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T H E F R O N T I E R S C O L L E C T I O N

Ole G. Mouritsen
Luis A. Bagatolli

LIFE –
AS A MATTER
OF FAT
L i p i ds i n a Membr ane
Biophysics Perspec tive

2nd edition

123
THE FRONTIERS COLLECTION

Series editors
Avshalom C. Elitzur
Unit of Interdisciplinary Studies, Bar-Ilan University, 52900 Ramat-Gan, Israel
e-mail: avshalom.elitzur@weizmann.ac.il

Laura Mersini-Houghton
Department of Physics, University of North Carolina, Chapel Hill,
NC 27599-3255, USA
e-mail: mersini@physics.unc.edu

T. Padmanabhan
Inter University Centre for Astronomy and Astrophysics (IUCAA), Pune, India

Maximilian Schlosshauer
Department of Physics, University of Portland, Portland, OR 97203, USA
e-mail: schlossh@up.edu

Mark P. Silverman
Department of Physics, Trinity College, Hartford, CT 06106, USA
e-mail: mark.silverman@trincoll.edu

Jack A. Tuszynski
Department of Physics, University of Alberta, Edmonton, AB T6G 1Z2, Canada
e-mail: jtus@phys.ualberta.ca

Rüdiger Vaas
Center for Philosophy and Foundations of Science, University of Giessen,
35394 Giessen, Germany
e-mail: ruediger.vaas@t-online.de
THE FRONTIERS COLLECTION

Series Editors
A.C. Elitzur L. Mersini-Houghton T. Padmanabhan M. Schlosshauer
M.P. Silverman J.A. Tuszynski R. Vaas

The books in this collection are devoted to challenging and open problems at the
forefront of modern science, including related philosophical debates. In contrast to
typical research monographs, however, they strive to present their topics in a
manner accessible also to scientifically literate non-specialists wishing to gain
insight into the deeper implications and fascinating questions involved. Taken as a
whole, the series reflects the need for a fundamental and interdisciplinary approach
to modern science. Furthermore, it is intended to encourage active scientists in all
areas to ponder over important and perhaps controversial issues beyond their own
speciality. Extending from quantum physics and relativity to entropy, conscious-
ness and complex systems—the Frontiers Collection will inspire readers to push
back the frontiers of their own knowledge.

More information about this series at http://www.springer.com/series/5342

For a full list of published titles, please see back of book or springer.com/series/5342
Ole G. Mouritsen Luis A. Bagatolli
•

LIFE – AS A MATTER
OF FAT
Lipids in a Membrane Biophysics Perspective

Second Edition

123
Ole G. Mouritsen Luis A. Bagatolli
Department of Physics, Chemistry Department of Biochemistry and Molecular
and Pharmacy Biology
MEMPHYS-Center for Biomembrane MEMPHYS-Center for Biomembrane
Physics Physics
Odense Odense
Denmark Denmark

ISSN 1612-3018 ISSN 2197-6619 (electronic)

THE FRONTIERS COLLECTION
ISBN 978-3-319-22613-2 ISBN 978-3-319-22614-9 (eBook)
DOI 10.1007/978-3-319-22614-9

Library of Congress Control Number: 2015946589

Springer Cham Heidelberg New York Dordrecht London

© Springer International Publishing Switzerland 2005, 2016
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part
of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations,
recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission
or information storage and retrieval, electronic adaptation, computer software, or by similar or
dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this
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The publisher, the authors and the editors are safe to assume that the advice and information in this
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Printed on acid-free paper

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Preface

The first English edition of the present book was published in 2005 and although
still in use around the world the text could be much improved by a timely update on
several counts. Since 2005 the field of membrane biophysics has expanded tre-
mendously and moved center stage in the study of biological membranes, not least
due to a tremendous development in the experimental techniques that can be
applied to membranes. It was deemed useful to prepare a revised edition of the book
by incorporating some of the advances in the field over the last decade. In addition,
the book now includes more tutorial material on the biochemistry of lipids and the
principles of lipid self-assembly.
The second edition greatly owes its materialization to the efforts of one of the
authors (LAB), who undertook a revision of the first edition (single-authored by
OGM) for preparation of a Spanish edition that was published in 2014. The present
second English edition is to a great extent a revised version of the Spanish edition.
The research work underlying the picture of fats, lipids, and membranes advo-
cated in the present book derives from many different scientists and students in
several laboratories across the world. The specific examples and data presented are,
however, biased toward the work carried out by the authors and their collaborators.
Therefore, the book should not be considered an authoritative monograph but more
a personal perspective on a diverse and rapidly expanding field of science.
The book is provided with a Bibliography containing a selected list of references
to books, review papers, and research articles accounting for most of the factual
statements made in the book. These references to the literature have been selected
according to a minimal principle. The rule has been adopted that references are
mostly made to recent publications and not necessarily to the original work. From
the references given, the interested reader should be able to track down the original
literature. The reader is furthermore referred to the list of specialized books and
review papers for a more comprehensive list of references. We apologize in
advance to those authors and colleagues who may feel that their original work
should have been referenced and discussed in more detail.

v
vi Preface

The authors have over the years benefited from stimulating interaction and
fruitful collaboration with a large number of colleagues and students, in particular
from the Center for Biomembrane Physics (MEMPHYS) at the University of
Southern Denmark.
The authors have drawn on the help from a number of colleagues and the
permission to use some of their graphic material for preparing many of the figures
of this book. A list of the sources for the figures is provided at the end.

Odense Ole G. Mouritsen

May 2015 Luis A. Bagatolli
Contents

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi

Prologue: Lipidology—The Science of Lipids . . . . . . . . . . . . . . . . . xiii

Part I The Overlooked Molecules

1 Life from Molecules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

1.1 The Three Kingdoms of Life . . . . . . . . . . . . . . . . . . . . . . . . 3
1.2 The Molecules of Life . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
1.3 A Brief Historical Overview of Membrane Theory . . . . . . . . . 6
1.4 Membranes in Cellular Systems . . . . . . . . . . . . . . . . . . . . . . 9
1.5 The Post-genomic Era . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.6 A Call for Physics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2 Head and Tail . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2.1 Fat Family: Fats and Fatty Acids . . . . . . . . . . . . . . . . . . . . . 19
2.2 The Polar Lipids—Both Head and Tail . . . . . . . . . . . . . . . . . 22
2.3 Cholesterol—A Lipid of Its Own . . . . . . . . . . . . . . . . . . . . . 28
2.4 Strange Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
2.5 Lipid Composition of Membranes . . . . . . . . . . . . . . . . . . . . . 30

3 Oil and Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

3.1 Water—The Biological Solvent. . . . . . . . . . . . . . . . . . . . . . . 31
3.2 The Hydrophobic Effect. . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.3 Mediating Oil and Water . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.4 Self-assembly and the Lipid Aggregate Family . . . . . . . . . . . . 35
3.5 Thermodynamics of Lipid Self-assembly . . . . . . . . . . . . . . . . 38
3.6 Plucking Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

vii
viii Contents

4 Lipids Speak the Language of Curvature . . . . . . . . . . . . . . . . . . . 45

4.1 How Large Is a Lipid Molecule?. . . . . . . . . . . . . . . . . . . . . . 45
4.2 Lipid Molecules Have Shape . . . . . . . . . . . . . . . . . . . . . . . . 47
4.3 Lipid Structures with Curvature . . . . . . . . . . . . . . . . . . . . . . 50
4.4 Microorganisms’ Sense for Curvature . . . . . . . . . . . . . . . . . . 52

5 A Matter of Softness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
5.1 Soft Matter. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
5.2 Soft Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
5.3 Forces Between Soft Interfaces . . . . . . . . . . . . . . . . . . . . . . . 60
5.4 Lipid Membranes are Really Soft . . . . . . . . . . . . . . . . . . . . . 61

6 Soft Shells Shape Up. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

6.1 Bending Interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
6.2 Spontaneous Curvature . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
6.3 Shaping Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
6.4 Red Blood Cells Shape Up . . . . . . . . . . . . . . . . . . . . . . . . . 72

7 Biological Membranes—Models and Fashion . . . . . . . . . . . . . . . . 75

7.1 What Is a Model? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
7.2 Brief History of Membrane Models . . . . . . . . . . . . . . . . . . . . 77
7.3 Do We Need a New Membrane Model? . . . . . . . . . . . . . . . . 79
7.4 Theoretical and Experimental Model Systems . . . . . . . . . . . . . 81

Part II Lipids Make Sense

8 Lipids in Bilayers—A Stress-Full and Busy Life . . . . . . . . . . . . . . 85

8.1 Trans-Bilayer Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
8.2 The Lateral Pressure Profile . . . . . . . . . . . . . . . . . . . . . . . . . 87
8.3 How Thick are Membranes?. . . . . . . . . . . . . . . . . . . . . . . . . 89
8.4 Lively Lipids on the Move. . . . . . . . . . . . . . . . . . . . . . . . . . 90

9 The More We Are Together . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

9.1 Phase Transitions Between Order and Disorder . . . . . . . . . . . . 95
9.2 Lipids Have Phase Transitions . . . . . . . . . . . . . . . . . . . . . . . 98
9.3 Mixing Different Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
9.4 Cholesterol Brings Lipids to Order . . . . . . . . . . . . . . . . . . . . 106

