for

accurate relative expression differences of
soluble proteins

OGE (off-gel electrophoresis)

Soluble protein separation in a gel/buffer

combination. Isoelectric focusing without
ampholytes

Separation of intact proteins. Used to

achieve moderately simplified protein
mixtures

Target affinity enrichment

Protein binds to an immobilized substance

selective for the protein of interest. Protein
binding is reversible

Known protein complexes MARS,

oxidations, glycosylations, phosphorlyations,
RNA/DNA. For highly specific protein
fractionation

Technique

Process

Application

SCX/HPLC (strong cation

exchange/HPLC)

On-line elution of tryptic digests

based on ionic strength followed
by reversed phase HPLC

Highly complex protein digest

mixtures

HPLC/CE

On-line elution based on

hydrophobicity and charge/mass
ratio

Low level, small volume protein

identification of digests

HPLC/CZE

On-line elution based on

hydrophobicity and size

Low level, small volume protein

Class enrichment

Affinity interaction of peptide

Preferential removal of modified

peptides

ICAT (isotope coded affinity tag)

Biotin derivatized cysteine

residues selectively isolated by
avidin column chromatography

Relative quantification and

identification of proteins based on
isotopic labels

AQUA (absolute quantitative

analysis)

Isotopically labeled peptides

quantified by the presence of a
stable isotopically labeled internal
standard

Absolute quantification of peptide

concentration

iTRAQ (isotope tagging reagents

for absolute quantification)

Isobaric labeling of primary

amines

Comparison of up to four samples

quantitatively in MS/MS mode