Life at the Cell and Below-Cell Level. The Hidden History of a Fundamental Revolution in Biology
by
Gilbert N. Ling, Ph.D.
Pacific Press
2001
ISBN 0-9707322-0-1

"Dr. Ling is one of the most inventive biochemist I have ever met."
Prof. Albert Szent-Györgyi,
Nobel Laureate

Chapter 6.

Colloid, the Brain Child of a Chemist
(p. 29-34)

The discovery that cells are made of protoplasm, a finding of critical im­portance to cell physiology, was promptly seized upon and pursued with vigor and insight—not by a cell physiologist but by Thomas Graham (1805-1869), a chemist. Graham was at the time Master of the Mint of England,53 p 183 an office once held by Isaac Newton toward the end of the 17th century.464 p 229

6.1 Colloid, the name-sake of gelatin—and  cogent model for protoplasm

Thomas Graham spent most of his life investigating the phenomenon of diffusion. He noted that substances like starch, gum and gelatin diffuse slowly and that they do not form crystals. Graham wrote in 1861: "As gelatine appears to be its type, it is proposed to designate substances of the class as colloids (from Greek κδλλη: glue, or gelatin, added by GL), and to speak of their peculiar form of aggregation as the colloidal condition of matter."53

 In naming the class of slow-diffusing substances colloids, Graham pointed out that "the plastic elements of the animal body are found in this class." By plastic elements he could only be referring to the soft tissues (in contrast to rigid elements like bones, horns, shells). These soft tissues are, according to Felix Dujardin, Hugo von Mohl, Max Schultze and others, made of protoplasm—even though Graham did not use this word.

 However, by introducing colloid chemistry, Graham not only opened the door to investigating protoplasm from a chemical point of view, he also gave impetus to the deeper understanding of cell membranes. Thus Graham also invented dialysis, using a colloid-"sized" (stiffened) membranes for separating colloids from water and from substances readily dissolved in water, including salts and sugars. Unlike colloids, salts and sugars diffuse much faster and do form crystals. For them, Graham gave the collective name, crystalloids.

 In 1857, Michael Faraday introduced to the Royal Society a substance he called colloidal gold. (Interestingly enough, four years before Graham was to introduce the new word, colloid.54 p 472) Faraday showed that colloi­dal gold solutions are (like all other normal solutions) totally transparent "when a light is looked at through the fluid." However, "if a cone of sun's rays be thrown by a lens into the fluid, the illumination of the particles within the cone shows their presence as undissolved bodies."54 p 472 This phenomenon is known as the Tyndall phenomenon, on the basis of which, an optical instrument called the ultramicroscope was constructed.64 p 87 The ultramicroscope enables one to see otherwise invisibly small colloidal par­ticles.

Martin Fischer, whose important contributions to cell physiology will be reviewed below, defined colloids in these words: "colloid systems result whenever one material is divided into a second with a degree of division coarser than molecular."64 p 5 Ross Gortner, whose important work will also be presented below, offered a modification: "colloidal systems result where one material is divided into a second with a degree of subdivisions either (a) coarser than molecular or (b) where the micelles exceed 1-1.5 millimicra (10-15 Ǻ) in diameter." Gortner further pointed out that an ul­tramicroscope makes visible colloid particles from 10 Ǻ to 1000 Ǻ in di­ameter.64 p 5 Wolfgang Ostwald set the limits of colloids between 10 Ǻ and 10,000 Ǻ.65 p 24 But another definition given by H. Staudinger poses a special problem.

 Staudinger believed that only molecules larger than 1250 A can be considered true colloids (or eucolloids)65 pp 23-24; 66 He also regarded colloids and macromolecules as synonymous. However, he was also the author of the macromolecular hypothesis, in which macromolecules are long chains of repeating units or monomers joined end-to-end by covalent bonds.67

 Yet well-known colloids like Faraday's colloidal gold and Traube's copper ferrocyanide gel are not macromolecules but big aggregates of smaller units. They are definitely not small units joined together by cova­lent bonds. Thus notwithstanding the wide practice to equate the two, col­loids and macromolecules are not the same. I shall return to this subject below in [11.3(2)].

By inventing colloid chemistry, Graham has brought together two sub­stances outstanding in the history of cellular and subcellular physiology. They are copper ferrocyanide and gelatin. We already know how copper-ferrocyanide had launched the membrane theory. In the next section I shall review how colloid chemists have discovered more and more intimate rela­tionships between gelatin and protoplasm. However, as will be made clear in [11.3(2)] following, the time to construct a (plausible) theoretical expla­nation for this intimate relationship was not to come until much later.

