The Physiology of Transport Substances in the Blood (Sodium)


By Professor Marcel Uluitu, M.D. Ph.D.

Spiru Haret University

Bucharest, Romania


Co-Authored by  Diana Popa (Uluitu), M.D.

Department of Microbiolgy, Immunology and Molecular Genetics

University of Kentucky, Lexington, Kentucky, USA


[Editor’s Note: This paper is presented as Part I of a series of chapters from the new book “The Physiology of Transport Substances in the Blood (Sodium)”; subsequent chapters will be featured in upcoming issues of this Journal. This segment features Chapter One (of six chapters) and Section 1 of Chapter Two]



Measure what is measurable
and make measurable what is not,
   -- Galileo Galilei (from Ev.Z. 2008)




General mechanisms regulating functions: Body unity, control and coordination of its functions are achieved by means of nervous, humoral and physico-chemical mechanisms.
The nervous path is fast and turns on as a result of the analysis and control of the functioning level of organs and tissues.


The humoral mechanism regulating the functions of the body recognizes the transport of substances as a means by which it provides for the needs of every tissue metabolic activity, synthesis of own substances,catabolites removal, etc. The humoral pathway is enabled as a result of the analysis by the nervous system of the internal environment, through feedback mechanisms, of the level of normal concentrations of constituents of the body and their balance within the three spaces, including nutriments, new compounds synthesized in some specific tissues, catabolites, etc. Humoral pathway activation mechanisms depend on the role of these substances in the economy of the body (composition, physico-chemical structure, functional role, the energy structure of the body, etc.).

The physico-chemical pathway includes the body composition and the circulating and structural substances which interact by means of trace energy and have significance in the processes of transport in the blood. The physico-chemical route of regulation depends on the composition and structural-functional lability of proteins. These substances are in constant motion, ensuring a local and global uniformity. 

Transport of substances in plasma is in a state of trace energy interaction ionic, electrostatic interactions, Van der Waals forces, hydrogen bonds, London forces of dispersion, cation-pi interaction, etc. These chemical interactions decrease the activity of electrolytes and of other compounds. The result of these interactions is a ligand-carrier complex whose unbinding issubject to mass action law.     

Disorders in the substance transport mechanisms in the blood generate a special pathology whose recognized causes are deficiencies (anemia ,acidosis, etc.) ,genetic disorders (Wilson’s disease involving Cu),endocrine diseases involving Ca ,abnormal excitability neuromuscular disorder, behavioral-type constitutional disorders (involving the transport of sodium).


Chapter 1


The General Structure of the Body.

Transport of Substances in the Blood.

Sodium is a chemical element in Group I of the periodic table. Very widespread in Nature, it is at the same time the monovalent cation best represented in the blood. It is extensively studied in both kingdoms because of its association with multiple processes.

It is associated with metabolic and enzymatic processes acting as cell activator. It is mainly involved in the cell membrane functions. It is an extra cellular cation and is present more especially in blood. In the excitable cells, the action potential is also identified under the Name of sodium potential. It is an activator of the heart, in vitro. It has an important role in the electrical manifestations of the excitable membranes of cells; neurons, cell rods, excitoconductor cardiac tissue, etc.


At the synapse level it acts in conjunction with acetylcholine. A seriously low concentration of sodium is not compatible with life. Sodium blood homeostasis is assured by a very complex regulatory system starting with food intake and continuing with transport in the blood, sodium distribution, and elimination by the kidney, the digestive tract, the skin, etc. Such mechanisms are nervously and hormonally regulated (adrenal cortical, natriuretic hormone), osmoregulatory mechanisms, mechanisms of cellular and molecular emergency. In case of acute intake increased, sodium is retained temporarily in the connective tissues, interstitial tissue and fatty tissue in order to maintain sodium concentration.

In the cell membranes, it works through sodium ion channels interacting with the anionic sites of the proteins in their structure. Thus, the information conveyed by the cation depends on the local ion concentration and on the size of their ionic radii, varying according to the number of hydrating water molecules. Its action can be antagonised in the ion pore region of the Na channel, by neurotoxines.
Its passage through cell membranes depends on co-transporters that have, however, a small importance compared to the passage using the ion channel and gradient mechanisms. Sodium has an insignificant role in the genesis and maintenance of the resting potential. These processes depend on the restoration metabolism and on potassium. Na+ is not involved in the functioning of non-excitable membranes. It is a vitally important cation. One more reason for this is its value as a biological constant of the blood.

The regulation of sodium activity in the blood depends on its interaction with proteins. Sodium chemical activity determines the capacity of response of nervous structures by the generation of an action potential. The fraction of chemically active sodium in the blood highlights the role of physical and chemical interactions in the body. Determination of total blood sodium with classical methods (physical, chemical, etc.) after removal of macromolecular organic compounds has the same value as the basic analysis of the composition of living organisms. It is a sort of "Chemical anatomy." The physiological role of Na+ can be established only by determining its chemically active form while maintaining the full composition of plasma. There are multiple interactions among plasma constituents. Despite the small energy values of such interactions, their number is particularly important.