10 Lipids in Flatland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

10.1 Gases, Liquids, and Solids in Two Dimensions. . . . . . . . . . . . 111
10.2 Langmuir and Langmuir-Blodgett Films. . . . . . . . . . . . . . . . . 113
10.3 Pattern Formation in Lipid Monolayers . . . . . . . . . . . . . . . . . 116
10.4 Lipids Make the Lung Work . . . . . . . . . . . . . . . . . . . . . . . . 119
Contents ix

11 Social Lipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

11.1 Lateral Membrane Structure . . . . . . . . . . . . . . . . . . . . . . . . . 125
11.2 Imaging Lipid Domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
11.3 Lateral Membrane Heterogeneity and Domains . . . . . . . . . . . . 132
11.4 Membrane Domains Carry Function . . . . . . . . . . . . . . . . . . . 138
11.5 Active Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140

12 Lively Lipids Provide for Function . . . . . . . . . . . . . . . . . . . . . . . 143

12.1 Leaky and Thirsty Membranes . . . . . . . . . . . . . . . . . . . . . . . 143
12.2 Repelling Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
12.3 Enzymes Can Sense Membrane Transitions . . . . . . . . . . . . . . 147
12.4 Lipid Thermometer in Lizards . . . . . . . . . . . . . . . . . . . . . . . 149

13 Proteins at Lipid Mattresses . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151

13.1 Coming to Terms with Lipids. . . . . . . . . . . . . . . . . . . . . . . . 151
13.2 Anchoring at Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
13.3 Spanning the Membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

Part III Lipids in Action

14 Cholesterol on the Scene . . . . . ............. . . . . . . . . . . . . . 163

14.1 Molecule of the Century . ............. . . . . . . . . . . . . . 163
14.2 Evolutionary Perfection of a Small Molecule . . . . . . . . . . . . . 164
14.3 Cholesterol Fit for Life . . ............. . . . . . . . . . . . . . 168
14.4 Cholesterol as a Killer . . . ............. . . . . . . . . . . . . . 170

15 Lipids in Charge. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

15.1 Lipids and Proteins Match Up . . . . . . . . . . . . . . . . . . . . . . . 173
15.2 Stressing Proteins to Function. . . . . . . . . . . . . . . . . . . . . . . . 179
15.3 Lipids Opening Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
15.4 Lipids Mediate Fusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

16 Being Smart—A Fishy Matter of Fat . . . . . . . . . . . . . . . . . . . . . . 189

16.1 The Essential Fatty Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . 189
16.2 Evolution of the Human Brain . . . . . . . . . . . . . . . . . . . . . . . 191
16.3 Lipids at the Border of Madness . . . . . . . . . . . . . . . . . . . . . . 193

17 Liquor and Drugs—As a Matter of Fat . . . . . . . . . . . . . . . . . . . . 195

17.1 Lipids Are Targets for Drugs . . . . . . . . . . . . . . . . . . . . . . . . 195
17.2 Alcohol and Anesthesia . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
17.3 Poking Holes in Membranes. . . . . . . . . . . . . . . . . . . . . . . . . 198
17.4 Gramicidin—The Portable Hole . . . . . . . . . . . . . . . . . . . . . . 200
x Contents

18 Lipid Eaters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

18.1 Enzymes that Break Down Lipids in Crowds . . . . . . . . . . . . . 203
18.2 Watching Enzymes at Work . . . . . . . . . . . . . . . . . . . . . . . . . 206
18.3 Lipids Going Rancid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208

19 Powerful and Strange Lipids at Work . . . . . . . . . . . . . . . . . . . . . 211

19.1 The Impermeable Barrier—Lipids in the Skin . . . . . . . . . . . . . 211
19.2 Surviving at Deep Sea and in Hot Springs . . . . . . . . . . . . . . . 215
19.3 Lipids as Messengers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218
19.4 Lipids as a Matter of Death . . . . . . . . . . . . . . . . . . . . . . . . . 219

20 Survival by Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223

20.1 Lipids for Smart Nano-technology. . . . . . . . . . . . . . . . . . . . . 223
20.2 Lipids Deliver Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
20.3 Liposomes as Magic Bullets . . . . . . . . . . . . . . . . . . . . . . . . . 229
20.4 Lipids Fighting Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233

Epilogue: Fat for Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

Sources for Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273

Titles in this Series . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295

Abbreviations

Fatty Acids
AA Arachidonic acid
DHA Docosahexaenoic acid
DPA Docosapentaenoic acid
EPA Eicosapentaenoic acid

Lipid Polar Head Groups

DGDG Digalactosyl diglyceride
PA Phosphatidic acid
PC Phosphatidylcholine
PE Phosphatidylethanolamine
PG Phosphatidylglycerol
PI Phosphatidylinositol
PS Phosphatidylserine

Lipids
DAG Di-acylglycerol
DAPC Di-arachidoyl PC
DCPC Di-capryl PC
DLPC Di-laureoyl PC
DMPC Di-myristoyl PC
DMPE Di-myristoyl PE
DMPG Di-myristoyl PG
DOPC Di-oleoyl PC
DPPC Di-palmitoyl PC
DPPE Di-palmitoyl PE
DSPC Di-stearoyl PC

xi
xii Abbreviations

DSPE Di-stearoyl PE
POPC Palmitoyl-oleoyl PC
POPE Palmitoyl-oleoyl PE
SM Sphingomyelin
SOPC Stearoyl-oleoyl PC

Others
ATP Adenosine triphosphate
DNA Deoxyribonucleic acid
ER Endoplasmic reticulum
GUV Giant unilamellar vesicle
HDL High-density lipoprotein
LAURDAN 6-Dodecanoyl-2-Dimethylaminonaphthalene
LDL Low-density lipoprotein
RNA Ribonucleic acid
ROS Reactive oxygen species
Prologue: Lipidology—The Science of Lipids

To many people, fats are something vicious that are dangerous to our health and
well-being and therefore should be avoided. Some people know that fats are
essential ingredients of the diet and furthermore provide for tasty meals. Others
acknowledge that certain fats like cholesterol are required for the body’s production
of important hormones, such as sex hormones, as well as vitamin D and the bile you
need in your stomach to break down the food. Many of us appreciate that the
unsaturated fats present in seafood provide for good health and longevity. However,
few realize that fats are as important for life as proteins and genes. And probably
very few people know that in terms of mass, fat is the most important part of our
brain and the second most important of all other soft tissue.
In the sciences, fats are called lipids. Lipids are studied by nutritionists who
investigate how the intake of fats in our diet affects the composition of various parts
of the body, e.g., the heart, the liver, and the brain. You are what you eat! Lipids are
also studied by biochemists who investigate the synthesis and the breakdown of
lipids as a source of energy and building material for cells. Among molecular
biologists and physical scientists lipids are less appreciated, although lipids are
listed along with proteins, nucleic acids (genes, DNA, and RNA), and carbohy-
drates (sugars) as the fundamental building blocks of all living matter. Proteins and
genes are known to be very specific to the functions they perform. The same is true
for the carbohydrates that are used by the living cell to recognize foreign substances
and to identify other cells.
Lipids as structure builders and fat depots are, however, most often characterized
by terms like variability, diversity, plasticity, and adaptability. Hence, lipids appear
to play a fairly nonspecific role, being rather dull and anonymous compared to
fashionable stuff like the proteins that catalyze all biochemical reactions and the
genes that contain the information needed to produce the proteins. There are no
genes coding for lipids as such, only for the enzymes that build and modify the
lipids which we, for example, obtain from our food.
There is a particular reason why lipids are considered as being dull and less
interesting. This reason has to do with the powerful concept of molecular structure
that has permeated life scientists’ way of thinking throughout the twentieth century.

xiii
xiv Prologue: Lipidology—The Science of Lipids

The central dogma is that molecular structure controls function. Lipids apparently
do not have the intriguing molecular structures which important molecules like
proteins and DNA have. Moreover, lipids do not as easily as proteins and DNA
lend themselves to revealing the secrets of how molecular structure leads to bio-
logical function. In fact, lipids are often tacitly assumed to constitute a structureless
fatty material which at best is organized in a membrane structure that plays the role
of a passive container of the cell and an appropriate solvent or template for the
important molecules of life: lipids grease the functional machinery of the cells
controlled and run by proteins and DNA.
The traditional viewpoint on lipids as dull molecules has changed considerably
over the last couple of decades and their importance in cell function and health is
becoming more recognized. This is what the present book is about. Lipids are
proposed to be as important for life as proteins and genes. The book will point out
that lipids lead to interesting and intriguing structures with very unusual and subtle
materials properties that have been optimized by evolutionary principles over bil-
lions of years. These properties are consequences of the fundamental physical
principles of self-organization that rule when many molecules act in concert. A key
player in this concert is water that functions as the unique biological solvent. Water
is omnipresent in all functioning biological systems. The peculiar properties of
water force lipid molecules to self-assemble and organize into subtle structures. In
particular, lipids in water can form lipid bilayers and membranes. Lipid membranes
are extended thin layers which are only two molecules thick. These layers constitute
the backbone of all biological membranes. Membranes are a ubiquitous structural
element in all living cells.
And cells are literally packed with membranes. In a human being which is
composed of about 4
1013 cells, the total surface of all plasma cell membranes
can be estimated to cover an area of about 12,000 m2, corresponding to almost two
football fields. Counting also internal membranes, the total area is probably a
couple of orders of magnitude larger. An important membrane function is purely
topological: membranes compartmentalize living matter into cells and subcellular
structures. Furthermore, membranes present themselves to macromolecules as
highly structured interfaces at which important biochemical processes are carried
out and catalyzed. Obviously, the structure and molecular organization of the
lipid-bilayer component of membranes hold the key to understanding the func-
tioning of membranes.
Due to the fact that lipids form membranes by self-assembly processes that do
not involve strong chemical forces, membranes are pieces of soft matter. Softness is
a materials feature that lipid membranes share with other forms of condensed matter
like polymers and liquid crystals. During evolution, Nature has evolved biological
membrane structures as an optimal form of micro-encapsulation technology that on
the one hand imparts the necessary durability to the particular soft condensed matter
that membranes are made of, and on the other hand sustains the lively dynamics that
are needed to support and control the mechanisms of the many essential cellular
functions associated with membranes.
Prologue: Lipidology—The Science of Lipids xv