6.2. Coacervates

(1) History

In 1902, Pauli and Rona added neutral salts to a solution of gelatin at 30°C and observed the separation of the solution into two distinct layers. The bottom layer is rich in gelatin; the top layer is gelatin-poor.58 In 1929 Bungenberg de Jong (1893-1977) and H. Kruyt coined the term coacervation for the phenomenon (from the Latin acervu meaning aggregation and the prefix ñî, meaning together).59 Coacervate is also used to designate the colloid-rich phase of the separated liquid. In the case where salt-linkage formation between fixed anions and fixed cations of the colloids plays a significant role in the colloid structure, the coacervate is referred to as a complex coacervate.

Colloid chemists in the past had called all proteins colloids. With this in mind, one may think that all proteins can form coacervates. This is not true. Only what Bungenberg de Jong called linear proteins such as gelatin form coacervates.61 p 185, p 239 Under conditions that promote the formation of co­acervates from linear proteins, most globular proteins form crystals in­stead. This is a very important distinction to keep in mind. Most isolated native proteins are globular. Gelatin is therefore unique or almost unique in maintaining on a permanent basis a linear, or what I call fully-extended conformation [11.2]. Why does gelatin assume and sustain such a fully-extended conformation—a hitherto unanswered question—was given a possible explanation on the basis of new knowledge unknown until re­cently. And it will be reviewed in [11.3(2)] to follow.

(2) Bungenberg de Jong's two views on the physical state of water in coacervates

Bungenberg de Jong offered not one, but two theoretical interpretations for the structure of coacervates and the physical state of water in the coacervate. In the old interpretation, individual small colloidal particles with a diffuse solvate coating first join together into larger particles with a clear boundary during the preparative process. When these larger particles in turn join together to form coacervate, their individual solvation shells merge to form an overall shell with a concrete outer boundary (though no explanation was given why a concrete boundary is formed). Note that in this (old) model, all or nearly all of the water in a coacervate is not normal liquid water but hydration water61 pp 245-246, p 249

However, the new interpretation of coacervates, in Bungenberg de Jong's own words, "stand(s) diametrically opposed to this (old) original idea" of water in coacervates just described. Indeed, in the new model, "by far the larger part (of water in the coacervate) is to be regarded as occlusion-water61 p 249 which is "Not bound to the macromolecules,"61 p 371 and therefore normal liquid water caught in between the network of macromolecules. This new definition leaves one with the impression that there is minimal interaction between the macromolecules and water in a coacervate—quite the opposite of the old model. Fortunately, Bungenberg de Jong and his coworkers have also left puzzled readers like myself some quantitative data, which permits a deeper look into the subject.

Holleman, Bungenberg de Jong and Modderman studied the equilib­rium distribution of sodium sulfate (Na2S04) in a simple coacervate of gelatin + Na2S04 at 50°C.70 At a gelatin concentration of 27.2%, the con­centration ratio of sodium sulfate in the coacervate water and in external solution is 0.62. This partial exclusion of Na2S04 indicates that of the 1 - 0.272 = 0.728 or 72.8% of water in the coacervate, 1 - 0.62 = 0.38 or 38% has no solubility for sodium sulfate. Dividing the total amount of this water (equal to 0.728 × 0.38 = 0.277) by the percentage of gelatin, one ob­tains (0.277 / 0.272) = 1.02 grams of "non-solvent water for sodium sul­fate" per gram of dry gelatin. This figure is between 3 to 4 times larger than the conventionally accepted (total) hydration water on native globular pro­teins (i.e., 0.2 to 0.3 grams/gram of dry protein).155 Table 5 Nonetheless, there is also a difference between this realistic 38% "hydration water" and the 100% "hydration water" as implied in the old model. On the other hand, if the hydration water has higher than zero solvency or q-value for Na2S04 {see [11.3(4)]}, the departure from the old model could become smaller. We shall return to this interesting subject in [11.3(3)] below.

