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Volume 77, 1948-49
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The Adsorption of Water Vapour on Casein

[Read before the Auckland Institute, May 14, 1947; received by the Editor, June 23, 1947; issued separately, February, 1949.]


The isotherms for the adsorption of water vapour on several preparations of casein are measured by means of the isopiestic method of Robinson and Sinclair. The multimolecular adsorption theory of Brunauer, Emmett and Teller is used to analyse the curves, and an attempt is made to show how the derived constants may be related to the amino-acid composition of the casein.


Careful measurements of the adsorption of water vapour on proteins are essential to an evaluation of the hypothesis put forward by Pauling (1945) concerning the relation of water adsorption to molecular structure. A number of proteins studied by Bull (1944) provided the original experimental basis for this hypothesis. It has since been shown (Green, 1948) that adsorption isotherms are rapidly and accurately determined by means of the isopiestic method of Robinson and Sinclair (1934). The present paper describes the application of the method to the study of casein, a protein not included in Bull's investigation.


Casein was purified by the method of Cohn (1930) and dried by washing first with alcohol and then with ether. It was then placed in a vacuum desiccator over a saturated solution of magnesium nitrate, which produces an atmosphere of approximately 51 per cent, relative humidity. When the ether had been completely removed, the casein was spread out in the air of the laboratory overnight and a number of samples were weighed out.

The moisture content of these samples was measured in several ways—by dehydration over concentrated sulphuric acid in vacuo in a rocking desiccator at room temperature, by heating at 100° in vacuo over phosphorus pentoxide, and by heating at 103° in an air oven. The rates of loss of water are shown in Table I.

Table I—Loss in Weight of Casein on Drying.
Room Temperature 100°in Vacuo 103° in Air Oven
Time Loss Time Loss Time Loss
1 day 10.63% 1 day 11.57% 3 hours 11.68%
3 day 10.82 2 days 11.73 16 " 12.09
5 day 10.98 3 " 11.67 1 day 12.31
9 " 11.13 4 " 11.67 2 days 12.56
11 " 11.08 5 " 11.73 3 " 12.77

These figures confirm for the globular protein, casein, what had already been observed in the case of the fibrous proteins, collagen and elastin, namely, that the measured water content is dependent on the temperature of drying. The continuous slow loss in the third experiment suggests that radical decomposition of the protein is going on and demonstrates the unsuitability of oven-drying for determining water content.

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The ash content of the dry casein was found to be 3·31 per cent. Adsorption isotherms at 25° were measured by the isopiestic method for casein prepared in the following ways:


Partially dehydrated over 70 per cent, sulphuric acid.


Completely dehydrated at room temperature.


Dried at 100° in vacuo over phosphorus pentoxide.


Dried in air oven for 1 hour at 103°.

Water activities (aw) were measured by a control solution of sulphuric acid using the data of Shankman and Gordon (1939). The results are shown in Table II as gm. water per 100 gm. dry casein. “Dry casein” refers to room temperature drying for A and B and to high temperature vacuum and air drying for C and D respectively.

Table II—Adsorption of Water Vapour on Casein at 25°
aw Sample A Conditioned over 70% H2SO4 Gm. H2O per 100 gm. Sample B Dried over conc. H2SO4 Dry Casein Sample C Dried in vacuo at 100° Sample D Dried in air oven at 103°
0.0450 2.55 2.00 1.83 1.47
0.0805 3.27 2.78 2.53 2.12
0.1216 4.07 3.73 3.43 2.92
0.1675 4.73 4.45 4.07 3.64
0.1766 4.88 4.65 4.26 3.75
0.2021 5.36 5.07 4.70 4.21
0.2483 5.95 5.67 5.23 4.77
0.2839 6.43 6.19 5.85 5.39
0.3196 6.96 6.77 6.47 6.00
0.3924 8.03 7.87 7.67 7.22
0.4812 9.51 9.41 9.30 9.05
0.5525 10.52 10.40 10.32 10.17
0.5984 11.48 11.37 11.30 11.25
0.6710 12.93 12.93 12.93 12.90
0.7453 14.61 14.69 14.55 14.60
0.7957 16.44 16.11 16.09 16.15
0.8779 20.12 20.05 19.86 20.02
0.9560 29.91 30.25 29.47 29.51

It is seen that for values of aw below 0·40 the course of the iso therm is strongly influenced by the method of drying the adsorbent, but that at higher water activities the isotherms for the several casein preparations coincide.