Lipids are now known not only to be structural builders of the cell and an energy
source for cell functioning. In the form of membranes, lipids are crucial for con-
trolling indirectly a great variety of biological functions that take place at or are
mediated by membranes. Evidence emerging from recent research has shown that
the functional role of membrane lipids may be as important as that of proteins. The
lipids not only act as a passive solvent for the proteins and as a means of com-
partmentalization; they are an integral part of cellular function. Many lipid species
are now known to play a very active role, serving as so-called second messengers
that pass on signals and information in the cell. Lipids also play the roles of
enzymes, receptors, drugs, as well as regulators of, e.g., neurotransmitter activity.
Lipids are known to modulate the expression of genes. Disorder in the lipid
spectrum of cells has been related not only to atherosclerosis but also to major
psychiatric illnesses. Finally, polyunsaturated lipids via the diet have been proposed
to provide an evolutionary driving force supplementing Darwinian natural selec-
tion. All these factors emphasize a necessary shift from a one-eyed genocentric
approach to membrane science toward a more balanced organocentric view. This
puts the study of the physical properties of membranes at a very central position.
We have just witnessed a scientific revolution, which at the end of the twentieth
century took us through the genomics era with focus on sequencing genes and
mapping gene products. We are passing through the proteomics era where the
multitude of proteins that are coded for by the genes are identified and their
functions being unveiled. We are in the middle of the lipidomics era. In a not
too distant future we can imagine all the ‘omics’ to converge into the grand
metabolomics era where the interplay between all the constituents of living matter is
being studied. This development mirrors a gradual anticipation of the actual
complexity of the problem.
Some have claimed that life sciences, due to the strong focus on sequencing and
mapping of genes and proteins, have been turned into a data-driven information
science. It is certainly true that modern biology has for ever been changed by the
tremendous wealth of information that has been derived from the genomics and
proteomics programs. However, a deep insight into the workings of living systems
requires more than collection of data and information processing. Science and
scientific thinking is by nature driven by hypotheses and it goes beyond stamp
collection. This is certainly also true of lipidomics. A mapping of all lipid species in
all cell types is undoubtedly useful. However, the real fundamental science of
lipids, i.e., lipidology, has to go beyond that and approach cellular functioning from
a more holistic perspective. And it will have to be hypothesis-driven. Lipidology
must involve a quantitative experimental and theoretical study of, e.g., lipid and
membrane self-assembly, lipid–protein interactions, lipid–gene interactions, and the
biophysical properties of lipid structure and dynamics.
A deeper knowledge of the physical properties of lipids is obviously useful for
understanding the living world and its modes of functioning. It may also be useful
for revealing the causes of malfunctioning and diseased conditions and how such
malfunctioning can be restored by medical treatment or by altered living conditions.
The insights into the physics and the inner workings of the cellular machinery,
xvi Prologue: Lipidology—The Science of Lipids

including the role of lipids, can furthermore be lessons for technology. Examples
include development of futuristic soft and biocompatible materials, functionalized
surfaces and sensors, design of new and effective drugs and drug-delivery systems,
for e.g., cancer therapy, as well as design of new enzymes for cheap and clean
production of drugs and chemicals.
The present book presents a personal and multidisciplinary perspective on the
physics of life and the particular role played by lipids and the lipid-bilayer com-
ponent of cell membranes. The emphasis is on the physical properties of the lipid
membrane seen as a soft and molecularly structured interface. By combining and
synthesizing insights obtained from a variety of recent studies, an attempt is made
to clarify what membrane structure is and how it can be quantitatively described.
Furthermore, it will be shown how lipid membrane structure and organization can
control functional properties of membranes. The strategy of the book is to provide a
bridge between, on the one side the microscopic world of membranes, i.e., the
world of the molecules, and on the other side the macroscopic world, i.e., the world
as we observe and sense it. This involves unravelling the organizational principles
which govern the many types of structure that arise on length scales from the size
of the individual molecule, across molecular assemblies of proteins and lipid
domains in the range of nanometers, to the meso- and macroscopics of whole cells.
A note on length scales, forces, energy, and temperature
The picture of lipids described in this book takes its starting point in the molecular
world where the molecules move around due to influence of temperature. It is hence
useful to describe the various entities on scales described in units of nanometers.
One nanometer (nm) is 109 m, i.e., one millionth of a millimeter. Cell sizes are
conveniently given in units of micrometers. One micrometer (µm) is 1000 nm. The
appropriate unit of force for biological molecules on the nanometer scale is
pico-Newton (pN). One pN is 1012 N. These units sound terribly small. It is
helpful to combine them in the form of the energy that corresponds to the thermal
energy at room temperature (Troom ¼ 293 K ¼ 20  C)

Thermal energy ¼ kB Troom ¼ 4:1  nm  pN; ð1Þ

where kB is Boltzmann’s constant which is a universal number. Most of the phe-

nomena characteristic of biomolecular structure and dynamics are strongly influ-
enced by temperature and it is therefore of importance whether the involved
energies are smaller or larger than the thermal energy in Eq. (1). As a rule of thumb
one would say that if a characteristic energy of some association, e.g., a binding
between two molecular-scale objects, is of the order of a few kB Troom or less,
thermal agitation should be significant, and the lifetime of the association can be
short. A couple of examples can serve as illustration. A covalent chemical bond
C–C between two carbon atoms represents an energy of the order of 100 kB Troom
(with a force equivalent around 5000 pN) and is therefore very stable at room
temperature. A typical hydrogen bond amounts to about 10 kB Troom (with a force
equivalent of around 200 pN) and is therefore often influenced by thermal agitation.
Prologue: Lipidology—The Science of Lipids xvii

Turning then to weak physical interactions, the van der Waals interaction between
two methane molecules, or other hydrocarbon moieties, represent about 1 kB Troom
(with a force equivalent of around 40 pN). Even lower energies and correspond-
ingly lower forces govern the weak molecular associations in biological systems,
e.g., the binding of a small enzyme to a membrane surface amounts to about 20 pN,
the force required to pull out a single lipid molecule of a membrane is only about
2 pN, and finally the motor proteins that function, e.g., in muscle contraction exert
forces as small as 1 pN. In all these cases, thermal agitation is of major importance.
 Part I
The Overlooked Molecules
Chapter 1
Life from Molecules

1.1 The Three Kingdoms of Life

Living organisms are divided into three kingdoms, the eukaryotes, the eubacteria, and
the archaebacteria. The eubacteria and the archaebacteria, which among themselves
differ as much as they do from eukaryotes, are conventionally grouped together
as prokaryotes. The bacteria common to most people, e.g., E. coli bacteria or the
bacteria in sour milk, are eubacteria. Archaebacteria are typically found in rather
hostile environments, such as in hot springs, at the bottom of deep sea, or in the very
acidic milieu of the cow’s stomach. These bacteria do not tolerate oxygen and they
often present a health hazard to humans. Eukaryotes are animals, plants, and fungi
and include also single-cell organisms like yeast. Figure 1.1 gives examples of single
cells from the three kingdoms.
Despite their difference in appearance and functioning, archaebacteria, eubacteria,
and eukaryotes are all made from the same basic molecular building blocks and they
are all based on the same chemistry. Although it is generally believed that all cells
have a common ancestor, the ancient evolutionary history of the different cell types
is subject to considerable dispute. The three kingdoms and their interrelations are
often represented by a so-called universal phylogenetic tree as shown in Fig. 1.2.
Although the origin of life on Earth is a controversial and unresolved problem, it
is a reasonable assumption that the first cellular living systems on Earth were assem-
bled from four types of molecular building blocks: (i) information-storing molecules
capable of reproduction, (ii) enzyme-like catalysts encoded by that information and
able to enhance reproduction rates, (iii) molecules capable of storing energy and
using this energy to convert molecules into organized assemblies of biologically
active molecules, and (iv) special boundary-forming molecules capable of encap-
sulating and protecting the former three types of molecules. The last category of
molecules is the focus of the present book.