The method for determining the chemical activity of blood Na+ described in this monograph has as the reference system the energy of interaction, E, between serotonin (5-hydroxitriptamin = 5HT) and the polyanion heparin (Natural components of blood). If there is Na+ in the solution, whose interaction with heparin is accomplished by energy EE, the interaction 5HT-heparin no longer takes place and 5HT goes out of the dialysis pouch
The protein anionic groups present in the blood do not interact with serotonin, (as this is Naturally transported by platelets), but they diminish Na+ interaction with heparin in a similar manner with the one in the cation hydration process described in the text and induce E values that are lower than E, thus conserving the interaction of heparin with 5HT. The intensity of such interactions depends on the structure and function of protein in solution. Because Na affinity for heparin is limited by interactions with other plasma compounds (proteins), the amount of dialysable 5HT decreases proportionally to the number of molecules bound to heparin. Serotonin is not appropriate for a blood test because it is attached to platelets that are the amin Natural carriers. It was thus established that plasma Na+ has a very low chemical activity both in humans and in laboratory animals (rats). Individuals presenting such a picture have normal excitability.

In the normal groups of both species studied, there are subjects in which the chemical activity of cations (Na+ up to 95%) antagonizes the interaction between heparin and 5HT, the latter leaving the dialysis pouch.

Prof. Alexandre Monnier (biophysicist at the Sorbonne) said that throughout his entire life he strongly believed that Na+ is circulating in the blood in a state of interaction with proteins, but he never had a method to prove it. The statement is repeated in a letter presented in facsimile.




Individuals in whom Na+ antagonizes the reaction heparin/5HT present with increased neuro-muscle hyper excitability expressed in the form of behavior disorders of a constitutional type in humans, state of alert on EEG, poor concentrated attention, good distributive attention, disorders in the regulation of the cardiac-homodynamic function, while rats are prone to audiogenic convulsions, hyper mobility in open areas, exaggerated intake of Nail, mineral-corticoid function deficiency.

The body can be divided into interdependent compartments in point of transport mechanisms: INTRAVASCULARLY, INTERSTITIAL ENDOCELULAR.

This division suggests the existence of two major types of transport of substances: transport of the substance within the same space (blood, interstitial, intracellular) and another transport between the three COMPARTMENTS, across separator walls consisting of semi permeable membranes and epithelia. The intravascular space communicates through capillary walls with the interstitial space and the latter with the endocellular space through cell membranes. Transmembrane transport, well-studied in both living and artificial membranes takes place under multiple influences: blood pressure, osmotic pressure and colloid osmotic pressure, concentration gradients, electrochemical gradients, etc.

The term "space" includes multiple interdependent structures, having close embryonic origin. The embryonic origin of interdependence is preserved by the chemical composition whose origin is the secretion of macromolecules in the cells of every compartment. Macromolecules are secreted by vascular endothelial cells and by other host tissues as well. Thus, a local, complex functional structure is created. The structure acts in an integrated manner. This phenomenon is known to organize and include the three spaces. In each of the three anatomical structures the three spaces communicate among them. The chemical composition of the compartments is maintained constant within the general concepts of "internal medium" and "homeostasis". In this context, the segment medium is maintained constant by the transport of substances between the compartments. The substances in the three spaces have oligoenergetic interaction. The complex connection between compartments is mediated by semipermeable mentioned structures. The knowledge of the physical and physico-chemical force enables a more precise understanding of the fundamental state of structural elements and their functions. The following will deal with transport processes, mainly in the blood, with particular focus on the relationships between transport of Na+ in the blood and functional status of the excitable and unexcitable structures.



Chapter 2:


The Blood-Vascular Space

2.1. The Capillaries.

The vascular tree is made up of the arteries, the capillaries and the veins. The vascular wall (artery) is made up of three layers: the outer layer - tunica adventitia, the second layer - tunica media, consisting of elastic and smooth muscle fibers and, finally, tunica intima. Typically, the capillary wall is made up of only a unicellular layer of endothelial cells which is surrounded by a basement membrane on the outer side. At this level exchanges are taking place between blood and the extra vascular space. It is an active physical and chemical space separated by a cellular layer and an extra cellular layer having a restrictive selective action for interstitial and blood components. For every organ and tissue there are structural and functional features of the capillaries that control the local exchanges.

2.2. Blood.

Blood is the circulating tissue that irrigates all body cells. It circulates in a closed system at variable speed given by the activity of heart pump, the elasticity of vessels and size variations in different parts of the tree. Blood is composed of a cell system (red cell, white blood cells, platelets) associated with respiratory functions, antimicrobial defense, haemostasis and a liquid intercellular system where the cells are floating – the plasma. Between the dissolved substances in the blood there are chemical, physical interactions resulting in the particular distribution of such substances. One should note leukocytes and platelets contain 5-60 times more amino acids than plasma and erythrocytes. Serotonin is also virtually wholly carried on platelets (219,220)


The blood tissue communicates with the extra vascular interstitial space and through it with all body cells, sustaining them and participating in the regulation of their function. In their turn they affect the biochemical composition and physico-chemical properties of the blood. This maintains the chemical and physiological homeostasis of the inner medium within limits compatible with life. Blood plays a key role through its circulating function as a transporter and participates alongside the nervous system to maintaining, adjusting and adapting the body functions.

