Colloid & Polymer Science Colloid & Polymer Sci 263:396-398 (1985)

COLLOID SCIENCE

Non-freezing water in protein solutions

S. All and E A. Bettelheim

Department of Chemistry, Adelphi University, Garden City, New York, USA

Abstract: Differential scanning calorimetric studies of protein solution provided the freezable water content as a function of concentration. Total water content was obtained by vacuum dehydration. The difference between the two gave the non-freezing water content in protein solutions. The values obtained by these techniques were 2-3 times lar- ger than those obtained on the basis of NMR measurements on the same solutions.

Key words: Bound water, differential scanning calorimetry, free water, proteins.

The properties of supercooled water are currently generating great interest [1]. Derbyshire [2], upon ex- amining a number of polymeric aqueous solutions, has claimed that, to a first-order approximation, the amount of "non-freezing water is independent of the particular systems examined, it is usually in the range of 0.4 to 1.0 g of water/g of macromolecule." Simple systems such as agarose [3] and iota carrageenan [4] show concentration independent non-freezing water contents of 0.59 and 0.43 g water per g polymer, re- spectively. Mostly based on NMR studies, others, notably Kuntz [5], have assigned hydration (non- freezing water) values to amino acid residues and been able to predict reasonably well the hydration of pro- teins on the basis of their amino acid compositions.

Based on our own studies on cataract formation in lenses, we proposed a syneretic mechanism [6] by which water of hydration is released upon the collapse of the protein network; this released water enters the bulk (free water) phase. Such syneresis enhances the refractive index fluctuations and thus contributes to the development of opacities. We have shown that syneresis is involved in aging [7] and in nuclear cata- ract [8] formation in human lenses (as shown by light scattering analysis). NMR studies of the lenses [9,10] supported our conclusion. Finally, direct measure- ment of freezing and non-freezing water contents [11] in normal and cataractous lenses proved the loss of bound water and the increase in free water content upon cataractogenesis. However, the numerical values of the non-freezing water content measured by NMR

EM909

were lower than those obtained by thermal analysis. Therefore, it was desirable to investigate if such discre- pancies between NMR and thermal analysis also exist in simple systems such as protein solutions.

Purified casein was acquired from Fisher Scientific Company (Lot 755727). Egg white lysozyme (EC.3.2.1.17), three time crystallized, and ovalbumin (Grade III crystallized and lyophilized) were obtained from Sigma Company. Bovine serum albumin, Frac- tion V, was purchased from Baker Chemical Com- pany. A mixture of lens crystallins (ce and 3) was obtained from isoelectric precipitation (pH 4.5) of water soluble crystallins from calf cortex homoge- nates. Extensive dialysis against distilled water yielded a residue that contained a mixture of a and 3 crystallins in 62:38 weight ratio [12].

Homogeneous aqueous protein solutions were obtained with varying concentrations between 1- 20 %; samples ranging from 6 to 12 mg of protein solu- tion were hermetically sealed into pre-weighed alumi- num pans. The heat of fusion of each sample was obtained m a differential scanning calorimeter (DuPont 990). First, the sample and the reference pan (empty aluminum pan) was cooled to - 3 0 ~ by external dry ice acetone bath. The heating rate was 3 ~ During the heating, a flow of nitrogen (50 cm3/min) was provided. The instrument was calibrat- ed with a sapphire disk. The differential scanning calo- rimeter records endotherms of differential heat flow (Aq) as a function of time. The area under the endo- therm provides the heat of fusion. All of the protein

All and Bettelheim, Non-freezing water in protein solutions 397

b-

I=

24

2o

16

12

0 f | t 0 4 8 12 16

% PKOTEnl

Fig. 1. Non-freezing water content of protein solutions (in terms of percent of total water) as a function of protein concentration. �9 �9 crystallins; [] [] lysozyme; O O casein; �9 �9 serum albumin; �9 �9 ovalbumin

solutions had only small melting point depression (AT -0.3 ~ hence the heat of fusion of water could be used to convert the experimental heat of fusion to freezing water content.