© Springer International Publishing Switzerland 2016 3

O.G. Mouritsen and L.A. Bagatolli, Life – as a Matter of Fat,
The Frontiers Collection, DOI 10.1007/978-3-319-22614-9_1
4 1 Life from Molecules

Fig. 1.1 Examples of cells from the three kingdoms of life. a An archaebacterium: Methanococcus
jannischiiwas. Diameter about 2 µm. b A eubacterium: Escherichia coli. Size about 2–3 µm. c
Eukaryotes: Human red and white blood cells shown together with a platelet. The diameter of the
red blood cell is about 6 µm. The cells from the three kingdoms are not drawn on the same scale

Fig. 1.2 Phylogenetic tree with the three kingdoms of life: eubacteria, archaebacteria, and eukary-
otes. The relative distance between the organisms is proportional to the evolutionary distance as
determined by ribosomal-RNA nucleotide sequencing

1.2 The Molecules of Life

All cells are built from small organic molecules that are based on the chemistry
of carbon. These small molecules belong to essentially four classes: the sugars, the
amino acids, the nucleotides, and the fatty acids. Examples of these small elementary
building blocks of living matter are given in Fig. 1.3.
1.2 The Molecules of Life 5

Fig. 1.3 Examples representing the four classes of small organic molecules that are the building
blocks of all living matter. a Sugar: glucose. b Amino acid: alanine. c Nucleotide: adenosine.
d Fatty acid: oleic acid

The small organic molecules are combined with other molecules from the same
class or with molecules from the other classes to make larger entities, so-called
macromolecules or macromolecular assemblies. There are basically four classes of
these larger entities, the poly-saccharides, the proteins, the nucleic acids, and the
fats (lipids and membranes), as illustrated in Fig. 1.4.
Proteins are also called poly-amino acids (or poly-peptides), and nucleic acids are
called poly-nucleotides, reflecting the fact that proteins and nucleic acids, just like
poly-saccharides, are biopolymers, i.e., long-chain molecules composed of many
monomers that are bound together by strong chemical bonds.
Since there are about twenty different types of amino acids in Nature and since a
protein can consist of up to several hundreds of amino acids, a very large number of
different proteins can be perceived. Similarly, the five different nucleotides used by
Nature allow for an immense richness in different nucleic acids that make up DNA
(deoxyribonucleic acids) and RNA (ribonucleic acids). Like proteins, the nucleic
acids are linear molecules, and it is the particular sequence of the monomers that
determines the properties of both proteins and nucleic acids. DNA and RNA contain
the genetic information that is organized in genes. The entire DNA string of an organ-
ism is termed the genome which can contain millions of nucleotides. For example the
human genome includes about 27,000 genes composed of over 3 billion nucleotides.
In addition to encoding genetic information, nucleotides also perform functions as
energy carriers (ATP, adenosine triphosphate), catalysts, and messengers.
When it comes to the sugars, living organisms exploits a large number of different
mono-saccharides. Hence it is not uncommon to find hundreds of different poly-
saccharides in a cell. Sugars allow for additional complexity in the type of materials
6 1 Life from Molecules

Fig. 1.4 Examples representing the four classes of larger macromolecular entities of which all living
matter is composed. a Poly-saccharide: cellulose. b Protein: myoglobin. c Nucleic acid: DNA in the
form of a double helix. d Lipid assembly: lipid bilayer membrane. The different macromolecules
and assemblies are not drawn on the same scale

that can be built because they can combine into branched macromolecular networks.
Such networks are responsible for forming biological fibers and scaffolding and they
are also an important part of the cell’s recognition system.
We are then left with the fatty acids and the lipids. In contrast to the sugars, the
amino acids, and the nucleotides, lipids do not link chemically to form ‘poly-lipids.’
No such thing exist under natural conditions. Instead they form ‘loose’ macromolec-
ular (or supra-molecular) assemblies of which the lipid bilayer membrane shown in
Fig. 1.4d is the most prominent example. In some cases, lipids combine chemically
with proteins and sugars. However when forming living matter, lipids usually main-
tain their molecular integrity. The lipid bilayer is the core of all biological membranes.
A lipid bilayer membrane contains billions of lipid molecules and a cell membrane
often contains hundreds of different kinds of lipids.

1.3 A Brief Historical Overview of Membrane Theory

The idea of the existence of a membrane covering the surface of cells has its origin
in the discovery of osmosis. This phenomenon was originally described by Jean-
Antoine Nollet in 1748. Nollet immersed in water a pig’s bladder filled with water
1.3 A Brief Historical Overview of Membrane Theory 7

and alcohol and discovered that water moved across the bladder wall to the inside
of the bladder containing both water and alcohol, but alcohol did not transport to
the outside containing only water. In the following years, Nollet’s observations were
further tested in plant cells by René Dutrochet (1828) and other scientists such as
von Nagli (1855) and W. Hofmeister (1867). The word ‘osmosis’ descends from the
words ‘endosmose’ and ‘exosmose,’ which were coined by René Dutrochet from the
Greek words νδoν (endon: within), ξ o (exo: outside), and ωσ μoς (osmos: push,
impulsion).
A simple but historical discovery was made in 1867 by an amateur research sci-
entist from Berlin named Moritz Traube. He observed that when a drop of copper
sulfate solution is brought into contact with a drop of potassium ferrocyanide solu-
tion, a thin layer of reddish-brown copper-ferrocyanide precipitate forms at, and
blankets, the entire boundary. After that, no further formation of precipitate occurs.
Thus the thin layer of precipitated material formed between the two drops of solu-
tions has stopped further passage to the other side of the copper ion as well as the
ferrocyanide ion. The relevance of these observations was acknowledged by Wil-
helm Pfeffer who employed the copper ferrocyanide membrane to demonstrate the
osmosis phenomenon by placing sucrose solutions of different strengths on either
side of the membrane. Specifically he saw water moving from the dilute to the con-
centrated side—similar to what Nollet witnessed across his dead animal membrane
and what Dutrochet observed in and out of living mature plant cells. Importantly,
Pfeffer also found that this osmotic water movement could be brought to a stop by
applying to the side containing the more concentrated sucrose solution a pressure
of just enough strength (to be referred to as osmotic pressure). Pfeffer stablished
important relations among the osmotic pressure, the concentration of sugar, and the
temperature in this system, observations that were brought to the attention of the
physical chemist Jacobus H. van’t Hoff. For the origin of the osmotic pressure, van’t
Hoff introduced his bombardment theory, pointing out the analogy of the ideal gas
law and the well-known van’t Hoff equation for solutions (i.e., V = R  T , where
is the osmotic pressure, V the volume, T the temperature, and R  a constant).
Regarding this analogy, van’t Hoff wrote: “In the former case (the ideal gas law), the
pressure is due to the impacts of gaseous molecules on the walls of the containing
vessel, and in the latter to the impacts of the molecules of dissolved substance on
the semipermeable membrane.” Thus Pfeffer’s accurate study of osmotic pressure
using the copper ferrocyanide membrane paved the way for what is often referred to
as van’t Hoffs solution theory. Continued investigations on both model systems and
living (mature) plant cells led Pfeffer to ideas regarding living plant cells, which were
later referred to as Pfeffers membrane theory. It is worth mentioning that this line of
thinking was proposed earlier by Theodore Schwann (1839), who also suggested the
essence of what would become known later as the membrane theory, namely, that
both plant and animal cells are membrane-enclosed cavities filled with clear liquid
water.
Pfeffer published his major work in 1877 where its main theme was that a periph-
eral layer of the protoplasm called the plasma membrane covers not only the outer
surface of the cell but any exposed surface of protoplasm where it comes into contact
8 1 Life from Molecules