The total volume of blood is 5.5 liters in adults, of which 3.5 liters (55%) is represented by plasma, and 45% of volume contains blood cells (haematocrit). The volume of blood varies with sex, age. The haematocrit varies with physical effort, environment temperature, altitude, age of pregnancy, etc. The color of blood is red, with shades depending on its gas content and Nature: oxygen, carbon dioxide, carbon oxide- accidentally. Density varies with gender (1061 in men and 1057 in women) consistent with haematocrit values. Viscosity, the resistance against the flow by friction with the neighboring areas of blood components and the vascular wall has values of 4.7 to 4.4 in men and in women, respectively.

2.3. Blood plasma

Plasma represents the liquid phase of the blood. It is a transparent, slightly yellow solution wherein blood cells are floating. It is a complex solution with heterogeneous composition of inorganic and organic substances which are interacting among themselves, with the solvent (water) and the vascular wall and their components. The solvent and the compounds with low molecular weight are in balance with the substances of the same kind in the extra vascular space.


2.3.1. Composition of plasma
Table 1. The classification of different substances in plasma (17).

2.3.2 Plasma properties
Plasma is a real solution of crystalloids and soluble macromolecules. It has some characters of a colloidal solution conferred by macromolecules, expressed in that it does not pass through the membrane, the Tyndale phenomenon, without expressing a condition of colloidal type in solution. The macromolecular substances in plasma, in particular proteins, are amphoterious, having anionic and cationic groups which interact with ions. The chemical composition of plasma presents variations from one organ or tissue to the other according to their functional or anatomical structure and their role. Temperature

Organs with intense metabolic activity (permanently or occasionally) have intense thermo genesis and efferent blood temperature is higher. This increase in temperature affects the stability of plasma solution and therefore the physical properties of plasma. Blood temperature is also variable (37.7 C - 40 C) in various organs, depending on the intensity of the metabolic processes specific to their function (liver, brain) or on functional requirements (muscles). Density and viscosity.

The lower density of plasma (1027) is due to the protein. It is low in hypoproteinemia (hepatitis, nephropathy with intense albuminuria, inanition, consumptive diseases).

Viscosity is defined by Newton as resistance to slipping between adjacent layers, in the case of plasma between macromolecules and between the latter and the vascular wall surface. It expresses the forces of cohesion and adhesion that oppose blood flow and contribute to peripheral resistance together with the vascular factor. Viscosity value is 1.86. The viscosity coefficient”"depends on the concentration of proteins. The viscosity plasma is identified as "abnormal" or "structural" because viscosity is not subject Newton’s laws. Osmotic pressure

It is a colligative property (68) representing the force that the particles of the solvite are exerting on the vessel wall. The chemical composition of blood varies between certain limits in different regions in point of concentration of protein, gas (O, CO, etc.) H, various electrolytes, some catabolites resulting from the tissues activity, etc
Osmotic pressure is best seen in a semi permeable membrane that separates an aqueous environment (pure solvent) from the solvent solution. Its value is given by the number of particles in the solution, not their Nature. Osmotic pressure is described by Van t'Hoff equation:
 v = niRT, the
  ni = number of particles dissolved.
   = Osmotic pressure.
   R = gas constant.
   T = absolute temperature

Another property of a colligative solution depends on the concentration of particles in the solution. This property is the "freezing point" or "cryoscopy point."

Table. 2: Cryoscopic point value in different tissues (129).


Cryoscopic point


0,55C - 0,57C


0,59C - 0,62C

red cell








Osmotic pressure varies with the intensity of tissue metabolism. It is 6.7 atmospheres (matching cryoscopic point is 0.560 C) and is given by the concentration of particulate substances (dissociated plus non dissociated) in plasma: Na, K, Ca, glucose, etc.. Under normal circumstances this is given, up to 90% by Na. Osmotic pressure is a condition for cell division in the processes of excitability. Colloid-osmotic pressure.

The plasma colloid osmotic pressure (PCO) is produced, in addition to the substances mentioned by dissolved proteins. They do not pass through the capillary membrane. Plasma protein content is 2 -3 times higher than the interstitial content (7-8 gm vs. 2-3 gm). The contribution of proteins to promote PCO is unequal as shown in Table 3.

Table 3 The value of PCO induced by plasma protein fractions (78).


molecular weight























At the level of capillaries, local forces are acting that create a slight pressure imbalance according to Table. 4

                        Table 4 Average expulsion forces of the capillary liquid (78).

Average capillary pressure


Negative interstitial pressure

3, 0


8, 0

Total exit forces

28, 3

Average entry pressure in the vessel(PCO)

28, 0

Effective exit pressure from  capillary

0, 3
 DonNan’s equilibrium.
We have shown that biological membranes have low, selective permeability retaining proteins and allowing passage of inorganic substances (electrolytes, water) and of organic substances with small molecule.

The proteins with amfoter character but with a dominant negative charge can form compounds with cations of the R – Na+ type. These types of compounds prevent the uniform distribution of electrolytes on both sides of the semipermeable membrane in accordance with the laws of osmosis. This action of the resulting compound is proportional to its concentration and may even stop the passage of electrolyte when R concentration is very high. This special behavior of osmosis, induced by non-diffusible proteins is identified as "Donnan’s equilibrium", expressing the unequal distribution of ions on both sides of the membrane. This balance explains the quantitative differences of electrolytes between blood and CSF, between red blood cells and plasma, etc.    The decrease in protein content alters the allocation of water and electrolytes. The unequal distribution of undiffusible ions creates a difference in the membrane potential, and the unequal distribution of electrolytes explains the existence of membrane potential both at rest (resting potential) and in action (action potential.








