New age of water
Water has come of age. It is cool on everyones lips. Decades of
research on water is giving us remarkable insights into its dynamic collective
structure, and changing our big picture of life and living process.
Organisms are seventy to eighty percent water. Is this water necessary
to life? What vital functions does it serve?
Entire biochemistry and cell biology textbooks are still being written
without ever mentioning the role of water. It is simply treated as the inert
medium in which all the specific biochemical reactions are being played out.
Instead, recent findings are raising the possibility that it is water
thats stage-managing the biochemical drama of life. Water is life,
it is the key to every living activity. Some people will even say it is the
seat of consciousness.
ISIS brings you the latest revelations on water in this extended series
that starts from the basics. The articles will not be circulated consecutively,
so do watch out for them.
ISIS Report 30/06/04
Water Forms Massive Exclusion Zones
Water, the most abundant constituent of living organisms, is
associated with an enormous amount of surfaces inside cells and in the
extracellular matrix. Is all of this biological water different from water in
bulk? The answer is definitely yes, if the incredible new findings are to be
taken on board. Dr. Mae-Wan Ho
A fully illustrated version of this article with
sources is posted on ISIS Members website. Details here.
What is biological water?
"Biological" water includes practically all the water in living
organisms, inside the cell as well as in the extra-cellular matrix, except,
possibly, for large reservoirs or conduits such as the bladder, gut, stomach
and vacuoles inside some cells. Biological water is rarely far from the surface
of a membrane or a macromolecule such as proteins, nucleic acids and
polysaccharides like starch and glycogen.
Inside the cell, the concentration of proteins in cytoplasm is between
170 to 300 mg/ml, which suggests that 7 to 9 shells of water (hydration shells)
coat the available surfaces, corresponding to a distance of 4 to 5nm
(nanometre, 10-9m) between the
surfaces. A substantial fraction of the water is quite closely associated (at a
distance of less than 0.5nm) with the proteins, nucleic acids, polysaccharides
and assemblies of smaller molecules that make up an organism, and is essential
for their functioning.
The idea that cell water is distinct from bulk liquid water goes back a
long way to pioneers like Gilbert Ling and Albert Szent-Györgyi in the
1960s and 70s; to many physicists and chemists in the latter half of the
19th century fascinated by the distinctive properties of
protoplasm inside living cells.
Since the 1970s, many physical and physiological techniques have
demonstrated that cell water behaves very differently from bulk water. It is
dynamically ordered or oriented, and exhibit restricted motion compared to
water in the bulk.
More recently, ordered interfacial water have been found to be
associated with pure protein or DNA crystals obtained at cryogenic (very low
freezing) temperatures. These ordered water molecules do not form the typical
ice structure, but are involved in many different forms of hydrogen bonding
networks with the macromolecule and with each other.
A major uncertainty is what fraction of the water in living organisms
and cells is distinct from bulk water, and to what extent water is essential
for different living functions.
Using sophisticated techniques with big machines, such as NMR and more
recently, neutron diffraction, no more than one or two layers can be detected
to have altered properties, which would imply that a substantial part of the
water inside cells and in the extracellular matrix is still bulk water.
But other scientists, notably, Gilbert Ling, who emigrated to the United
States on a Boxer Fellowship from China, has been insisting since the 1960s
that practically all the water in the cell is in an altered state
different from bulk water (see SiS review).
Interfacial water as model of biological water
Water generally forms ordered layers over solid surfaces, and this
ordered interfacial water can tell us a great deal about water in
Interfacial water has different properties from bulk water; for example,
certain solutes that dissolve in bulk water are excluded from interfacial
water, or fail to dissolve in it.
Interfacial water is generally thought to be no more than one or at most
several layers of water molecules thick. But several reports published in the
1990s suggested that hydrophilic (water-loving) surfaces could extend their
influence over much larger distances from the interface.
Small experiments that tell a big tale
Gerald Pollack and Zheng Jian-ming in the Department of Bioengineering,
University of Washington, Seattle in the United States decided to do some
simple elegant experiments to find out exactly how far such hydrophilic
surfaces can extend their influence; and came up with some startling
They used as solutes, microspheres 0.5 to 2 mm in diameter, which can be seen with the ordinary light
microscope. For the hydrophilic surfaces, they employed several common
hydrogels known to interact strongly with water.