with another aqueous solution. The plasma membrane exhibits properties similar to
those of the copper-ferrocyanide precipitation membrane endowing all living cells
with its semipermeable properties and osmotic behavior. Additionally, the lipid nature
of the cell membrane was first intuited by G. Quincke (1888) and further extended
by evidence from the study of anesthetics performed independently by Hans Meyer
and Ernest Overton (1899, known as the ‘lipoid theory of narcosis’). Based on this
evidence and further experiments, it was concluded that the cell membrane might be
made of lecithin (phosphatidylcholine) and cholesterol.
The long tradition of visualizing living cells as fluid-filled vesicles, supported by
the elegant solution theory of van’t Hoff, provided the foundation for the membrane
theory. Further evidence came from osmotic studies and from investigators in other
specialized fields of cell physiology, such as J. Bernstein, author of the membrane
theory of cellular electrical potentials (1902) and F. Donnan, author of the theory
of membrane equilibrium of ionic distribution and electrical potential (1911). Those
theories and their corroborative evidence indeed offer support for the membrane
theory, follows from the fact that they are based on the same fundamental assumption
that living cells are membrane-enclosed dilute solutions. This is perhaps one of the
weakest points of this model considering the highly crowded environment exist-
ing in the cytoplasm of any cell. The interlinking theories mentioned above have
also conjointly made the membrane theory the first coherent general theory of cell
physiology. In other words, the membrane theory can explain four major subjects of
cell physiology: (i) cell volume control, (ii) selective solute distribution, (iii) selective
solute permeability, and (iv) cellular electrical potentials.
It is important to mention, however, that the membrane theory is not the only
model capable of explaining the four major subjects of cell physiology mentioned
above. During the early times a different view that diverges from the membrane
theory was developed, focused in the colloidal properties of the cell cytoplasm. In
1835 the French zoologist Felix Dujardin had already described a water-insoluble
and gelatinous material that emanated from within a crushed protozoon. This mate-
rial was definitely not the clear watery liquid that Schwann described and gave the
impression that he (Schwann) had observed in many animal and plant cells. In 1861
Max Schultze, professor of botany in Bonn, pronounced his protoplasmic doctrine,
according to which a living cell is a membrane-less lump of protoplasm containing a
nucleus, a view that was supported by Thomas Huxley, the one-time opponent to the
concept of protoplasm, who stated that protoplasm is the physical basis of life. With-
out going in deep details, this view known as protoplasmatic theory evolved along
the years having a strong foundation in the physico-chemical properties of colloidal
systems, i.e., sustaining the premise that the behavior of most solutes inside cells
does not necessarily resemble their behavior in dilute solutions. In this model, cells
are conceived as dynamical colloidal systems which respond to fluctuations, either
by damping them or amplifying them cooperatively, and therefore their properties are
considered as emergent properties of organized supramolecular systems. Importantly
and in contract to the membrane theory, the cellular interior accommodates solutes
based on adsorption sites and solubility properties of its ‘colloidal’ water and it is kept
organized by central metabolism in a low-entropy state, which becomes responsive
1.3 A Brief Historical Overview of Membrane Theory 9

to environmental factors in very specific ways. Although other important contribu-

tions were made to these ideas during the 20th century, e.g., by Dmitrii Nasonov
and Afanasy S. Troshin, the most complete version of the protoplasmatic theory was
provided in 1965 by Gilbert N. Ling, called association-induction (A-I) hypothesis.
Ling’s A-I hypothesis strongly challenges the modern mainstream consensus model
of cellular membranes based on the fluid-mosaic model, to be described later, that
envisions a lipid bilayer separating the inside from the outside of cells with associ-
ated ion channels, pumps, and transporters giving rise to the permeability processes
of cells. In addition, Ling’s A-I hypothesis is not assigning any relevant role to lipids
or to their supramolecular structures, emphasizing that interactions among proteins,
water, salts, and relevant metabolites are the dominant parameters for cellular func-
tioning.

1.4 Membranes in Cellular Systems

All cells of living beings are confined and compartmentalized by a number of mem-
branes as illustrated in Fig. 1.5 in the case of eukaryotes. Common for all cells is
a cell-surface membrane called the plasma membrane. The plasma membrane is a
very stratified and composite structure whose central element is the lipid bilayer
as illustrated in Fig. 1.6 in the case of a very simple uni-cellular microorganism,
Escherichia coli. The lipid bilayer is extremely thin in comparison with the size of
the cell it encapsulates. A schematic cartoon of the plasma membrane of an eukary-
otic cell with all the other components it contains in addition to the lipid bilayer is
shown in Fig. 1.7. The plasma membrane is a unique composite of all the types of
macromolecules described above, except nucleic acids. Its molecular composition
depends on the type of cell. Carbohydrates is a minor component with less that 10 %
of the dry mass. The weight ratio of proteins and lipids can vary from 1:5 to 5:1.
Whereas prokaryotic cells only have a plasma membrane and some less structured
internal membrane systems, the eukaryotic cells have in addition a number of well-
defined internal membranes associated with the cell nucleus and the organelles (cf.
Fig. 1.5). The cell nucleus, which contains the cell’s genetic material, is wrapped in
a porous double membrane (the nuclear envelope). This membrane is topologically
connected to the membranes of the endoplasmic reticulum (ER) which is the major
site of synthesis of lipids and proteins. The Golgi apparatus contains a very convo-
luted agglomerate of membranes. This is where the newly synthesized molecules are
modified, sorted, and packaged for transport to other organelles or for export out of
the cell. The membranes of both Golgi and the ER are morphologically very com-
plex and exhibit substantial curvature. The mitochondria, which contain their own
DNA and RNA and produce their own proteins, contain two intertwined membranes,
an inner and an outer membrane. The outer membrane acts as a sieve retaining the
larger proteins within its compartment. Lysosomes are rather small organelles bound
by a membrane. The lysosomes operate as the cell’s garbage and recycling system,
performing digestion, degradation, and export of unwanted molecules. Finally, there
10 1 Life from Molecules

Fig. 1.5 Schematic illustration of a generic eukaryotic cell which is drawn artificially to compare
an animal cell (left) and a plant cell (right). Plant cells, as well as bacterial cells, have an additional
outer cell wall

is a bunch of vesicles that support the extended trafficking needed by the cell to
transport material within the cell and across the plasma membrane.
Membranes thereby become the most abundant cellular structure in all living
matter. They can be considered as Nature’s preferred mode of micro-encapsulation
technology, developed as means of compartmentalizing living matter and protecting
the genetic material. The biological membrane is the essential capsule of life. Many
important biological processes in the cell either take place at membranes or are
mediated by membranes, such as transport, growth, neural function, immunological
response, signaling, and enzymatic activity. An important function of the lipid bilayer
is to act as a passive permeability barrier to ions and other molecular substances and
leave the trans-membrane transport to active carriers and channels.
1.4 Membranes in Cellular Systems 11

Fig. 1.6 Electron microscopy picture of the bacterium Escherichia coli. This Gram-negative species
is encapsulated in an inner bilayer cytoplasmic membrane, an intermediate peptidoglycan layer, and
an outer bilayer membrane. Also shown is a schematic illustration of the 5 nm thick lipid bilayer
which is the core of the membrane as illustrated in more detail in Fig. 1.7

Fig. 1.7 Schematic model of the plasma membrane of a eukaryotic cell which highlights the mem-
brane as a composite of a central lipid bilayer sandwiched between the carbohydrate glycocalyx
(which consist of poly-saccharides) on the outside and the rubber-like cytoskeleton (which is a poly-
meric protein network) on the inside. Intercalated in the lipid bilayer are shown various integral
proteins and poly-peptides. The membrane is subject to undulations and the lipid bilayer displays
lateral heterogeneity, lipid domain formation, and thickness variations close to the integral pro-
teins. Whereas the lipid molecules in this representation are given with some structural details, the
membrane-associated proteins remain fairly featureless. In order to capture many different features
in the same illustration, the different membrane components are not drawn to scale
12 1 Life from Molecules

Judging naively from Fig. 1.7, the role of lipids is much less glorious than that
played by proteins and nucleic acids. Whereas proteins possess a specific molecular
structure and order supporting function, and DNA has a very distinct molecular
structure and order encoding the genetic information, lipids appear to be characterized
by disorder and a lack of any obvious structural elements. The lipid bilayer is a
molecular mess and it is hard to imagine any structural order in and among the lipids
that is specific enough to control a delicate biological function.
This is the reason why lipids were not among the favorite molecules of the 20th
century’s molecular and structural biologists. The lipids became the most overlooked
molecules in biology.
In many ways this is a paradox. Without comparison, lipids are the most diverse
class of molecules in cells. Prokaryotic cells typically contain a hundred different
types of lipids, and the larger eukaryotes cells many thousand different kinds. More-
over, the results of genomic research have revealed that more than 30 % of the genome
codes for proteins that are embedded in membranes. A possibly even larger percent-
age codes for proteins that are peripherally attached to membranes. If one adds to this
the observation that the membrane-spanning parts of the proteins contain some of
the evolutionary most conserved amino-acid sequences, it becomes clear that lipids
are indeed very important molecules for life. This insight calls for a deeper under-
standing of how membrane proteins and their function are related to the properties
of the lipid bilayer membrane.
In order to understand the implications of disregarding lipids in the study of the
physics of life and which challenges it has left us with, we have to make a status on
life sciences upon the entry to what has been called the post-genomic era. This era is
characterized by an almost complete knowledge of genomes of an increasing number
of different species ranging from bacteria, fungi, insects, worms, and mammals,
including mouse and man.