2.3.3. The stability of plasma solution.
The solubility of macromolecular compounds and consequently of plasma compounds is preserved with no addition of stabilizer agents as required by colloids. The stability of the plasma solution is provided by water and by two other categories of factors: the chemical and the physical factors that condition its physiological properties Chemical factors of stabilization.

The solubility of proteins depends on their Nature and chemical structure, the solution pH, the degree of ionization, the ionic composition of the environment (salt concentration and Nature of salts), the dielectric constant of the environment, the ionic composition of the environment, including the type of side groups of protein. The role of the solvent: water is bound by polar interactions to the ionized groups and by the hydrogen bond to the peptidic bond. Water binding is minimal at the izoelectric pH of proteins.

Electrolytes in low concentration favor protein solubilisation and stabilize the solution. If the electric charges on the protein molecules are neutralized by the addition of salts, proteins precipitate by salefier (178). Electrolytes in high concentration destroy the water interaction with the protein molecule and separate them. Non-electrolytes may also dehydrate macromolecules through interaction with water and decrease the solubility of proteins through a process of pseudo-hyper concentration.

The ability of electrolytes to influence water dissolving power follows Hofmeister's liotropic series. (52):
                          Li> Na> K> Rb> Cs

The solubility of the proteins increases in solutions with high a dielectric constant (52) and in the presence of dipole ions.

An important role is also played by the attraction and repulsion forces existing on the surface of molecules in solution. In the two segments, arterial and venous, there occur composition changes of water, electrolytes, various other molecules that can cross the capillary wall as well as a result of loading with catabolites and other products of local secretion. Also at this level, there occur variations in the concentration of macromolecules retained by the capillary wall, as, for example, the increase by 20% of the  concentration of protein in the blood during the formation of primary glomerular ultra filtrate. H-ions concentration increases in venous plasma. Physical factors of plasma stability.

There are many physical conditions of stability. Among them one could list Brownian motion, continuous flow of blood, rheology factors depending on the type of blood flow in vessels, etc. The flow of a liquid through a closed system of straight, cylindrical tubes is laminar. The liquid is moving in parallel, immiscible layers alongside the vessel wall. Otherwise, the layers are mixed, form whirling areas where plasma is shaken. In these areas the lumen of the vessels is altered naturally (the valvular cardiovascular blood pressure, fast-growing areas of vascular bed, and under conditions of temporary exertion of some organs in the precapllary region), or in pathological conditions. An important physical factor is represented by the large specific surface of the solvite substances (relative to the surface, the particle size with an average of 3.5 mμ of a gram of substance). The surface grows even larger as a result of hydration (178).


2.3.4. Plasma functions (78, 136) are listed only:
  (A) - maintain the physical and chemical properties of blood.
  (A1) - maintain colloid osmotic pressure
. (A2) - monitor the exchange of electrolytes between plasma and interstice.
  (A3) - maintain blood viscosity.
  (A4) - maintain acido-basic balance and the pH of blood.
  (A5) - maintain blood volume.

B) - the body's defense mechanism and the immune response.
  (C) – own enzyme function: coagulation, fibrinolysis, etc..
  (D) - ensure common fund of proteins and amino acids and ensure balance with tissue proteins.
  (E) - transport function for amino acids, polypeptides, fats, carbohydrates, vitamins, minerals, electrolytes, hormones, bilirubin, metabolites, gas, etc.
  (F) regulatory function:
  (F1) - maintain the intra- and extra-vascular hydro - electrolytic equilibrium and general homeostasis.
  (F2) – ensure humoral unity of the body alongside the nervous path.

2.3.5. Plasma proteins. General information
          Proteins are chemical compounds with high molecular complexity and variation (1o-10 types of proteins in the living world) and special significance (69). The fundamental elements of proteins are amino acids, the peptidic bond, polypeptide chains and functional groups alongside the polypeptide chain. The polypeptide chain is composed of a skeleton consisting mainly of carbon chains of amino acids associated through peptidic bonds.
The chain variation is given by radicals "R" of amino acids. The uni-dimension chains are flexible as the covalent bond allows free rotation of the carbon atoms. This allows a spontaneous conversion to a three-dimensional structure typical of a specific sequence of amino acids. Spatial organization of constituents of proteins is achieved by noncovalent interactions, which also involve groups in the peptidic bond and other groups in the side chain of the catena: Coulomb interaction, Van der Waals'force, London's dispersion forces, hydrogen bonds, and some configuration of the polymers The hydrogen bonds are established following privileged directions imposed by the strongest direction to the colinearity of the three atoms involved in the link. Most proteins are spontaneously refolding after their prior unfolding (denaturizing), which shows that all the molecular information is contained in the sequence of amino acids.


The folding of the polypeptide chain is conditioned by the amino acid composition and the distribution of polar and unpolar groups (Table No. 11). They control the folding of protein molecules (160124,15,16,17). Hydrophobic sites are accommodated inside of the molecule thus avoiding contact with water and creating globulin structures. Hydrophilic polar side chains are distributed on the external surface where they can interact with water and other polar molecules. The hydrogen bond is important in keeping together the various segments of the folding molecule.