Total water content of the protein solutions was obtained by vacuum dehydration. The samples, in weighing bottles, were placed in a vacuum desiccator over P205 and the desiccator placed in an oven at 60 ~ and evacuated (i x 10 -3 torr). This procedure was repeated until constant weight was established. The amount of non-freezing water was obtained by the dif- ference between total and freezing water content.

Figure 1 shows the variation of non-freezing water for the five protein solutions as a function of concen- tration.

Using only the approximate straight line portion of the curves at low concentrations (1-5 %), one finds the

following values for non-freezing water expressed in g water/g protein: 1.14 for crystallins; 1.14 for lysozyme; 1.08 for casein; 0.87 for serum albumin and 0.72 for ovalbumin. These values are two or three times larger than those obtained or calculated by Kuntz (5) on the basis of NMR measurements.

Differences in non-freezing water content meas- ured by DSC and NMR were found also in lens tis- sues. For example, 24 % of the total water was found to be non-freezing water in normal human lenses by NMR measurements [9,10], while DSC and total wa- ter measurements gave a value of 60 0/0 [11]. Therefore, the discrepancy between DSC measurements and NMR data is not due to the complexity of the lenses under investigation, but it is a systematic discrepancy.

Some people may interpret the non-freezing water content in terms of kinetics, i. e., at low temperature the solution becomes freeze concentrated and due to the viscosity, supersaturation may occur which will allow crystallization only over infinitely long periods. In this concept, the system is metastable and thus the non-freezing water content cannot be identified with a special kind of water (bound water or water of hydra- tion). Without entering into a theoretical discussion on the nature of non-freezing water content, we side with the majoritiy of the papers in the literature which regard non-freezing water as bound water [1- 3,9,10,13].

The purpose of the present paper is to show that dif- ferent techniques under the same boundary conditions (lowest freezing temperature) give different non-freez- ing water content. This cannot be due to the different interpretations of the nature of non-freezing water content. The phenomena under investigation in NMR studies involve different molecular motions from those associated with absorption of heat. It seems that while the non-freezing (bound) water molecules are less likely to have translational motions than "free" (freezing) water, they have greater potential for re- orientation due to the protein surface in their vicinity.

Acknowledgements

This work has been supported by a grant from the National Eye Institute, EY 02571.

References

1. Franks F (ed) (1982) Water: A comprehensive treatise Vol 7. Water and aqueous solutions at subzero temperatures, Plenum Press, New York

2. Derbyshire W (ed) (1982) Franks F, The dynamics of water in heterogeneous systems with emphasis on subzero tempera- tures, Water, Vol 7, Plenum Press, New York

398 Colloid and Polymer Science, VoL 263 �9 No. 5 (1985)

3. Derbyshire W, Duff ID (1974) Disc Faradays Soc 57:243-251 4. Baghdadi SMA (1977) PhD thesis, University of Nottingham 5. Kuntz ID Jr (1971)J Amer Chem Soc 93:214-218 6. Bettelheim FA (1979) Exp Eye Res 28:189-197 7. Siew EL, Opalecky D, Bettelheim FA (1981) Exp Eye Res

33:603-614 8. Bettelheim FA, Siew EL, Chylack LTJr (1981) Invest Ophthal-

mol 29:348-354 9. Racz P, Tompa K, Pocsik I (1979) Ezp Eye Res 28:129-135

10. Racz P, Tompa K, Pocsik I, Banki P (1983) Lens Res 1:199-206 11. Bettelheim FA, Christian S, Lee LK (1983) Current Eye Res

2:803-808 12. Bettelheim FA, Wang TJY (1977) Exp Eye Res 25:613-620

13. Ohno H, Shibayama M, Tsuchida E (1983) Makromol Chem 184:1017-1024

Received July 16, 1984; accepted January 9, 1985

Authors' address:

E A. Bettelheim, S. All Department of Chemistry Adelphi University Garden City, New York 11530, USA