The State of Water in Biological Systems

doi:10.1016/S0074-7696(08)60530-6

International Review of Cytology

Volume 192, 1999, Pages 281-302

https://doi.org/10.1016/S0074-7696(08)60530-6 Get rights and content

This paper addresses the issue of how the aqueous cytoplasm is organized on a macroscopic scale. Mitochondria were used as the experimental model, and a unique experimental approach was used to probe the properties of water in the mitochondrial matrix. The results demonstrate aqueous phase separation into two distinct phases with different osmotic activity and different solute partition coefficients. The larger phase, designated “normal water,” is osmotically active and behaves in every respect like a bulk, dilute salt solution. The smaller phase, designated “abnormal water,” is osmotically inactive and comprises the water of hydration of matrix proteins. It is, nevertheless, solvent water, with highly selective partition coefficients, and behaves like a Lewis base.

Section snippets

Introduction*

This review deals with the time-averaged, equilibrium properties of water in cells. In this domain, one is dealing with familiar macroscopic properties such as osmotic activity and solute activity coefficients.

In the early 1970s, I put forward a novel hypothesis for the macroscopic state of water in biological systems: that biological water spontaneously separates into two (or more) phases with distinct solvent properties (Garlid, 1976, Garlid, 1978, Garlid, 1979). This hypothesis was both

Mitochondria—The Experimental Model

The mitochondrion is an excellent experimental model for studies of biological water. It is structurally simple and contains a single osmotically active compartment. Under controlled experimental conditions, this compartment retains its ionic contents during wide volume changes. Mitochondria behave as osmometers (Tedeschi and Harris, 1955, Bentzel and Solomon, 1967, Beavis et al., 1985, Garlid and Beavis, 1985), and they can undergo very large amplitude swelling without rupture, a consequence

Theory

Osmotic equilibrium is the state in which, in the absence of pressure gradients, water activity, a_w, is equal in the two phases. Thus, osmolality, ϕ, is equal in the two phases, and $g_{i} s_{i} = ϕ_{0}$

where s_i designates the ideal osmolality of the internal phase; s_i = Σv_j m_j where ν_j = the number of particles into which solute j dissociates. The variable ϕ₀ is the (measured) osmolality and g is the osmotic coefficient. (It must be emphasized that g is a property of the solution and not of the individual

Theory

When solute x is at equilibrium between two homogeneous phases, the following relationship holds: $m_{x 1} = f m_{x 0}$

where f is the thermodynamic partition coefficient, given by $f \equiv Y_{x 0} / Y_{x 1}$

where the Ys are molal activity coefficients. The internal concentration, m_x1, cannot be measured directly. Instead, we know internal solute content, X_i, and total matrix water content, W_i, which leads to an average concentration, [X_i]: ${[X]}_{i} \equiv X_{i} / W_{i}$

Note that [X]_i is a derived, rather than measured, quantity. Solute distribution

Experimental Evidence for Phase Heterogeneity in Mitochondria

Equation (14) provides a rationale for measuring distributions as a function of matrix volume, and it contains no assumptions about aqueous phase structure in the matrix. Equation (14) also provides a direct test of the single-phase theory. If one homogeneous phase exists, then the slopes, Q, of data plotted according to Eq. (14) must equal the slopes plotted according to Eq. (13) (Fig. 5).

The results plotted in Fig. 6 clearly demonstrate that this is not the case. The slopes of Fig. 6 differ

The Evidence for Aqueous Phase Separation in the Mitochondrial Matrix

The hypothesis that biological water separates into two distinct phases was introduced more than 20 years ago (Garlid, 1976, Garlid, 1978, Garlid, 1979) and has recently been revived (Walter and Brooks, 1995). Despite an extensive literature of studies on cell water, the experimental approach used to test the hypothesis remains unique. This approach is also powerful, because it provides decisive evidence. It was possible to exclude the alternative hypothesis that matrix water comprises a single

Concluding Remarks

Walter and Brooks (1995) have suggested that cellular proteins segregate into two or more phases, much like macromolecules in the test tube, as described by Albertsson, 1971, Albertsson, 1986. Similar forces may be involved in cells, but I do not believe that proteins segregate randomly, by a purely physicochemical process. Rather, I believe that proteins undergo “channeling” of their own, on their passage from the ribosomes to their target destinations. In this view, the overwhelming majority

Acknowledgments

The author wishes to acknowledge the expert technical assistance of Mr. Craig Semrad and to express special gratitude to James S. Clegg for his encouragement of these efforts in the early years. This work was supported in part by NIH grants GM31086 and GM55324 from the National Institute of General Medical Sciences.

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The State of Water in Biological Systems

Section snippets

Introduction*

Mitochondria—The Experimental Model

Theory

Theory

Experimental Evidence for Phase Heterogeneity in Mitochondria

The Evidence for Aqueous Phase Separation in the Mitochondrial Matrix

Concluding Remarks

Acknowledgments

J. Biol. Chem.

Cell Biol. Int.

Curr. Top. Cell. Regul.

J. Biol. Chem.

J. Biol. Chem.

Comp. Biochem. Physiol. A

Annu. Rev. Biochem.

J. Biochem. Biophys. Methods

Partition of Cell Particles and Macromolecules

Partition of Cell Particles and Macromolecules

Osmotic properties of mitochondria

J. Gen. Physiol.

Water, free and bound

Quantv. Biol.

Protein solvation in allosteric regulation: A water effect on hemoglobin

Science