The additional covalent bonds between the chains and disulfuric bridges contribute to the stabilization of the three-dimensional structure of the extra cellular proteins. Molecular specificity results from the interactions between their various structural elements and the molecules in their environment. They are based, in addition to covalent bonds, on hydrogen bonds, phosphate bonds, saline bonds, etc.The distribution of various atoms on the surface of the molecule, makes each protein unique and able to interact with specific molecular and other surfaces and with some smaller molecules.

Each protein has different segments that are repeated in the macromolecule. The polypeptides, in the solution, take a unique spatial arrangement as a result of noncovalent interactions in the protein so that its energy could be minimal. Noncovalent bonds are established very quickly with no catalysts and they are effective by aggregation. Inside the protein molecule there is a dielectric environment which favors interactions, is associating to reduce contact with water, forming a large number of contacts unpolar. The structure of the protein.
Each species is unique protein in the primary structure and conformation, and the radicals compete to achieve the structure of tertiary and quaternary field. The complexity of proteins required recognition of several steps of organization structure (53,160): (1) primary, (2) secondary (3) tertiary, (4) and quaternary. The primary structure.
The primary structure defines the number and sequence of the amino acids residue, the totality of peptide bonds in the polypeptide chain. It expresses the message described in DNA. The primary genetic structure is unique and compatible with a specific function of the protein. If an amino acid is replaced with another one in the same group (conservative substitution) or from another group (unconservative substitution), homologous proteins appear. Molecular polymorphism defines the existence of multiple molecular structures for the same function in the same species.

 Inter-atom measurements of distances and angular values connecting the amino acids in the polypeptide chain established the following primary characteristic of structure:
- The peptide bond has the character of double bond by the conjunction of orbitals p.
- Free rotation around the peptide bond and carbon can take an equal footing in the plane of the bonds.
- Natural peptides in the configuration trans are more stable than in the configuration "cis"
 - the group = NH forms hydrogen bonds with the group C-O from other peptide bond to form NH ... O = CH. The secondary structure.
A polypeptide chain can exist in two configurations: and.  The helicoid- structure results from spinning of the polypeptide chain to the peptide bonds so that the groups O=C and  =N-H are adjacent and can form hydrogen bridges and helicoid repetitive units. A group =N-H forms hydrogen bonds with group O=C of the fourth residue of amino acid in the same chain of linear sequence. The peculiarities of the alpha-helix structure are determined by the bonds angles, interatomic distance, co linearity of the hydrogen bonds, series optical levogyrous. These features are: (1) With every amino acid residue the length increases by 1.47 Ǻ. (2) the helix pitch is 5.21 Ǻ and contains six amino acid residues. (3) diameter of the helical cylinder where the carbon atoms are located is 10.1 Ǻ. (4) the sense of rotation is from left to right. (5) radicals "R" of all amino acids are oriented towards the outside of the helix, (6) all =N- and =CO groups form hydrogen bonds. (7) polypeptide chain has the shape of a stick with a diameter of 10.1 Ǻ for 300 amino acid residues.

The residue of proline prevents the - spinning, and the chain is bent at an angle of 130. The residue glycocol (no side chain) confers fexibility interrupts the structure , and changes slightly the chain direction. Other amino acids: valine, isoleucine, treonine induce steric disturbances. Serine forms hydrogen bonds at alcoholic group and prevents the helix stability. Cysteine creates disulphur briges and a rigid polypeptide chain, hindering formation of the helix. The - structure has the shape of a folded sheet. In this structure the maximum of hydrogen bonds is achieved between CO and NH. The hydrogen bonds are intercatenary and polypeptides are arranged in the shape of sheets. The most stable configuration is the structure with antiparallel chains (chains are directed from the "N" terminal to the C terminal and the other vice versa).

Radicals "R" are arranged on alternating sides of the polypeptide chain. Between sheets there are hydrogen bonds connecting the "R" groups. Distances between the sheets are 5.7 and 3.5 Ǻ  alternatively. The structures are frequent in globular proteins, especially in immunoglobulins. A simple - structure is formed of a polypeptide chain bent on itself, forming two antiparallel segments identified as the -tower. Tertiary structure.

This includes the secondary structures as spatial units: domain and motifs. The packaging is noncovalent and by carbon interactions between the radicals "R" at C of the structure, or unorganized. The bonds are determined as follows: (1) hydrogen bonds between the OH groups of the amino acid residue tironyl, tirosyl, with amide groups of glutamyl and asparaginyl (2) ionic bonds between radicals with negative charge of lisinyl, arginyl, histidyl with and without polar radical charge (3) hydrophilic interaction between the residue unpolar amino acids valine, leucine, isoleucine, alanine, phenylalanine (4) links between the cysteine residue distant covalent regions of the molecule.

A polypeptide chain adopts the configurations  and  until it satisfies all the affinity of radicals "R" under steric conditions influences when the final conformation results, the best possible compromise of stable energy and space. Therefore, the decisive factor to obtain the tertiary conformation given is again its genetically induced primary structure whose primary spontaneous structure depends on the chemical composition. The plasma globular proteins have on polar groups on their surface. The unpolar radicals are included within the protein molecule.