The equations of Brunauer, Emmett and Teller (1938) for multimolecular adsorption were applied to the isotherms in Table II, using the range between aw = 0·05 and aw = 0·40 to calculate the constants vm and C by the method of least squares. vm is the weight of adsorbate required to fill the first molecular layer of 100 gm. of adsorbent and C is related to the heat of adsorption of the first layer. The total number of molecular layers of adsorbate (n) was calculated in the higher-humidity region. Table III shows the results obtained, and Fig. 1 shows the experimental isotherm for sample B together with theoretical B.E.T. curves calculated for n = 6 and n = 7.

Table III—B.E.T. Constants for Casein.
Sample A Conditioned over 70% H2So4 Sample B Dried over conc. H2SO4 Sample C Dried in vacuo at 100° Sample D Dried in air at 103°
vm 5.44 5.53 5.49 5.43
C 13.7 10.3 8.3 6.3
n 6–7 6–7 6–7 6–7
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Fig. 1—Adsorption of water vapour on casein dried over concentrated sulphuric acid. ⊙ Experimental points. The curves correspond to values calculated on the Brunauer, Emmett and Teller theory, assuming six, seven and an infinite number of layers.

The value 6–7 for n appears to be a general one for this type of adsorption, as has previously been shown for collagen and elastin. The decrease in C with increasing severity of drying is also in agreement with the results for the fibrous proteins.

We may seek an interpretation of the value of vm in the aminoacid composition of the protein.

Casein resembles other proteins in possessing a number of polar side-chains derived from the amino-acid residues. The principal aminoacids which can give rise to polar groups in the side-chain are serine,

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threonine, tyrosine, tryptophan, cysteine, hydroxyproline, aspartic acid, glutamic acid, hydroxyglutamic acid, histidine, arginine and lysine. In addition, the protein contains some ammonia which is believed to be present as the amide of the carboxylic acid groups in the side-chains.

As a basis for calculation, we may assume that every polar sidechain adsorbs one water molecule in the first layer. When this, hypothesis is tested on a number of proteins it is often found to give results much higher than the observed value of vm. This suggests that the carboxylic amide groups may be too weakly polar to adsorb water, and a recalculation in which the number of ammonia equivalents is subtracted from the number of polar groups yields much better agreement. Another variant in the method of deriving vm was introduced by Pauling. He considers that carbonyl groups of the backbone contribute to vm only when there are tertiary nitrogen atoms from proline and hydroxyproline in the vicinity. According to Pauling, since tertiary nitrogen cannot saturate a carbonyl group by forming a hydrogen bond, the latter groups are left free to co-ordinate water. He therefore counts proline residues once and hydroxyproline residues twice when reckoning the number of adsorbent points in the molecule. Table IV shows values of vm calculated by these methods for five proteins. The values of vm for insulin and plasma albumin were measured by Robinson (1947). The analytical figures used in calculating the number of polar groups are taken from McLaughlin and Theis (1945) for collagen and elastin, except that the ammonia figures are taken from Jordan Lloyd and Shore (1938). The composition of casein and insulin is given by Cohn and Edsall (1943) and of plasma albumin by Brand, Kassell and Saidel (1944).