1.5 The Post-genomic Era

Molecular and structural biology have been some of the most successful sciences
of the 20th century. By focusing on the concept of structure—from the genes to the
workhorses of living beings, the proteins—first revealing the genetic code, then the
structure of many proteins, and finally the whole genome of several species, including
that of man, these sciences have had an enormous impact on life sciences and society.
Structure, in particular well-defined atomistic-level molecular structure, has been the
lodestar in the quest for unravelling the genetic code and the properties of DNA, for
understanding transcription of the code into protein synthesis, and for determining
the properties of the proteins themselves. The relationship between macromolecular
structure and function is simply the key issue in modern biology. The human genome
project is probably the most monumental manifestation of the conviction among life
scientists that gene structure is the Holy Grail of life.
Another random document with
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gleaming, murderous eye and pierced to the brain.
The brute gave a few convulsive struggles and then straightened out
beside its mate.
The situation had changed with the quickness of a kaleidoscope.
The would-be killers had themselves been killed just at the moment
that their victory had seemed assured.
But by whom had their death been brought about? Were these
newcomers friendly or hostile to Bomba? The thought came to the
boy that they might be of the party of headhunters. If so, he might
have escaped death in one form to meet it in another even more
terrible.
But as he hesitated, he heard their voices more clearly and
recognized them. Then he waited no longer.
He slid hurriedly to the ground and came face to face with Ashati and
Neram, the former slaves of Jojasta, the medicine man of the Moving
Mountain!
 CHAPTER XIII
THE MAD MONKEY

Ashati and Neram dropped to their knees before Bomba, clasping

his hands and bowing their black heads before him. Neram, bending
lower, took one of Bomba’s sandaled feet and placed it on his neck
as a sign that he was slave and Bomba master.
The heart of the lonely boy swelled at this sign of gratitude and
affection. He stooped and raised the kneeling men, and made them
stand on their feet before him.
“You have saved Bomba’s life,” he said with deep feeling. “If you had
not come when you did the jaguars would have killed him. Bomba
will not forget.”
“Ashati and Neram would have been nothing now but bones buried
in the heart of the Moving Mountain if you had not come to their
help,” replied Ashati, who seemed to be the spokesman for the two.
“You saved them from death, and freed them from the cruel yoke of
Jojasta, the medicine man. Ashati and Neram have no master but
Bomba, and will go anywhere in the jungle at the side of Bomba as
his slave. Their lives belong to Bomba.”
“You shall go with me wherever I go,” replied Bomba. “But you shall
go as my friends and not as my slaves. Bomba has nothing to offer
you but friendship. If you will take that and go with him, he will be
glad.”
So it was settled, and with many more expressions of gratitude and
devotion on the part of the ex-slaves of Jojasta, Bomba and the two
men set to work to skin and quarter one of the dead jaguars that had
unwittingly furnished them a feast.
While engaged in this and the building of a fire for the roasting of the
best portions of the meat, Ashati and Neram imparted to Bomba
news that he was anxious to hear.
In their wanderings they had noted the bands of Nascanora and his
half-brother, Tocarora, heading in the direction of the Giant Cataract.
This had been only two days before and not far from the place where
they were now standing. From their hiding place in the brush, the
slaves had seen that each party had with it a number of captives.
“Was there a white man among them?” asked Bomba eagerly.
“Yes,” replied Ashati; “an old man, very thin and with white hair.”
Bomba’s heart leaped. Casson then was still alive! He had not
succumbed to the hardships of the journey.
“Can you take Bomba to the place where you saw them and point
out to him the way they were going?” he asked.
“Yes,” replied Ashati. “But they must be a long way from there now,
for they were going fast. And they kept looking behind them as
though they thought men were coming after them.”
The news set Bomba on fire with impatience to be off. Under his
urging and example, the roasting of the meat was swiftly completed.
He divided the food equally between the three of them. Whatever
might happen to them in the future, it was certain that they would not
starve.
After they had got fairly started, with Ashati as guide, toward the spot
where the path of the slaves had crossed that of the savages,
Bomba narrated to his companions some of his experiences since
he had left the hut of Pipina.
When he came to the part that concerned the loss of his bow and
arrows, Ashati insisted that the boy should take his and that he,
Ashati, could do very well with his hunting knife. Besides, he would
make another set of weapons at their first resting place.
Bomba would have refused, but seeing that he could not do so
without hurting the feelings of the devoted fellow, at last accepted the
gift.
“It was an arrow from that bow that found the heart of the jaguar that
would have sunk its claws in your flesh and ripped it from the bones,”
said Ashati, as he handed it over.
“Bomba has not asked you yet how it was you came just in time to
save his life,” said the lad.
“We hunted Bomba day and night,” Ashati replied. “Our lives were
yours, for you had saved them. We came on your trail in the jungle,
and followed after. Ashati saw you in the tree as the jaguar lifted its
paw to strike. Then Ashati prayed to the Spirit of the Jungle and shot
his arrow. The Spirit made it go straight, and the jaguar died.”
It was now late afternoon, and Bomba and his followers had not
traveled far before the swift tropic night descended on them and
forced them to rest for the night.
Bomba chafed at the necessity, though he himself was almost
exhausted in mind and body by the stirring events of the day that
had taxed both to the utmost.
He was up with the dawn, however, and, rousing Ashati and Neram,
summoned them to share his hasty breakfast and start on the day’s
journey. They obeyed with willingness, though no more than half
awake. Their bodies wasted and their strength sapped by years of
deprivation and torments at the hands of Jojasta could not throw off
the fatigue as readily as the healthy jungle lad, whose veins were
pulsing with vitality.
But Bomba’s quest of Nascanora could not wait. Delay, however
slight, might result in the death of his friends, if indeed they still lived.
If Ashati and Neram could travel at Bomba’s pace, he would be glad
to have them with him, for they were companions in his loneliness
and allies in case of danger.
But if they could not keep up with him, he would have to go on
ahead, leaving them to join him when they could.
But once roused, they seemed as eager as himself to continue the
journey. Even the prospect of an encounter with Nascanora and his
braves did not deter them, as long as they were under Bomba’s
leadership. What they had already seen of him had led them to
attribute to him almost magical power. Their anxiety to please and
serve the lad in every way they could deeply touched Bomba, in
whose life loyalty and service of any kind had been so sadly lacking.
They stamped out the embers of the fire they had built to keep off
the jungle beasts during the night, ate of their supply of jaguar meat,
and struck onward through the forest in the direction of the Giant
Cataract.
The sun rose higher, and with full daylight came a fresh burst of
speed on the part of Bomba. If Ashati and Neram found it hard to
keep up with him, they did not murmur. They would have suffered
any hardship rather than be left behind by him whom they had
chosen as their master.
They traveled all day without meeting with any unusual adventure,
pausing only briefly at noon to roast some jaboty eggs they found in
the forest that gave a welcome variation to their meat diet.
The shadows were beginning to gather when they came at last to the
spot where the paths of the slaves and the two parties of
headhunters had crossed.
The trail of the Indians was cold, but it was not difficult for one so
versed in woodcraft as Bomba to pick it up. There was still a little
daylight remaining, and he persisted in utilizing every moment of it to
gain another mile or two before he called a halt for the night.
On and on they went, although by this time they were nearly
stumbling with fatigue. They were penetrating a part of the jungle
that was new to Bomba. Pools, swelled by the recent rain, were
frequent, some of them so deep that it was necessary to cross them
by notched trunks of trees, the crude bridges of the jungle.
Crossing one of these, Ashati, wearied almost to fainting, stumbled
and would have fallen had not Bomba seized him and dragged him
to the safety of the further bank.
They had gone but a few yards farther, Bomba’s eyes straining to
detect the faintly marked trail, when there was a thud, and on the
ground before them, directly in their path, appeared a figure so
grotesque in form and ugly in face that Bomba took a startled step
backward and the two slaves fell to the ground in a fit of shuddering
terror.
“The mad monkey!” chattered Neram, and then, as the creature
advanced on them, uttered an ear-piercing shriek.
Gibbering and mouthing ferociously, froth slavering from its jaws, the
huge ape sprang toward Bomba and the cowering slaves.
Bomba was paralyzed at first by the hideous appearance of the
beast and infected to some degree with the superstitious terror that
animated Ashati and Neram. He seemed bereft of the power of
movement.
Then gathering together his forces, he sprang backward swiftly and
fitted an arrow to his bow.
 CHAPTER XIV
BESET BY ENEMIES