The organization of the "domains" is a structural intermediate form between the secondary and tertiary structures. Because of this, it is identified as "secondary superstructure" or "superstructure domain." Domain "is a continuous piece of the primary structure of polypeptide chain, packed up as a functional entity with its own secondary and tertiary structure. The "domains" are part of the less organized part of the chain, allowing flexible dynamic segments of their field. "Domains" are packagings of -helix and - sheet, forming globular compact units. It contains a number of 50 - 350 amino acid residues. Protein may be constituted in a domain or more connected with each other through long chains of open polypeptides. Through these connections large molecules are formed, identified as “protein ensembles” or “protein complexes” in which the subunits are linked with a large number of noncovalent bonds. In the extra cellular environment they are often stabilized by disulphur bridges. So a "domain" of protein appears as a basic core of a protein which is composed mainly of a - sheet,-helix or both. These structures allow long hydrogen bonds that stabilize the inside of the molecule where water has no access to form hydrogen bonds with the oxygen or polar carbon with the hydrogen of peptide bond. Proteins can be formed through recombination of pre-existing polypeptides "domains" with supramolecular structures of complex type enzyme, protein filaments, membranes, etc. Domains are the underlying structure of proteins diversification. They are the basis of "analogous" proteins (different proteins with similar functions) and of "homologous" proteins (similar proteins with different functions). Quaternary structure.
This represents associations of protomers (polypeptide chains with their own structures: primary, secondary and tertiary) identified as "oligomers". The specific function of an oligomer protein is manifested only at the level of quaternary structure. The separated protomers are inactive.

The assembling of the protomers to oligomers is accomplished only when the contact areas are complementary and have a large number of atoms, and the bonds are close to the level where the Van der Waals are active. The protomers of homologue proteins of different species do not associate although they contain the information to be decoded. The interaction capacity explains the formation of supramolecular structures. Perturbation of a protomer annihilates the oligomer function. Electric charge of proteins.
The presence of amino and carboxylic groups of amino acid residues gives proteins their amphoteric properties. The solution can be split as acids or bases as having both negative and positive charges. Sign and size of the proteins charge are not constant but depend on the composition of the environment and in particular on the pH of the solution. The behavior of protein macromolecules [Michaelis] in water can follow two schemes:

(a) in an alkaline environment H+ is released, so it acts as an acid (see also acid-basic equilibrium):
[R-(NH) (COOH] + Na + OH [R-(NH) (COO] Na + HO
(b) in an acid environment(HCl) with an excess of H+ which are additional, the amine group as a base is loaded positively:
[R-(NH) (COOH] + H + Cl -------- [R-(NH) (COOH1 + Cl

In both cases a reaction of neutralization is described, with formation of salts: Na-protein ate, in the first case (178, 68), protein clorhydrate (macrocation) in the second case.
This is also seen during the electrophoresis of the protein solutions. It suggests the representation of proteins as ion amphoters or amfions [R. (NH) (COO)] --------- [R - (NH) (COOH), the two forms being in equilibrium.In a neutral state, the linear amino acids are in the form of amfions. The behavior of amfions can be written like this:
               R (NH +) (COO) + OH------------ [R-(NH) (COO] + HO in an alkaline environment. In an acid environment, H+ in excess H is in added to the NH grouping which is loaded positively.




R-(NH2) (COOH) + H --------- [R - (NH)(COOH)]
The isoelectric point represents the state of electric potential "zero". In solutions of proteins it depends directly on the total environment pH and indirectly on the concentration of other ions in solution. Note that the isoelectric pH of the protein molecule is not constant and depends on the Nature of the amino acid composition of the protein molecule. Also, even when number of NH = COOH, the isoelectric pH is slightly on the acid side. Electrochemically, proteins are macromolecular ampholytes. The most important contribution to the acidic and basic charges belongs to the residues of glutamyl, aspartyl, lizyl, arginyl and histidyl. The contribution of terminal "C" and "N" is reduced as the length polypeptide chain increases. These electrical charges can be modulated in solution to be separated electrophoretically.


I2.3.5.4. Protein denaturizing
The denaturizing of the protein is a structural modification where molecular weight and covalent bonds are preserved. Denaturizing is of interest to noncovalent bonds specific to the Native conformation. Denaturizing is induced by physical agents (temperature above 60C, with high frequency radiation as ultraviolet rays, X-rays, strong shaking, hydrostatic pressure, etc.), chemicals (acids, bases, organic solvents, detergents, etc.), and biological stimuli acting in vivo. By denaturizing, the solubility of the protein decreases to the level of precipitation, it also lowers the colloid-osmotic pressure, their viscosity, optical activity, there is increased sensitivity to the action of enzymes some of their biological functions are inactivated (enzyme, hormone replacement therapy). The mechanism of denaturizing depends on the Nature of the agent. After removal of the agent (in vitro), the protein returns to its original state. During stimulation of excitable structures in vivo and during transmission of information message, there occur changes in the status of physical-chemical properties of cellular protein, identified as "Tran conformational changes" (16, 17) which are reversible after a while by metabolic and genetic mechanisms.

Such changes occur during the reflex stimulation of the cerebral cortex in rats (14, 16, and 17) as a decrease of protein solubility in the stimulated area, moving the buffer capacity of proteins towards the alkaline side, thus lowering the capacity of ionization of the carboxylic groups of protein from the stimulated cortex. The same changes occur in old, non-stimulated rats, compared with adult rats. Therefore, the processes of aging are accompanied by changes in the brain proteins and soluble endocellular potassium is depleted. Restoration of the structure of proteins takes place with a very small consumption of energy (15, 16, 17).

