Table Of ContentCELL-ASSOCIATED W A T ER
edited by
W. DROST-HANSEN
JAMES S. CLEGG
University of Miami
Coral Gables, Florida
Proceedings of a Workshop on Cell-Associated Water Held in
Boston, Massachusetts, September, 1976
ACADEMIC PRESS New York San Francisco London 1979
A Subsidiary of Harcourt Brace Jovanovich, Publishers
Academic Press Rapid Manuscript Reproduction
Copyright © 1979, by Academic Press, Inc.
ALL RIGHTS RESERVED.
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United Kingdom Edition published by
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Library of Congress Cataloging in Publication Data
Cell-associated water.
"A workshop... organized in connection with the First
International Congress on Cell Biology, Boston, September 1976."
1. Cell physiology-Congresses. 2. Water in the body-
Congressess. 3. Water-Physiological effect-Congresses. I. Drost-Hansen,
W. II. Clegg, James S., Date III. International Congress on Cell Biology
1st, Boston, 1976.
[DNLM: 1. Histocytochemistry-Congresses. 2. Body water-Congresses.
3. Water-Congresses. QH613 W926c]
QH631.C453 574.8'7 79-241
ISBN 0-12-222250-4
PRINTED IN THE UNITED STATES OF AMERICA
79 80 81 82 9 8 7 6 5 4 3 2 1
CONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors' contributions begin.
Paula T. Beall (271). Department of Pediatrics, Baylor College of Medicine,
Houston, Texas
P. Belouschek (3), University of Wurzburg, Federal Republic of Germany
James S. Clegg (363), Laboratory for Quantitative Biology, University of Miami,
Coral Gables, Florida
Frank M. Etzler (125), Laboratory for Water Research, Department of Chemistry,
University of Miami, Coral Gables, Florida
Joel L. Fisher (415, 431), Science Advisory Board, U.S. Environmental Protection
Agency, Washington, DC
Keith T. Garlid (293), Department of Pharmacology, Medical College of Ohio,
Toledo, Ohio
Marcel P. Gingold (53), Departement de Biologie, Service de Biophysique,
Cen-Saclay, Gif-Sur-Yvette, France
W. Drost-Hansen (115, 125), Laboratory for Water Research, Department of
Chemistry, University of Miami, Coral Gables, Florida
Carlton F. Hazlewood (165), Baylor College of Medicine, Department of
Pediatrics and Physiology, Houston, Texas
Ricardo Μ. Hurtado (115), Laboratory for Water Research, Department of
Chemistry, University of Miami, Coral Gables, Florida
Gilbert N. Ling (261), Department of Molecular Biology, Pennsylvania Hospital,
Philadelphia, Pennsylvania
Jean E. Morel (53), Departemente de Biologie, Service de Biophysiqu Cen-Saclay,
Gif-Sur-Yvette, France
G. Peschel (3), Department of Physical Chemistry, University of Essen, Federal
Republic of Germany
Albert Szent-Gyorgyi (1), Laboratory of the Institute for Muscle Research, Marine
Biological Laboratory, Woods Hole, Massachusetts
Philippa M. Wiggins (69), Department of Medicine, University of Auckland School
of Medicine, Auckland, New Zealand
PREFACE
This book was inspired by a Workshop, devoted to ''Cell-Associated Water,"
organized in connection with the First International Congress on Cell Biology (Bos
ton, September 1976). Many of the papers in this volume were read at that Work
shop; in addition, a few papers have been solicited from authors unable to attend the
meeting.
The state of cellular water continues to be the subject of considerable activity and
controversy. For this reason, as Editors, we have stressed the desirability of each
author ''setting the stage" for his discussion by the presentation of appropriate
review material, but apart from that we have urged each author to discuss freely his
views and opinions, however controversial. As a result, the views expressed in this
volume do not necessarily form a conceptually integrated overview of cell-
associated water but rather offer various new insights and, in some cases, novel
approaches to the topic. By the same token the Editors disclaim responsibility for
the personal opinions expressed by the individual authors. On the other hand, we
believe with Dr. Ralph Home that we "would like to see scientific texts written
as if the authors cared about the subject matter, as if they thought it important
enough to venture an opinion. Opinions as well as facts belong in scientific papers,
monographs, and texts." To this we may well add T. H. Huxley's observation, "It
is a popular delusion that the scientific enquirer is under an obligation not to go
beyond generalization of observed facts—but anyone who is practically acquainted
with scientific work is aware that those who refuse to go beyond the facts, rarely get
as far."