Even as Bomba drew his arrow to its head he found that he felt a
strange unwillingness to inflict injury on this antagonist.
The monkeys were his friends. Often they had helped him when
other foes, much more like Bomba in form and appearance, had
sought to take his life. He could not forget how the swarm of
monkeys had turned the tide of battle in his fight with Nascanora and
his braves when they had attacked the cabin.
So Bomba called to this slavering, hideous object in the language of
the monkeys, trying to show that he was not an enemy.
But if anything, the aspect of the ape became still more fierce and
threatening. It uttered a shrill cry and sprang at the lad with hairy
arms outstretched to grasp him.
Ashati and Neram gave a shout of warning, and seeing that their
young leader was in grave danger, conquered their fear and sprang
to his help.
The bow of the jungle boy sang with a twanging sound as the arrow
sped from it. But the attack of the monkey was so swift that the
arrow, instead of entering the heart as Bomba had intended, pierced
the flank of the brute.
With a howl of pain and rage, the great ape plucked out the arrow,
and swung around upon Bomba with the bloody point of the weapon
upraised.
But Bomba was quicker than the ape. He sprang aside and, drawing
his machete from his belt, struck the animal’s arm a blow that cut
deep and caused the blood to spurt into the distorted face of his
assailant.
The arrow dropped clattering to the ground, and with a weird and
terrible howl the ape swung itself with its uninjured arm into the
branches of a tree and vanished into the depths of the jungle.
Trembling, Ashati and Neram faced Bomba in that uncanny twilight.
For a while they said nothing, but stood staring solemnly at each
other.
It was Bomba who first broke the silence, and in the deep stillness of
the shadow-filled jungle his voice sounded hollow in his own ears.
“The mad monkey! If he is alone, all will be well. But if there are
others——”
“There are others,” interrupted Ashati. “One mad monkey needs but
bite another, and that one too will become mad. It is in that way the
evil spirits get possession of a flock of monkeys and set loose a
thousand demons upon the jungle.”
“And the bite of one sets loose an evil spirit within ourselves, and we
become even as the mad monkeys,” said Neram, his teeth
chattering.
Bomba knew that he spoke truly. A kind of hydrophobia would
sometimes be communicated by a snake-bitten monkey to its mates,
and by them to any human being that came within their reach while
the epidemic was raging. He had known of whole flocks of monkeys
having been decimated before the terrible disease had run its
course. And at such times there was no inhabitant of the jungle to be
so much feared as a mad monkey.
“We cannot stay here,” said Bomba, looking about him. “We must go
on. Perhaps we shall find shelter, a cave or an abandoned hut of a
caboclo, where we can spend the night and leave this terrible place
with the daylight.”
“Yes, we must go on,” agreed Ashati, and Neram nodded his head in
agreement. “To stay here would be to bring that hurt monkey back
for his revenge.”
“With good fortune we may escape the notice of others of the flock,
since it is night and they may sleep,” suggested Neram.
“Then we must make no more noise than the foot of the jaguar,”
warned Bomba, and, turning, he sped silently and swiftly from the
place, followed closely by his companions.
They had proceeded only a short distance when a horrible chattering
overhead caused them to look up, and in the branches of a tree they
saw two big apes gibbering and grinning at them, with the same
awful look in their eyes that had marked the first one they had
encountered.
As the little party moved swiftly on, a large castanha nut struck
Ashati on the shoulder with such force that he was felled to the
ground.
Bomba lifted him to his feet and hurried him onward just as a
bombardment of the heavy missiles came pelting down. A hideous
wailing and a sharp, crackling sound like a crazy laugh followed the
three fugitives as they raced onward.
By this time, superstitious terror had taken complete possession of
Ashati and Neram, and Bomba himself could feel the hair rising on
his head. The swift-falling darkness, the knowledge that danger was
all about them, that insane enemies were skulking perhaps behind a
tree, leering at them from branches overhead, crouching in ambush
behind a concealing bush or shrub, at any moment to reach out a
hairy arm—all these things combined to fill them with terror.
Their flight was unreasoning. They plunged through thorn bushes
that tore at their flesh, and felt no pain. They stumbled and fell into
black ooze that might hold writhing snakes, and scarcely thought of
it. To put distance between themselves and this nightmare became
their only aim.
Once a terrible figure dropped upon the neck of Ashati from the
branches above. The native gave a dreadful shriek and threw
himself upon his face.
The mad beast catapulted from the shoulders of Ashati and fell at
the feet of Bomba. It was the work of a moment for the lad to sink his
machete to the handle into the hairy body. The thing crouched as
though to spring, then gave a ghastly screech and sprawled upon
the ground.
“He is dead?” asked Neram, coming forward, unbelieving.
At the words, the prostrate Ashati raised himself on hands and knees
and crawled over to where the lifeless brute was lying.
“Dead!” he gasped, and dragging himself to his feet stared hard at
Bomba.
For it was a superstition among the natives that a mad creature was
possessed of an evil spirit that made it immune to death. The fact
then that Bomba had killed one of the mad monkeys so easily filled
them with surprise and hope. If he had killed one, he might kill many.
Their weapons were not as powerless as they had feared.
But their relief was swiftly changed to fear by a strange, weird
rustling among the trees that swelled in volume as they listened, as
though a great storm had entered the jungle and was sweeping all
before it. But there was no storm nor sign of one.
“The mad monkeys!” whispered Bomba. “They have told each other
that we are here. They know that we are few and they are many.
Come!”
If their flight had betrayed panic earlier, it was nothing to the fear that
now gave wings to their feet. They might hold their own against a
few. They could not face an army, such as, from the sounds, now
seemed to be in pursuit of them.
But even then Bomba had no hate against his pursuers, such as he
felt toward jaguars and snakes. He pitied them as the suffering
victims of a terrible disorder. None the less, he knew that they were
bent on taking his life and that if it came to a combat it was a case of
kill or be killed.
There was but one hope, and that a slender one; the hope that they
might find some cave or other shelter in which they could barricade
themselves and hold the maddened animals at bay. He knew of no
such place in that vicinity, and even if he had, it would have been
difficult to find it in the dark.
Ashati and Neram, with what breath they had, were muttering
prayers to the Spirit of the Jungle. If ever help was needed, it was
needed then.
Onward they plunged through the black night of the jungle, that
terrible rustling as of a mighty wind coming closer and closer with
every moment.
As the pursuing monkeys drew closer to their prey, they began to
howl and jabber horribly. It seemed to Bomba that the whole jungle
was one hideous jangle of sound.
Neram screamed. A hairy arm had reached from a low-hanging
branch, encircling his neck. He struck at it with his knife and
stumbled after Bomba and Ashati.
They were panting, spent. In another moment that awful swarm of
maddened beasts would descend upon them.
Stumbling blindly on, Bomba felt his foot slip into a hollow at the
same time that his body struck violently against a hard substance.
He stretched out his hands and felt rather than saw in the darkness
that he had come to the entrance of a cave.
“Quick!” he gasped, stepping back and nearly falling over the
cowering form of Neram. “Into the cave! Quick!”
 CHAPTER XV
LYING IN AMBUSH

The two slaves seemed not to hear Bomba.

