The Interaction Of Serum Albumins With Calcium

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Vol. 47
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The Interaction of Serum Albumins with Calcium
BY N. H. MARTIN AND D. J. PERKINS (Smith Research
Fellow)
Department of Chemical Pathology, St
G(eorge'8
Ho8pital Medical School, London, S.W.
1
(Received
1 March 1950)
Pribram in 1871 postulated that some part of the
serum calcium was bound to the serum proteins.
Rona & Takahashi (1911) produced experimental
evidence justifying Pribram's suggestion. Numerous
authors (Loeb, 1926; Marrack & Thacker, 1926;
Greenberg & Greenberg, 1931; McLean & Hastings,
1935; Weir & Hastings, 1936; Drinker, Green &
Hastings, 1939; Ludewig, Chanutin
& Masket, 1942)
have published papers on qualitative and quan-
titative studies of the distribution of calcium in
human serum, and the extent to which it may com-
bine with human and other proteins.
McLean & Hastings (1935) gave tables of pK
values for calcium proteinate based on a hypo-
thetical mass law relationship
[Ca++] [Protein--]
K
[Ca protein]
and in the same paper developed a nomogram based
on this mass law to explain the relationships in
human serum. Weir & Hastings (1936) produced
further data to support this relationship, but revised
the pKvalues. Ludewig et al.
(1942) re-examined
the
relationship of calcium to protein in human serum,
using the ultracentrifuge to study the equilibrium.
They concluded that the system was too complex
for the simple mass law to be applied.
Recent developments in the technique of low-
temperature fractionation of proteins (Pillemer &
Hutchinson, 1945; Cohn, Strong, Hughes, Mulford,
Ashworth, Melin & Taylor, 1946; Kekwick &
MacKay, 1949) offered fresh opportunity for the
examination of the relationships of calcium to the
individual serum proteins. Of these, albumin
seemed the most satisfactorily characterized for
careful study. In the present investigationwe started
with the object of discovering whether the calcium-
binding capacities of albumins of different species
are the same, and whether albumins from the same
species, separated and purified by different tech-
niques, have the same calcium-binding capacity.
EXPERIMENTAL
The albumins examined had been prepared by the methods
of Cohn et al. (1946), Pillemer & Hutchinson (1945) and
Kekwick & MacKay (1949). The purity of the specimens was
judged by electrophoretic analysis carried out in the 11 cm.
analytical cell in phosphate buffer, ionic strength 02, at
pH 8-0, using the moving-boundary technique of Tiselius
(1939).
The Ca
distribution
was studied
by dialysis. These
dialyses were carried
out at 20 in a two-chambered system
separated by a semi-permeable membrane.
The time taken
to establish
equilibrium
was
measured
by controls
run in
parallel. Though equilibrium in the majority of
cases was
established
in 72 hr. all specimens were left for 6 days
before
analysis of the
ionic distribution between the two chambers
was carried out. During this period the samples were stirred
frequently
to
help
to
establish equilibrium. The ratio of the
volume of fluid in the protein-free compartment
to that in
the
protein-containing compartment
was
15: 1.
Determinations of pH
were carried out on all
samples at
the completion of each experiment, using
a
glass electrode
and the Cambridge
potentiometer.
Ca determinations
on
the fluid in each compartment
were
made by
a
modification of the method of Trevan & Bain-
bridge (1926). Fig.
1 shows the
percentage deviation of Ca
estimated by
our
method, from Ca
concentrations estimated
gravimetrically
on
large samples.
Chloride determinations
were carried out on the fluid
of
both compartments
by
the method of Van
Slyke (1923).
The protein concentrations in the inner compartment
were
determined
at
the
completion
of each
equilibrium by N
determination using the standard micro-Kjeldahl technique.
In each
instance the fluid in the outer
compartment
was
examined,
and any
experiment
in which transference of N-
containing materials could be demonstrated
was
rejected.
The albumins had been
dialysed against 015m-NaCl prior
21-2

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