 Methods of dosing proteins.
In order to individualize blood proteins molecular parameters are used: size, molecular weight, electric charge, chemical bonds, the complexity of structural complexes with other substances, conformation, the Nature and number of reactive groups, behavior in different environments, ion concentrations at various temperatures, the identification of their function as transporters, the biological properties as antigenicity and their immune characteristics.  About 100 protein fractions were identified in blood. Below is a brief review of some methods used in clinical practice and research.
Methods of quantification: Kjeldal methods (determine total nitrogen in protein), colorimetric methods (burette reaction), spectrophotometric methods based on protein interaction with light (especially ultraviolet), spectrophotofluorimetric methods based on stimulated emission of light by proteins that contain aromatic amino acids, refractometric methods. Methods of separation Precipitation with neutral salts of different concentrations: NaCl, PO K, Na SO (SO (NH), by precipitation to isoelectric point, or ethanol in the cold, etc... Methods of electrophoretic separation and characterization.
These methods are based on the property of proteins to become selectively ionized in various conditions of pH, and thus to migrate to a variable electric field. This method can be combined with other procedures thus enhancing its capacity of providing information. It can use various supports.




Electrophoresis in a liquid phase (Moving boundary electrophoresis Tselios). It was used by us as well for research activity. It requires a Tisselius apparatus. It is using 1 -2 ml of blood serum that is diluted with 2 ml.buffer Michaelis-Wideman, pH = 7, 4, ionic strength = 0.118. First, it is dialyzed in buffer at 40 C for three hours. Dialysis continues for a second time for another hour after changing the buffer. Direct current of 150 volts is used.

Two photographic exposures of the two branches of the device are performed, upward and downward, after 45 min. and 75 min. migration. One calculates the mobility (fig. 1) of the fractions obtained, with the equation:
= V / E = ds / dt where:
= Electrophoretic mobility of the "i" fraction.
S = space covered by the fraction "i" in the 30 min. interval
E = electric field strength in the cell (30 V / cm).
, represents the average mobility in the two branches

Figure 1.Electrophoretic mobility (=cm/V)of three plasma protein fractions of normal children and children with behavioral constitutioNal disorders and transport of Na in the ionic state. (230)


Figure 2.Schematic representation of the serum electrophoresis obtained by four methods: (a) Tisselius electrophoresis, (b) electrophoresis on paper (c) gel electrophoresis in starch (d) immunoelectrophoresis (160).

In Figure 2, vertical arrows indicate the point of departure in gel electrophoresis in starch but moving in the field of departure in each case. Globulin remains the starting point in the gel electrophoresis of starch but is shifting in the domain, in other metods.Other methods use starch or agar gel as support, with good results in imunoelectrophoresis.Imunoelectrophoresis allows identification of the areas having globulin immunochemical specificity.

   Centrifugal analysis of serum.
The method takes into account the molecular weight and environment density. The method allows a good separation, if centrifugation is performed in solution with gradient of concentration. It is especially useful to separate lipoproteins.

 Chromatographic Methods
These methods are based on the adsorption properties of hydrophilic ion exchangers. Given the ionic properties of proteins, exchange and buffers for adsorption and elution are almost invariably used. Adsorption chromatography involves substance deposition on solid surfaces, followed by differential elution. Between the solids (bed selected) and proteins various interactions are established: ion, hydrophobic, hydrogen bonds, etc. The most important are the exchanger-ion interactions. Elution is performed by washing or by lowering the pH, which alters the number or sign of protein (or adsorbent) charge, or by increasing the concentration of salts which decrease the strength of bonds between proteins and adsorbent. In other systems hydrogen bonds are generated between the carboxylic group of proteins and the non-ionized carboxylic groups on the ion exchanger. Their affinity is controlled by adjusting the pH to adjust the number of non-ionized carboxyl on protein and resin. Resins (Amberlite XE) have very high density of non-ionized carboxylic groups, compensating to some extent the fact that only groups on the surface are available to bind the proteins. Hydrophobic bonds appear less frequently, but they strengthen the bonds of the primary adsorbents, ionic or hydrogen bonds. If many interactions are formed between the adsorbent and the protein, the latter will not move from where it is connected as long as conditions remain unchanged, and the likelihood of these links to simultaneously dissociate is small. Therefore affinity of the protein for the adsorbent will gradually decrease to zero and therefore one obtains a range of protein unbinding. For several proteins, there appear several equilibrium constants. The affinity of a protein for an ion exchange is expressed by the number of links between proteins and adsorbent and these are useful for separating large molecules.Amberlite XE-64 is a cationic exchanger that allows the exchange with negative proteins. In amphoteric hydrophilic gels the binding forces are weak. So the number of charges on the molecule and their space distribution are criteria of differentiation of proteins.

Different proteins have different ways to change the variations of pH. The adsorbents can be very different, and eluents infinitely increase the technical possibilities. Ion exchange resins (Dowex 1 and Dowex 50) have ion charges on the surface of the calcium phosphate gel type. The cellulose ion exchange has ionizing groups with affinity for many proteins. The covalent bonds allow cellulose stability ionizing groups. An example of the group is DEAE cellulose and carboxymethyl cellulose, an ion exchanger for basic proteins. Phosphorilate cellulose (P cellulose) and sulfoethyl cellulose (SE-cellulose) are cationic exchangers with stronger acidic groups that retain the negative charges to very low pH. Other derivatives are guanetidil cellulose (G-cellulose) for very high pH, triethylaminocellulose anionic exchanger, sefadex is an ion exchanger with cross-links. Chromatography with molecular sieve or gel filtration uses sefadex, gel of granulated polyacrilamid, agar and agarose. So this is a way of sifting molecules by size, along with electrochemical separation. The more sensitive methods used for detecting and dosing are the radioimmunologic (RIA) and the immunoenzymologic ones (ELISA).


