To: Professor pauling
From: R. A. Pasternak
Subject: The Average Number of Amino Acid Residues Contained in the Unit Cell of Collagen
I have collected experimental data for samples of untreated kangaroo tail tendon.
They are:
(1) unit cell dimensions
(2) density
(3) chemical composition
All the experiments were carried out with a batch of tendon equilibrated with a saturated
solution of K2CO3 (relative humidity about 45%).
(1) An X-ray photograph has been taken in the 10 cm. camera of an unstretched sample
of the material. Humidity equilibrium was maintained throughout the exposure. The
photograph was measured with a photometer.
Results:
(a) Meridional spacing: d = 2.88 Å.
(b) Equatorial spacing:
[Data Equations]
Since the 1. Order reflection is much sharper, double weight was given to it for the
average.
The volume of the unit cell, assuming that the equatorial reflection is the (1100)
of a hexagonal unit cell, is V = 2.88 x 11.542 x cos 30º = 443.3 A30
(2) The density of the material was determined by flotation. The density of the liquid
(bromo- and chlorobenzene) was adjusted approximately with some samples, and the final
adjustment was made with new samples; thus a possible change in hydration was reduced
to a minimum. In a second check, samples were evacuated for 15 minutes and the density
liquid was added before breaking the vacuum in order to get rid of the possible air
bubbles. No change in the density of the samples, however, was observed.
The density was found to be 1.32 gr. cm.-3. This is a lower limit, because air may be included in the samples.
From (1) and (2) one obtains as minimum weight of material in unit cells
M = V x d x N = 443.3 x 10-24 x 1.32 x 6.023 x 1023 = 352 gr.
(3) A part of the equilibrated material was sent to Dr. Elek for a microanalysis.
He carried out duplicate determinations of C, N, H, ash and water. The values reported
by him, and listed in column 1 of Table I, are referred to the equilibrium weight
of the sample. Elek reports the ash as sulfate. The composition of the ash was not
determined and for the calculations I assume that the amount of inorganic impurity
in the sample is one half of the ash. The same amount of water is, within the accuracy
of the analysis, lost by the sample at the three drying temperatures, 105˚, 125˚,
and 150˚. This is undoubtedly the total amount of water bound in my collagen sample.
This conclusion agrees with the observation of other investigators; samples used for
the determination of the amino acid composition were in general dried at 105° and
the amount of amino acid residues found added up to nearly 100% of the material. In
column 2 Elek’s results are recalculated for the dry, ash-free basis, i. e., 83.4
gr. = 100%.
In column 3 the values for C, N, H, calculated from the amino acid composition as
reported in the literature are given, in parentheses the observed nitrogen value cited
by Tristram is added; it is appreciably higher than the calculated one because the
latter does not include amide nitrogen. I took the data of Tristram (Adv. Prot. Chem.
15, 143 (1949)) which are based mainly on Bowes’ results because his collagen samples
are probably least denatured of all samples investigated.
Our value for nitrogen compares well with the observed nitrogen of Tristram; our carbon
value, however, is significantly higher than the calculated one. This high value may
be caused by a non-collagenous component in our sample. One cannot, however, exclude
the possibility that the calculated value does not represent the real composition;
oddly enough, I was not able to find in the literature an experimental determination
of the carbon content of collagen.
Average number of amino acid residues in the unit cell. I assumed in this calculation that all the organic material of the sample is collagen
of an average residue weight of 93.3 as given by Tristram.
In our sample, 16.3% water + cations are associated with 83.7% protein; 93.3 gr. protein
would hold 93.3 x 16.6 = 18.6 gr. H2O, 83.4
i. e., the apparent molecular weight = 93.3 + 18.6 == 111.9 gr. mol. Total weight
of the unit cell = 352
[Data Equation]
The uncertainties in this value are
(1) average residue weight
(2) observed density may be low; this would increase n
(3) the presence of non-collagenous material would decrease n
I plan a series of experiments with gelatin designed to minimize the sources of error
(2) and (3).
[Data Table]