The material in this volume is primarily ordered according to the complexity of
the systems examined, starting with aspects of pure colloid and surface science and
ending with topics in zoogeography.
It is our hope that this volume will reach those scientists who are concerned with
cell biology in general and those in particular who are concerned with the events in
and the role of the aqueous phase of cells. It is also our hope that graduate students
Cell-Associated Water
WELCOMING ADDRESS
Albert Szent-Gyorgyi
Laboratory of the Institute
for Muscle Research
Marine Biological Laboratory
Woods Hole, Massachusetts
The biologists of the XIX century were unable to
approach the problem of the living state, being prevented
to do so by their cellular prejudice. They were convinced
that complex chemical processes could be performed only by
complete cells. It was E. Büchner who destroyed this pre
judice by showing that fermentation could take place in an
assembly of dissolved molecules. Hence, present-day biology
is a molecular biology. We have traded in our cellular pre
judice for a molecular prejudice. Our body being built of
molecules, we believe that all its reactions have to be
molecular.
There can be no doubt about the wonderful achievements
of the molecular approach. However, the molecular is but one
of the three dimensions involved in the mechanisms of life:
the molecular, the infra- and the supra-molecular. Life can
be understood only by their combination.
While, in theory, there is no dividing line between
these dimensions, there is a difference in the tools and
methods by which they can be studied. While in the mole
cular dimension the main tools are chemical, in the infra-
and supra-molecular dimensions the main tools are physical,
like NMR or ESR. In the everyday life of the scientist
there is also another difference between these dimensions.
The molecular outlook being the oldest, the physical approach
is not sufficiently represented on committees of granting
agencies and editorial boards, which favor the molecular
approach at the expense of the physical, a difference which
has to be corrected urgently.
Copyright © 1979 by Academic Press, Inc.
. All rights of reproduction in any form reserved.
' ISBN 0-12-222250-4
REVIEWS OF CLINICAL INFECTIOUS DISEASES, 1982
3. Maisch ΡΛ, Calderone RA. Role of Surface Mannan in the
Adherence of Candida albicans to Fibrin-Pi atelet Clots
Formed in vitro. Infect Immun 32:92, 1981.
The results indicate that cell surface mannan may play an
important role in the adherence of Candida to the fibrin-
platelet matrices which form in vivo in the endocardium of
heart valves. This mannan can be shown to be an important
component of a thick floccular material which is adherent
to the external surface of C. albicans cell walls.
4. Sobel JD, Myers PC, Kaye D, Levinson ME. Adherence of
Candida albicans to Human Vaginal and Buccal Epithelial
Cells. J Infect Dis 143:76, 1981.
This is an in vitro study of adherence. Factors that enhanced
germination and viability of the organism enhanced adherence.
There were significant differences between adherence with
cells of different volunteers, and adherence to buccal cells
was slightly greater than to vaginal cells. Pre-lncubation
of the cells with certain enzymes or sugars inhibited
adherence, as did precoating of the epithelial cells with
lactobacilli. They believe that adherence of C. albicans
is enhanced by a surface component of germinated yeast cells,
probably a surface protein, that binds to the epithelial
receptor, possibly to glycoprotein.
5. Sobel JD, Schneider J, Kaye D, Levinson ME. Adherence of
Bacteria to Vaginal Epithelial Cells at Various Times in
the Menstrual Cycle. Infect Immun 32:194, 1981.
They studied ten healthy sexually active medical students for
adherence of E. coli, lactobacilli, group Β streptococci,
Gardnerella vaginalis and N. gonorrhoeae to isolated vaginal
epithelial cells at various times during the menstrual cycle.
There were no significant differences in adherence for any
of the organisms at various times.