In the first experiment they put a small gel sample between two large
glass cover slips, and filled the space to either side with a suspension of the
microspheres, then sealed the chamber. The whole assembly was placed on the
stage of a microscope fitted with a camera to follow what happens.
In the second experiment, the gel was formed around a glass cylinder,
which was withdraw after the gel was formed, leaving a channel, l mm in
diameter, which is then filled with the suspension of microspheres and placed
under the microscope.
To their amazement, they found that the microspheres were excluded from
the gel surfaces in both experiments over distances of tens of
mm, and in extreme cases, up to 250mm or more. Such massive exclusion zones are totally
unexpected, and have never been reported before (see Fig. 1).
Microspheres were almost completely absent from the exclusion zone, and
the boundary between exclusion and non-exclusion rather sharp, of the order of
10% of the width of the exclusion zone. The zone forms rather quickly, and
appears 80% complete after 60s. Migration velocity was about 1.5mm per second, and microspheres near the boundary migrated
at the same speed as those far away from it. Once formed, the exclusion zones
remained stable for days.
Figure 1. Exclusion zone formed next to the surface of
polyacrylic acid gel.
Could this be an artefact? For example, could there be some invisible
threads sticking out from the gel surface to push the microspheres away? They
tested this by using the atomic force microscope and other sensitive probes to
detect such strands, but no protruding strands were detectable, not even after
they fixed and cross-linked the gel and washed it extensively, so no lose
strands could ever leak out.
Could it be that the gel was in fact shrinking away from the surface and
extruding water, and therefore squirting the microspheres away? But no such
shrinkage was detectable; the boundary did not shift appreciably as the
microspheres migrated away from it. Over a period of 120 minutes, the diameter
of the cylindrical hollow in the gel changed by less than 2mm. Thus, in the 2 min period during which the exclusion
zone was formed, shrinkage was insignificant.
Could it be that polymers were leaking out into the exclusion zone, and
pushing away the microspheres? They added a polymer to the microsphere
suspension, but this only narrowed the exclusion zone.
Yet another test was to continuously infuse microsphere suspension into
the cylindrical hollow in the gel under pressure at a speed of about 100mm/s,
so that any suspended invisible solutes ought to be washed out. But the
exclusion zones persisted, virtually unchanged even at the highest speeds.
The exclusion zones were not a quirk due to the particular gel used.
Polyvinyl alcohol gel, polyacrylamide gels, polyacrylic acid gels, and even a
bundle of rabbit muscle all gave similar results (Fig. 2); and microspheres of
different dimensions, coated with chemicals of opposite charge nevertheless
resulted in exclusion zones. Thus, exclusion zones are a general feature of
hydrophilic surfaces. One gel that did not show exclusion was when
polyacrylamide was copolymerised with a vinyl derivative of malachite green.
Figure 2. Exclusion zone next to surface of rabbit
Exclusion was most profound when the microspheres were most highly
charged, so negatively charged microspheres gave maximum exclusion at high pH,
whereas positively charged microspheres gave maximum exclusion at low pH. The
presence of salt tended to decrease the size of the exclusion zone somewhat.
The size of the exclusion zone also went up with the diameter of the
How could it be explained?
What could be the explanation for this strange phenomenon that has never
been observed; that apparently goes against all expectations based on data from
the latest big machines?
After ruling out several trivial explanations, Zheng and Pollack
considered whether it could be due to layers of water molecules growing in an
organized manner from the gel surface and extending outwards, pushing the
microspheres out at the same time. That would seem consistent with the
observation that the speed of migration of the microspheres is constant
regardless of distance from the boundary. It is also consistent with the
finding that larger microspheres give bigger exclusion zones.
The increase in exclusion zone with charge, too, is consistent with
their water-structuring hypothesis, as higher surface charge is known to be
associated with larger extent of water structuring. But, as they remark, "While
these several observations fit the water-structure mechanism, no reports we
know of confirm any more than several hundred layers of water structure at the
extreme, and not the 106 solvent
layers implied here."