Perhaps they were half dead from fright, or perhaps they thought
that the malady that had attacked the monkeys had disordered
Bomba’s mind as well.
Despite his command, they continued to crouch stupidly close to the
ground, striving to keep out of reach of the clutching hands that
swung from the branches overhead.
One great ape dropped to the ground and came swiftly toward them
on all fours, uttering howl after howl, so wild and eerie that it froze
the blood in the veins of the adventurers.
Finding that the slaves were too terrified to heed his orders, Bomba
seized Neram, who was nearer to him, and flung him bodily through
the opening of the cave. He took Ashati by the arm and dragged him
forward, shouting:
“The cave! The cave! Do you hear what I say? Get inside quick, and
I will follow.”
From inside the cave Neram reached out a hand and drew Ashati
within the sheltering blackness.
As Bomba was about to follow, the monkeys, following the
leadership of the great ape that had begun the actual attack,
dropped to the ground and swarmed toward Bomba.
The boy leaped for the gaping mouth of the cave just as the first of
the monkeys grazed his arm with its paw.
Neram and Ashati dragged him further into the depths of the cavern,
while, with a shriek of rage, the baffled monkey, seeking to follow,
dashed instead into the rock surface at the side.
Bomba stubbed his foot, bruising it as he fell forward into the cave.
He stooped down to examine the object and found that it was a great
rock.
“Come, help me!” he cried to the two slaves, who were fully awake
now to the fact that there was hope of escape.
They stooped to aid him, and as the monkeys recovered from their
surprise at the sudden disappearance of their enemies and began to
search out the entrance to the cave, those within rolled the great
stone to the entrance, fairly blocking it.
While they held that stone in place they were safe.
Bomba and his companions sank down on the ground, gasping for
breath, and pressed their weight against the rock.
The weird howling of the enraged monkeys outside their shelter
made them tremble even now, lest their cunning foes should find
some other entrance to the cave and come pouring in to indulge in
massacre.
But as time passed and they remained unmolested in their retreat,
while the awful howling of the monkeys died down to a mere jumble
of sound, they began to take courage and hope that all would still be
well with them.
Bomba had feared at first that the cave might be the lair of some wild
beast of the jungle.
But the fact that the stone was so near the mouth of the cave and
was evidently intended to block up the entrance seemed to argue
human occupation. Some native, perhaps, had become an outlaw
from his tribe and had chosen the cave as his home.
When it became certain that the monkeys had tired of their quest
and were straggling off again into the jungle, Bomba ventured to
make a cautious tour of the cave to make sure that his guess had
been correct.
Even then he was afraid that at any moment his fingers might touch
something warm and alive but not human.
However, the cave was a small one, and he soon found, to his great
relief, that, save for Ashati, Neram and himself, it was unoccupied.
Tired beyond words, but jubilant at their escape, Bomba returned to
the two, who still squatted on the ground close to the stone that
guarded the entrance to the cave.
Ashati was inclined to indulge in prophecy.
“The Spirit of the Jungle is good,” he stated, as Bomba threw himself
down beside them. “Twice it has saved the life of Bomba. The
captives of the wicked Nascanora and his half-brother Tocarora will
be spared. Bomba will live to seek out Sobrinini, and find from her
the secret that the poor old white man cannot tell. It is for that that
the Spirit of the Jungle has twice saved the life of Bomba.”
“Ahma,” said Neram, which meant “so be it,” as he bowed his head
reverently.
It was not long before all three were asleep, stretched on the ground
against the great rock, so that the slightest push on it would be
certain to rouse them.
But they were undisturbed all through the long hours of the night,
and at dawn awoke, refreshed and ravenous for the great chunks of
roasted jaguar meat they had prepared the day before.
They ate quickly, listening meanwhile for any sounds from the jungle
which might warn them of a further attack on the part of the mad
monkeys.
But, aside from the ordinary jungle noises, everything was as quiet
as they could wish. Unless they were still slyly watched by their
enemies, their escape from the cave should be a matter of no great
difficulty.
Nevertheless, both Neram and Ashati were reluctant to leave the
friendly shelter of the cave, and urged that Bomba rest there another
day and night until all danger should be gone.
But Bomba would not listen to this.
“You stay,” he said. “But Bomba must go. If, as you say, it was the
Spirit of the Jungle that twice saved his life yesterday, the Spirit
might be angry if Bomba were afraid and perhaps the next time
would not save him from the hairy arm of the great ape or the sharp
claws of the jaguar. No, Bomba must go.”
So, seeing that he was in earnest and thinking that if the lad were
under the protection of the Spirit of the Jungle they would be also,
Neram and Ashati said nothing more and helped Bomba roll the
great stone from the entrance to the cave.
“Where you go we will go,” said Ashati gently, as the welcome
sunlight streamed in. “Though you will not have us for your slaves,
you are our master. Let Bomba start and we will follow.”
Making no sound, they went swiftly through the jungle and did not
pause or stop to rest until they had put a great distance between
them and the scene of the fantastic nightmare adventure of the
evening before.
When the sun was high above their heads and all the jungle seemed
to fry and crackle beneath the heat of it, Bomba and his companions
sat down to eat the last of the jaguar meat and some berries and
nuts they had gathered by the way. Farther back they had found a
stream of clear, cool water, where they had slaked their thirst.
They resumed their route and had not gone far before they heard the
sound of rushing waters. The sound lent wings to Bomba’s feet, and
the faithful slaves kept pace with him, no matter how fast he went.
They came out soon on the banks of a river. The noise of the
foaming waters had been growing louder and louder until now it
smote upon their ears like thunder. A torrent of black water dashed
along the river bed and leaped angrily against the rocks that studded
its course, flinging a shower of spray upon Bomba and his
companions where they stood at the edge of the fringe of trees that
bordered the river.
“The River of Death!” muttered Ashati in hushed tones. “It is so that
our people call it.”
“And it is well named,” said Neram, making a cabalistic sign as
though to ward off evil.
“Beyond the River of Death,” said Bomba in a voice of impatience,
“though so far away that the eye cannot see it, is the Giant Cataract.
Come, let us be quick. Nascanora and his braves cannot be far
away.”
They started again, following the course of the stream. Suddenly
Neram paused with his head to one side as though he were
listening.
“Hark!” he said, when Bomba would have questioned him. “Someone
comes.”
As soon as the words had fallen from his lips, Bomba threw himself
upon the ground and put his ear to it.
Instantly he was on his feet again, drawing Ashati and Neram within
the shelter of the trees, which at that point were growing in great
profusion.
“We will watch as silently as the jaguar watches for his prey,” he
hissed. “Let no one move or make a sound.”
Hidden by the rank marsh grass that formed a perfect covert, the
three lay motionless, not a breath betraying their hiding place.
For what seemed a long time to the intent watchers, no one
appeared in sight. For all that could be seen, the jungle was free
from all human presence and given over to its animal inhabitants.
Unseen himself from the bank of the river, only a few yards away,
Bomba could yet have a perfect view of any living thing that might
pass by. Absolutely still, as motionless as though he were an image
of stone, his eyes alone moved to show that he lived and breathed.
The faint sound of footsteps that they had heard gradually became
more distinct, and at last a solitary traveler came into view. The man
was an Indian, but of a tribe with which Bomba was not familiar. He
was journeying swiftly when those concealed in the long grass
caught their first glimpse of him, glancing again and again over his
shoulder as though he feared pursuit.
Motioning to Neram and Ashati to remain silently where they were,
Bomba wriggled through the underbrush as sinuously as a snake.
Not a motion of the grass betrayed his presence.
Then, without a sound, as though he had risen from the heart of the
earth, Bomba leaped to his feet directly in the path of the
unsuspecting native.
The fellow started back and opened his mouth to yell, but before a
sound could issue from it Bomba had clapped one lean, brown hand
over his mouth and with the other had thrown him prone on the
ground.
Kneeling on the chest of his captive, who was too paralyzed by the
suddenness of the attack to make more than a feeble resistance,
Bomba called softly to Ashati and Neram. As though, like Aladdin, he
had conjured them by the rubbing of a lamp, they were instantly at
his side.
At their unexpected appearance, the feeble resistance of the native
ceased altogether. He seemed to be stupefied with terror, and stared
from one to the other of his captors with red and watery eyes that
begged for mercy.
“We will not hurt you,” said Bomba, taking his hand from his
prisoner’s mouth while at the same time he motioned Neram and
Ashati to grasp the fellow, should he seek to break away. “Only tell
us what you know and you shall go free. But lie to us,” he added,
with a significant motion toward his machete, “and you die!”
 CHAPTER XVI
THE ISLAND OF SNAKES

The native writhed and twisted, the eyes bloodshot with terror at the
threat.
Ashati and Neram came a step nearer with menace in their eyes,
and the captive subsided again, gasping and groaning beneath the
pressure of Bomba’s knee upon his chest.
“Do not kill me! Only do not kill me!” he jabbered. “I will answer you!
You have only to ask.”
“Then listen to me, and listen well.” The fierce, impatient note was in
Bomba’s voice again. “Nascanora, the great chief, the chief with a
black heart, is the man I want to find. Give me news of him and you
shall go free.”
A gleam of hope came into the prisoner’s eyes.
“Yes, yes, I will tell. I will tell you anything I know. But you must not
let Nascanora know that I have told, or he will cut my heart and fry it
over a fire.”
“Nascanora shall not know,” broke in Bomba, as he lightened
somewhat his pressure on the man’s chest and bored him through
and through with his eyes as though to wrench the truth from him.
“But if you speak with a forked tongue your place will be with the
dead.”
“I will make straight talk,” asseverated the native. “I do not want to
die.”
“Where is Nascanora? Quick!” demanded Bomba.
“In two days’ time,” replied the trembling native, “he will pass this
spot on the other side of the river.”
“I thought he had already passed,” said Bomba.
“He has,” returned the native. “But he turned back to burn another
village and take some more captives. He will move slowly, for he has
other enemies that he wants to capture near Snake Island, where
lives the old witch, Sobrinini——”
Bomba cried out in astonishment and quick hope.
“You know Sobrinini?” he asked eagerly.
The native marked his change of tone, and seemed encouraged by
it. His terror abated, and he spoke so swiftly that the words stumbled
over each other and Bomba could scarcely make out what he said.
“I know of Sobrinini. Who does not know of her who lives near the
Giant Cataract?” he cried. “But I do not go near her island, for it is full
of snakes and Sobrinini is a woman of evil whose frown means
death.”
“Where is this island?” Bomba asked in a fever of eagerness.
“It is called Snake Island. But to go there is to die.”
The native shivered with a superstitious terror as he spoke the
words.
Bomba glanced at Ashati and Neram, who had been listening with
absorbed interest to the story of the native and on whose faces was
reflected the same look of fright.
Bomba released his hold upon the prisoner and arose to his feet.
“There are two things I must know,” he said.
“Command, and you shall be obeyed,” returned the native humbly.
“Was there a white man among Nascanora’s captives?”
“I cannot say. He had many captives and he will return as I have
said. That is all that the jungle has told me.”
“How do you go to this island where the witch woman, Sobrinini,
lives with her snakes?” asked Bomba, feeling that the native had
spoken the truth.
The native looked at him with curiosity dawning in his reddened
eyes.
“You will not go there?” he cried in horror. “I tell you it would be better
for you to go to the giant anaconda and let him wind his coils about
your body than to seek out Sobrinini on her island that lies under the
curse of the gods. I tell you again that to go there is to die.”
“That is for me to say,” replied Bomba. “I do not fear Sobrinini. I do
not fear her snakes. I have come a far way to see her and I will see
her. Tell me what I want to know. Bomba does not like to ask a thing
twice.”
In response to this imperative demand, the native jabbered out
directions, telling Bomba the course he must take to reach Snake
Island if he did not wish to be swept to destruction by the rapids of
the River of Death.
When Bomba had extracted all the information he could, he
motioned the native to rise to his feet.
“Listen!” he said. “If I were Nascanora I would kill you, so as to be
sure that your tongue would be still. But Bomba’s heart is not black
like that of the chief of the headhunters. I am going to let you go free.
But if you tell anyone that you have seen me you will find that
Bomba’s knife is sharp and his vengeance is swift. Go now and
remember what I say.”
After the man had disappeared, Bomba wasted little time in
discussing the matter with his companions. For if he were to
accomplish all he hoped to before Nascanora should pass that way,
he would have to work hard and fast.
He was in a ferment of eagerness to visit Sobrinini and wrest from
the strange woman the secret of his birth. He was sure she knew.
Jojasta had said she could tell him. Casson’s queer actions when the
name of Sobrinini had been mentioned and his statement that “Nini
would know” had further confirmed his conviction.
As for the terrors that, according to the native, barred access to her,
he cared not at all. He was used to meeting and overcoming danger.