6. Lemberg Η, Jodal U, Svanborg-Eden C, et al. PI Blood Group
and Urinary Tract Infection. Lancet 1:551 (Letter), 1981.
The severity of a urinary tract infection correlates with
the ability of the infecting E. coli to adhere to human
uroepithelial cells and greater adherence has been recorded
for patients especially prone to UTI's. The authors have
suggested that glycolipids act as receptors for the organisms
on cells, and that the carbohydrate sequence gal-alpha 1-4
gal is recognized by the bacteria. Many individuals carry
a PI antigen on their red blood cells which contains the
specific sugar sequence mentioned. The authors considered
the question that such patients might have a similar
glycolipid composition in other cells such as epithelial
Cell-Associated Water
THE PROBLEM OF WATER STRUCTURE
IN BIOLOGICAL SYSTEMS^
G. Peschel
Department of Physical Chemistry
University of Essen
Federal Republic of Germany
Ρ. Belouschek
University of Wurzburg
Federal Republic of Germany
I. INTRODUCTION
The structure of water has aroused the interest of many
investigators since the turn of the century, but its highly
complex nature has prevented a good understanding of its
physical properties (61,49). The problem becomes even more
difficult if water is structurally modified by ions or by
proximity to a solid surface. Indeed, the effects of inter
faces on water structure is attracting considerable atten
tion as the results of such studies (on both pure water and
aqueous solutions) must be applicable to and necessary for an
understanding of the state of water in cellular systems. As
long as no theoretical model exists which rigorously reflects
all bulk properties of water, it appears an overwhelming task
to describe water structurally as influenced by the proximity
to a solid interface in terms of concrete structural para
meters. Efforts made in this direction were summarized ex
cellently by Drost-Hansen (45).
^We express our gratitude to the Deutsche Forschungsge
meinschaft and the Fonds der Chemischen Industrie for
financial support.
Copyright © 1979 by Academic Press, Inc.
J All rights of reproduction in any form reserved.
ISBN 0-12-222250-4
4 G. Peschel and P. Belouschek
It is commonly believed that the structure of water
near interfaces is different from the bulk, this change
originating from the first molecular adsorption layer which,
when being attached to an underlying hydrophilic surface,
is regarded to be strongly subject to restricted molecular
motions. This effect is envisaged to be transmitted over
many molecular diameters via hydrogen bonds deep into the
bulk phase.
It is still unclear whether there is an abrupt break
down of molecular surface orientation far from the inter
face, though some findings support this view (32,29)· More
over, there is a significant amount of evidence that the
degree of structural change in vicinal water depends sensi
tively on whether the interface is more or less hydrophilic
or even hydrophobic. Drost-Hansen (44) has adapted a fre
quently cited model for explaining the "structuring" of
aqueous surface zones. In his opinion, the adjacent solid
wall induces the formation of some sort of clusters,
clathrate cage-like or high-pressure ice polymorphs, all
being in equilibrium with monomeric water molecules. Exten
sive clathrate-like ordering was ascribed to the presence of
hydrophobic surfaces. This means that the surface proper
ties of water are also strongly directed into the third
dimension and, therefore, display "capillary-II effects" as
introduced by Shcherbakov (105). Former work in the field
of anomalous behavior of vicinal water was summarized by
Henniker (56).
Although it is well-known that water plays a major role
in a vast number of biological processes in living tissue,
this problem has until recently received little attention.
It is quite clear that cells and tissues are very rich in
interfaces and these must exert a marked influence on adja
cent water structure. The problem of elucidating water
structure in living tissue has chiefly been studied by NMR
methods which permit non-destructive experiments.
Important early work in this field was carried out by
Hazlewood, et al. (53) (see also chapter in this volume).
By aid of spin-echo decay measurements, they investigated
water in skeletal muscles from rats and mice and found that
the intracellular water experiences restricted molecular
motions. More recent work by Hazlewood, et αΙ· (54) re
vealed that water in cells might be composed of three dif
ferent fractions of non- (or slowly) exchanging water. The
authors believe that the structure of all or a very large
part of the cellular water is affected by the presence of
Problem of Water Structure *
the large number of interfaces encountered within cells.
