Percentage heat stable ribonuclease activity
below 5 It could only be assumed that lowering the pH in this
way protected the homogenate from enzymes that may have a deleterious
effect on sensitive components. It could also be recognised that 150
indigenous R.N.A. is precipitated normally at this pH and removed
with the first fraction at centrifugation. A deep smooth stirring
action was maintained to prevent turbulence. The inclusion of the
chelating agent EDTA ensured the removal of harmful divalent cations
that might be released into the medium by preferentially forming a
more stable complex with this agent. Precautions were exercised in
the choice of suitable utensils, dionised water and analytical grade
reagents.
0
Kaplan and Heppel made no mention of a homogenisation time.
42 55
The homogenization time varied from one paper to another . A
15 minute duration to "emulsify" with a Waring blendor as advocated 35
by Maver and Greco seemed too long and suggested excessive froth
production and the possibility of protein denaturation by surface
tension effects.. In the present work the tissue which was not
membranous or tough in any way, appeared adequately dispersed after
A O
homogenizing for 1 - 1-jjs- minutes . The sieving of the homogenate
at this stage removed blood vessels, particularly a bulky splenic
artery which often remained as a stringy mass and interfered with
centrifugation operations. The removal of these blood vessel fragments
produced a more uniform sample for the first salting out with
ammonium sulphate, especially when reproducibility had to be taken
into account. Removal by centrifugation of cellular particles and
debris, components precipitated at pH 3*5 and by the first salting out
with ammonium sulphate from the homogenate completed the extraction
(page
6 5
).3. Salting out and heat treatment
(l) The salting out technique
(a) Theory
The theoretical aspects of the salting out process and of the
variables which determine the solubility of proteins in salt solution
66 67
have been presented recently by Gzok and Bucher and Dixon and Webb 69
and earlier by Green and Hughes in comprehensive reviews of the
subject.
Electrolyte progressively added to a protein solution causes two
effects, as the ionic strength increases. First of all there is an
increase in solubility as the activity coefficient of the protein
decreases,i.e. a "salting in" effect. As the ionic strength becomes
higher, the second effect causes a diminishing of solubility and this
predominates after the solubility passes through a maximum then
decreases until precipitation is complete. In "salting out" the salt
anions are important while the cations play a secondary role. Salts
with di- and trivalent anions are especially effective for precipitating
proteins, e.g. R ® ^ ^ Exactly why
proteins are salted out is not completely understood. It has been
suggested ^ that the salt ions on dissolving become hydrated, thus
"removing" part of the water which is then "unavailable" as solvent
and "dehydration" of the protein molecule is brought about. The
solvent becomes organised preferentially about the salt ions to an
extent that its normal organisation around the protein molecule is
decreased. This decrease in solvation enables the protein molecules
to associate in the solid phase as a precipitate.
70
A direct relationship has been formulated by Cohn between
protein solubility and the ionic strength of the solution. In a
concentrated solution of electrolyte the decrease in the logarithm
of the solubility of a protein is a linear function of increasing
ionic strength. In mathematical terms this has been represented by
the formula t
log S • p - k£
where S - Solubility of the protein in g/Kg water
2 ■ Ionic strength in moles/litre of solution P and K are constants
K represents the slope of the linear function while P is the hypothetical
solubility, i.e. log S at zero electrolyte concentration. Temperature,
pH, the amount and type of salt, the amount and type of protein are
the important variables.
(b) Effect of pH, temperature and salt
Amount and type of salt, pH and temperature can all be adequately
controlled. Much less is known about how proteins behave when a
mixture is presented for fractionation by the "salting out" technique.
temperature and the amount of a particular salt on the intrinsic
properties of that protein. Exactly where the protein will
precipitate at salting out depends not only on these factors hut on 6T the nature of the salt and the amount of that protein in solution •
The effect of these variables can be briefly delineated.
(i) jog
At constant ionic strength protein solubility increases at pH
values acid or alkaline to the isoelectric points of the proteins. 6*7
Dixon and Webb illustrate the importance of this variable. They
show how the relative solubilities of ovalbumin and carboxyhaemoglo-
bin may vary very rapidly with pH. At constant ionic strength a
change from pH 5 "to
6
is sufficient to alter the ratio of the solubilities of these two proteins by several thousand fold. Toretain well defined, reproducible precipitation limits pH must be
strictly controlled.
(ii) Temperature
In dilute aqueous solutions of electrolyte most proteins have a
positive temperature coefficient and are more soluble as the tempera
ture rises from 0°. In more concentrated electrolyte solutions this
condition in some instances is reversed. Some proteins are less
soluble and can be precipitated when the solution temperature is
raised from 0° without an increase in salt content. This sensitive 54 effect to temperature change is illustrated in the report by Edsall
who states that myosin which is soluble at 800 mg of protein nitrogen/
litre at 0° under a standard condition of pH and ionic strength has
no measurable solubility at room temperature. The effect is used 71
to crystallise human haemoglobin by Green who shows that carboxy-
haemoglobin is ten times more soluble in an ammonium sulphate
solution at 0° than it is at 25°. From this it can be realised that
protein solubility in the salt fractionating concentration range is
affected by changes in temperature. The effects are different for
the different proteins of a mixture and the order of precipitation of
a series of proteins as the salt content is increased may be entirely
different at one temperature from that of another temperature. By
implementing a standard temperature condition and strict buffer
control of the pH from one fractionation to the next, reproducibility
with respect to these two variables is adequately controlled.
(iii) Type of salt
r Cn Z n
The formula • « is used to calculate ionic strength. - is the molar ionic strength, C is the molar concentration of each
2
2
ion n and Z the valency. For each ion of salt in solution CZ can be
calculated and the sum of these values for each ion present divided by
2 to give g • Bivalent and trivalent salt ions produce much higher
ionic strengths and are generally more effective as precipitants. 67
Bixon and Webb show that some are better than others in the
efficiency of precipitation. These authors illustrate that the salts
are more effective in the order potassium phosphate sodium sulphate
ammonium sulphate ^ s o d i u m citrate > magnesium sulphate when
precipitating carboxyhaemoglobin. All the chlorides are much less
effective. They maintain that the salt anion and its valency state
are more important than cations in influencing salting out and that
cations, particularly di- and trivalent cations, are more effective
in the salting in process, except at very low ionic strengths.
Chlorides are listed as extremely inefficient. Though sodium sulphate
is more effective than ammonium sulphate it is less soluble and has a
limited application. Magnesium sulphate is not very effective as it
causes the protein to precipitate over wide ranges of salt addition.
Phosphates are particularly good protein precipitants and act as 72
buffers at the same time. Pennell who notes that sodium chloride,
magnesium sulphate and various acetates and chlorides are considered
as specific precipitants for individual proteins, points out that it
has not been demonstrated that their use is more specific than that