 Classification of plasma proteins
At the basis of plasma proteins classification (62) are the results of research with all the methods applied, discussed above. There are holoproteins consisting of chains of amino acids only, such as albumin, and heteroproteins which have a prostetic fragment: carbohydrates, lipids, metals in glycoproteins, lipoproteins, metalproteins respectively. Electrophoresis classification.
Migration in the electric field is in Veronal buffer pH = 8.6. Other buffers can also be used, but the basic condition is that the pH should be as far away as possible from the piezoelectric point. Albumins also migrate in a pH = 7.4, equal to the normal pH of the blood.

Table 5. The main plasma proteins isolated by electrophoresis (175)

Electrophoresis fraction

values %






5, 2 – 5, 9

3, 5 – 5, 5


3, 5

0, 25-0, 35


8, 0 – 1o, o

0, 50-0, 75

- globulins

12, 0 -  14, 0

0, 80-1, 05

- globulins

16, 0 -  20, 0

1, 10-1, 50

Another classification is based on the rate of sedimentation in the centrifugal field in liquid medium with or without gradient density. The use of a large number of physico-chemical characteristics of the protein fractions allows the identification of fibrillary proteins that adhere to the vessel wall. Other classifications
A classification highlights their immunological value. Another class is active in the processes of haemostasis. Other proteins form the signalers group and yet another, the group of receptors. Classification as carriers
Many proteins are transporters, ensuring the takeover of ligands: hormones, amino acids, chemical mediators, electrolytes, Na. (92, 160). From the molecular parameters, (electrophoretic mobility, izoelectric point, molecular weight, the sedimentation constant, the diffusion constant, partial specific volume, viscosity, friction rate), one established molecular polymorphism ranging from the fibrillar elongated form, insoluble in water to hydrophilic, globular forms. Molecular weight of plasma proteins is also a very heterogeneous parameter; their values are between 44,000 and 1,000,000. The determination of molecular weight of plasma protein is difficult because they easily form aggregates, dimers with different weights, or they dissociate during their storage. When injected intravenously, small forms are easily removed in the urine. If they have more acidic groups, elimination is hindered.


Table 6 the percentage content of prostetic groups in certain plasma proteins (132)


% sugars

% lipids

physiological role


1, 3

0, 2

tyrosine fixing


0, 0

0, 0

Transportation substances


41, 4

0, 0



8, 5

0, 0

„Transport” ? 


18, 6

0, 0

Substance transport

Ceruloplasmin (content Cu =0, 84%)

7, 8

0, 0



5, 9

0, 0

Iron transport


1, 5

55, 0

Lipids transport


1, 7

90, 0

Lipids transport


1, 8

80, 0

Lipids transport


3, 1

0, 0


One notes the better expressed transporter role of proteins with sugars as a dominant group, the transport of lipids belonging more especially to lipoproteins.


[The remainder of Chapter Two will be featured in the upcoming September-October issue of this Journal.]



Professor Marcel Uluitu, M.D. Ph.D. began his scientific activity in Physiology in 1953 at the Physiology School (Medical School) Cluj-Napoca, in Romania. He continued his scientific activity in Physiology in Bucharest at the Institute of Physiology and Pathophysiology Daniel Danielopolu until 2004, at which time he retired as Director of the Institute; he had held this position since 1990. His research work includes studies of the central nervous system physiology relating to the transmission of information in the nervous centers; cerebral excitability, using methods, in their evolution, from the determination on reactive isolated organ, to the most complex physical and chemical methods, in the present. In his research of chemical mediation he studied acetylcholine, serotonin, and their connection with the cerebral metabolism.

Professor Uluitu has also investigated cerebral tissue excitability, studying the structure modification of the protein macromolecules, and the physiological and pathopysiological processes in which are involved Sodium and Lithium. He implemented an original method for physical and chemical processes which involve the chemic active sodium, in normal processes and in the cerebral excitability dysfunctions, in human and in experimental model (animal). These results of this work gave him the chance to outline the chapter herein relating to the physiology of substances transport in the blood. This is based on the physical and chemical interaction between blood components.

His papers are included in the collections of the U.S. National Library of Medicine and the U.S. National Institute of Health. He is a member of the Romanian Academy of Medical Sciences.


Dr. Diana Popa (Uluitu) is a researcher in the Department of Microbiolgy, Immunology and Molecular Genetics at the University of Kentucky in Lexington, Kentucky, USA. She attended Medical School at Vasile Goldis University in Arad, Romania, and graduated in 1998; during summer breaks at medical school she volunteered at the Institute of Physiology D. Danielopopu in the phyisiology and microbiology laboratories as well as in the clinic. After completing medical school and a one year internship, she worked as a Research Assistant at the Danielopopu Insitute for three years before coming to the University of Kentucky. In addition to her medical thesis, which focused on the actions of Lithium on the central nervous system, since coming to the United States she has published numerous additional papers relating to Lithium and other substances. 



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