This point, to be sure, is still a subject of active contro
versy.
Similar studies by Chapman and McLauchlan (14) lead to
the observation that two types of proton environments exist
in biological tissue. These findings are more or less con
firmed by a number of other authors using different tissue
specimens (10,18,71,46,20,9,26,79).
The NMR behavior of Na"^ ions in muscle, brain and
kidney tissue has been examined by Cope (19). The results
indicated that 60-70% of the Na"*" had a response time analo
gous to that of Na"*" complexed in an ion exchange resin.
Thus, Na"^ might be considered to be complexed or associated
with charged sites on macromolecules. The rest of the Na"*"
ions appear to be found within the structured vicinal water.
Further studies on Na"*" were carried out by Czeisler and
Swift (23) and Magnuson and Magnuson (72). Corresponding
tests with K"*" were made in bacteria (21) . It should be men
tioned that Cope treated his results in terms of semi-
conduction phenomena and solid state physics. The particular
importance of the NMR investigations of Damadian (24) lies
in his attempts to devise a method by which malignant tumors
in tissue can be detected via structured water NMR signals.
A number of studies on the structure of intracellular
water is based on freezing point changes which, according to
the nature of the different intracellular interfaces, are
lowered to variable extents. This suggests that at least
part of the intracellular water might be "anti-crystalline"
in the sense advanced by Ubbelohde (109). Basic work in this
field has been done by Mazur (73) and further evidence for
anomalous water properties in biological systems comes from
intracellular freezing patterns (98), indicating that intra
cellular water can undergo appreciable super-cooling. The
lowering of the freezing point is commonly ascribed to the
lack of intracellular nucleators of super-cooled water. Fur
ther details are summarized by Drost-Hansen (45).
Electrical conductivity studies by Carpenter^ et al.
(13) using Aplysia neurons suggest that the observed reduc
tion in conductivity is somehow connected with the presence
of structured water. Fernández-Moran (50) pointed out that
structured water might be an important constituent of biolo
gical membranes exhibiting a number of functional properties.
Particularly, the selective permeability of membranes should
be controlled by modified water existing chiefly in membrane
6 G. Peschel and P. Belouschek
pores. More detailed work in this field has been conducted
by Schultz and Asunmaa (103) who have developed molecular
models of lipid/water components of the hexagonal subunits
found in plasma membranes.
The most interesting finding, however, is the observa
tion of thermal anomalies in a vast number of kinetic pro
cesses in living tissue, including enzyme activities. These
anomalies, discussed in detail by Drost-Hansen (42,43,45)
occur in narrow temperature ranges around 15°, 30°, 45° and
60°C and are ascribed to structural transitions of surface-
ordered intracellular water which, in turn, may exert a
marked influence on the conformations of enzymes and proteins
in the cell. Abrupt changes in enz3mie activity near these
transition temperatures point to larger energy differences
between two forms of the enzyme, both being stabilized by
vicinal water structure.
Long-range structuring effects represented by particu
larly deep surface orientation of water between two fused
silica plates with extrema close to the temperatures cited
above have been observed by one of the present authors (85,
87). These results support the belief that most of the
thermal anomalies occurring in biological systems are due
to structural peculiarities of intracellular structured
water.
The cooperativity of surface-ordered water is also
stressed by Ling (66-69) (see also chapter by Ling in this
volume). He has advanced the "association-induction hypo
thesis" in which intracellular water is considered to exist
as polarized multilayers which are functionally linked to a
cooperative adsorption of ions on protein sites. In con
trast to the classical view which postulates that ion accumu
lation in cells occurs by the action of ion membrane pumps.
Ling provides experimental evidence that structured water is
an essential ingredient in this regard by exhibiting changed
solvent properties for the different electrolytes and non-
electrolytes encountered in biological tissue. Excellent
model investigations by Wiggins (116) with inanimate
materials have strengthened the view that intracellular
water structures play a major role in cellular solute dis
tributions. The serious drawback of all these experiments
lies in the fact that biological tissue has an extremely com
plex structure which makes it very difficult to elucidate
the basic characteristics of structured water.