Table IV—Calculation of vm from Protein Composition.
Protein Observed vm Calculated vm
I * II * III * IV *
Casein 6.47 8.21 6.52 5.37 6.55
Insulin 5.81 7.62 5.87 5.87 5.87
Plasma albumin 6.38 7.85 6.73 6.73 6.73
Collagen 10.1 5.65 5.23 5.23 9.95
Elastin 5.4 0.53 -ve -ve 2.45

It will be seen from the table that the calculated value of vm for casein is too large even when the correction for amide groups is applied. However, if we take the view of Nicolet and Shinn (1941) that casein contains little, if any, hydroxyglutamic acid and that serine and threonine are the only aliphatic hydroxyamino acids present in any quantity, the new calculated value, showing in column III. is almost identical with the experimental figure. Inclusion of proline as in Pauling's method again gives too high a result.

Insulin and plasma albumin, containing no proline, give good agreement.

The collagen figures appear to support Pauling's view, but the calculated value of vm for elastin is far too low by all methods. This led the present author (1948) to suggest that both collagen and elastin adsorb on the polypeptide backbone to an extent which is not

[Footnote] * Calculated from total polar side-chains.

[Footnote] * Calculated from total polar side-chains minus amide groups.

[Footnote] * As for II, but omitting hydroxyglutamic acid.

[Footnote] * As for III, but with the addition of proline and hydroxyproline.

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necessarily determined by, and may exceed, the content of proline rings in the molecule. The fact that proline seems to make no contribution to vm for casein would appear to support this view.

We may summarise by saying that vm can be accurately calculated for three globular proteins by postulating the adsorption of one water molecule per polar side-chain excluding acid amides. In the particular instance of casein, better agreement is obtained if the reported hydroxyglutamic acid is ignored. Calculated values of vm for collagen and elastin are much too low and we are led to the conclusion that adsorption in these two fibrous proteins may occur on the polypeptide backbone.

It was suggested in an earlier paper that changes in vm with drying might be due to blocking of polypeptide groups by cross-links. If this explanation were valid, it would be expected that vm for casein, which appears to be independent of backbone adsorption, would be unaltered by the method of drying. Table III shows that vm is indeed virtually constant over the four isotherms measured.


The isopiestic method has been used to measure adsorption isotherms for water vapour on casein.

The adsorptive power of the casein is diminished by complete dehydration at room temperature and still more by drying at 100° in vacuo or in the air oven.

Analysis of the isotherms by the method of Brunauer, Emmett and Teller shows that these changes in adsorptive power are associated with a smaller energy of adsorption rather than with a change in the area available. Several methods of relating vm to the amino-acid composition of the protein are compared.

Different procedures for drying casein have been investigated and it has been found that raising the temperature causes a greater final loss of water from the protein. Drying in the air oven is not suitable for measuring the water content of this material.


The author wishes to express his indebtedness to Dr. R. A. Robinson for his helpful interest in the work and to Mr. B. Cleverley for preparing the casein. He also wishes to thank the Chemical Society for a grant from their Research Fund.


Brand, E., Kassell, B., and Saidel, L. J., 1944. J. Clinical Investigations, 23, 437.

Brunauer, S., Emmett, P. H., and Teller, E., 1938. J. Amer. Chem. Soc., 60, 309.

Bull, H. B., 1944. Ibid., 66, 1499.

Cohn, E. J., 1930. Organic Syntheses, 10, 16.

Cohn, E. J., and Edsall, J. T., 1943. Proteins, Aminoaoids and Peptides, Reinhold Publishing Corp., New York, p. 368.

Green, R. W., 1948. Trans. Roy. Soc. N.Z., 77, 24.

Jordan Lloyd, E., and Shore, A., 1938. Chemistry of the Proteins, J. & A. Churchill Ltd., London, p. 136.

McLaughlin, G. D., and Theis, E. R., 1945. The Chemistry of Leather Manufacture, Reinhold Publishing Corp., New York, p. 32.

Nicolet, B. H., and Shinn, L. A., 1941. J. Biol. Chem., 139, 687.

Pauling, L., 1945. J. Amer. Chem. Soc., 67, 555.

Robinson, R. A., 1947. Private Communication.

Robinson, R. A., and Sinclair, D. A., 1934. J. Amer. Chem. Soc., 56, 1830.

Shankman, S., and Gordon, A. R., 1939. Ibid., 61, 2370.