 

Figure 5. The step-by-step illustration of the in vitro evolvement of hollow vesicles with bilayered membrane and several outstanding examples A. Diagrammatic illustrations of the morphological development of the hollow sphere with bilayer membrane (g) from a negatively charged (solid) coacervate drop of the complex gelatin-gum arabic type (a), b shows primary vacuolation. ñ and d are in the form of foam bodies as they are progressing through successive stages toward the hollow sphere (g). B. Actual photograph of coacervate at the foam-body stage corresponding to stage ñ in A. (203 × lin.) C. Actual photograph of foam bodies in stage f. Note the double-layered wall in hollow vesicle marked a in this picture (as well as in the diagrammatic illustration g in A.) (196 × lin.) D. Partial reproduction of an electron micrograph of phospholipid vesicles reconstituted from pure renal (Na+ and K+-ATPase by the cholate dialysis method, (× 290,000). Diameter of vesicle close to 900 Ǻ. E. Electron micrograph of proteinoid "microsphere," subjected to elevated pH. Scale bar: 1 um or 1000 Ǻ. Note that membrane can be formed from gelatin + gum arabic (A,B,C), from (Na+, K+) ATPase + cholate (D), or from pure proteinoids obtained by heating mixtures of pure amino acids (E). The only common denominator among these three preparations is proteins of one kind or another. (A, B and Ñ from Bungenberg de Jong," 61 p 460 D from Skriver et al.,487 reproduced from The Journal of Cell Biology by copyright permission of The Rockefeller University Press; E from Fox216 by permission of Springer-Verlag)

(3) Coacervate and protoplasm

If one mixes in the right proportion gelatin and gum arabica highly water-soluble, large complex polysaccharide from Acacia trees60 p 98—and allows the mixture to stand, two layers also separate out. If the test tube containing the layers is shaken, the gelatin-rich coacervate breaks up into many little balls or droplets61 p438 (see also Figure 5A), which stay undis-solved in the surrounding colloid-poor phase.

In 1926 W.W. Lepeschkin reported that the protoplasm oozing out from broken (young) cells of the plant, Bryopsis plumosa can also be shaken and broken up into many little balls.62 p 75 They too stay undissolved in the sur­rounding aqueous medium. As judged by these strikingly similar charac­teristics, the gelatinous materials emerging from crushed protozoa by Felix Dujardin and Willy Kiihne and from broken plant cells by von Nageli, von Mohl, Lepeschkin as well as Kuroda (who produced Figure 3) must all be coacervates. This is not a new idea. Lepeschkin was among the firsts to suggest that protoplasm is a coacervate.324

(4) Coacervate and the living cell

In a review written for the journal, Protoplasma,71 Bungenberg de Jong cited nine similarities between coacervates and what he called a static model of the living cell, including (i) water immiscibility, (ii) tendency to form vacuoles, (iii) tendency to engulf solid particles, (iv) behaviors under the influence of a direct-current (DC) electric field. 

Bungenberg de Jong then pointed out that the most basic difference between the living cells and his static model lies in the possession of mem­branes in the living cells but not in the coacervates ("Der wesentliche Unterschied der lebenden Zeile gegemiber unserem statischen Modell bezieht sich wohl aufdas Vorhandensein von Filmen oder Membranene in ersteren, die grundsatzlich Ungleichgewicht ermoglichen.").71 p 164

Why Bungenberg de Jong made this distinction is a mystery because he himself has shown how, under the right conditions, coacervate can form membranes too (Figure 5A and 5C and legend). In a following section I shall present the work and ideas of A. S. Troshin, who saw perhaps even greater significance in Bungenberg de Jong's work on coacervates than Bungenberg de Jong did himself.

Ðàçäåëû êíèãè
"Life at the Cell and Below-Cell Level.
The Hidden History of a Fundamental Revolution in Biology":

Contents (PDF 218 Kb)
Preface (
PDF 155 Kb)
Answers to Reader's Queries (Read First!) (
PDF 120 Kb)
Introduction

1. How It Began on the Wrong Foot---Perhaps Inescapably
2. The Same Mistake Repeated in Cell Physiology
3. How the Membrane Theory Began
4. Evidence for a Cell Membrane Covering All Living Cells
5. Evidence for the Cell Content as a Dilute Solution
6. Colloid, the Brain Child of a Chemist
7. Legacy of the Nearly Forgotten Pioneers
8. Aftermath of the Rout
9. Troshin's Sorption Theory for Solute Distribution
10. Ling's Fixed Charge Hypothesis (LFCH)
11. The Polarized Multilayer Theory of Cell Water
12. The Membrane-Pump Theory and Grave Contradictions
13. The Physico-chemical Makeup of the Cell Membrane
14. The Living State: Electronic Mechanisms for its Maintenance and Control
15. Physiological Activities: Electronic Mechanisms and Their Control by ATP, Drugs, Hormones and Other Cardinal Adsorbents
16. Summary Plus
17. Epilogue 

A Super-Glossary
List of Abbreviations
List of Figures, Tables and Equations
References (
PDF 193 Kb)
Subject Index
About the Author

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