Page 87 - Color Atlas of Biochemistry

P. 87

78 Biomolecules

Isolation and analysis of proteins spherical gel particles (diameter 10–500 µm)
of polymeric material (shown schematically
Purified proteins are nowadays required for a in 1a). Theinsides of theparticles aretra-
wide variety of applications in research, med- versed by channels that have defined diame-
icine, and biotechnology. Since the globular ters. A protein mixture is then introduced at
proteins in particular are very unstable (see the upper end of the column (1b)and elution
p. 72), purification is carried out at low tem- is carried out by passing a buffer solution
peratures (0–5 °C) and particularly gentle through the column. Large protein molecules
separation processes are used. A few of the (red) areunableto penetrate theparticles,
methods of purifying and characterizing pro- and therefore pass through the column
teinsare discussedon thispage. quickly. Medium-sized (green) and small par-
ticles (blue) are delayed forlongerorshorter
periods (1c). The proteins can be collected
A. Salt precipitation
separately from the ef uent (eluate)(2). Their
The solubility of proteins is strongly depen- elution volume V e depends mainly on their
dent on the salt concentration (ionic strength) molecular mass (3).
of the medium. Proteins are usually poorly
soluble in pure water. Their solubility in-
creases as the ionic strength increases, be- D. SDS gel electrophoresis
cause more and more of the well-hydrated The most commonly used procedure for
anorganic ions (blue circles) are bound to checking the purity of proteins is sodium do-
the protein’s surface, preventing aggregation decylsulfate polyacrylamide gelelectropho-
of the molecules (salting in). At very high resis (SDS-PAGE). In electrophoresis, mole-
ionic strengths, the salt withdraws the hy- cules move in an electrical field (see p. 276).
drate water from the proteins and thus leads Normally, the speed of their movement de-
to aggregation and precipitation of the mole- pends on three factors—their size, their shape,
cules (salting out). For this reason, adding and their electrical charge.
saltssuch asammoniumsulfate (NH 4 ) 2 SO 4 In SDS-PAGE, the protein mixture is treated
makes itpossibleto separateproteins from a in such a way that only the molecules’ mass
mixture according to their degree of solubility affects their movement. This is achieved by
(fractionation). adding sodium dodecyl sulfate (C 12 H 25 -
OSO 3 Na), the sulfuric acid ester of lauryl alco-
hol(dodecylalcohol). SDSis a detergent with
B. Dialysis
strongly amphipathic properties (see p. 28). It
Dialysis is used to remove lower-molecular separates oligomeric proteins into their sub-
components from protein solutions, or to ex- units and denatures them. SDS molecules
change the medium. Dialysis is based on the bind to the unfolded peptide chains (ca.
fact that due to their size, protein molecules 0.4 g SDS / g protein) and give them a strongly
areunableto pass through thepores of a negative charge. To achieve complete denatu-
semipermeable membrane, while lower-mo- ration, thiols are also added in order to cleave
lecular substances distribute themselves the disulfide bonds (1).
evenly between the inner and outer spaces Following electrophoresis, which is carried
over time. After repeated exchanging of the out in a vertically arranged gel of polymeric
external solution, the conditions inside the acrylamide (2), the separated proteins are
dialysis tube (salt concentration, pH, etc.) made visible by staining. In example (3), the
will be thesameas in the surrounding solu- following were separated: a)a cell extract
tion. with hundreds of different proteins, b)a pro-
tein purified from this, and c)amixture of
proteins with known masses.
C. Gel filtration

Gel permeation chromatography (“gel filtra-
tion”) separates proteins according to their
size and shape. This is done using a chroma-
tography column, which is filled with

82 83 84 85 86 87 88 89 90 91 92