Water Forms Massive Exclusion Zones

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press-release wrote:30 Jun 2004 18:32:59 -0000

 

Water Forms Massive Exclusion Zones

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ISIS Press Release 30/06/04

 

 

Water Forms Massive Exclusion Zones

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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 reports

 

A fully illustrated version of this article with sources is

posted on ISIS Members' website

http://www.i-sis.org.uk/full/WFMEZFull.php.

Details here

http://www.i-sis.org.uk/membership.php.

 

 

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 living organisms.

 

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

results.

 

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 muscle.

 

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 microsphere.

 

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. "

 

 

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