In this article we will discuss about:- 1. Denaturation of Proteins 2. Important Properties of Proteins 3. Classification.
Denaturation of Proteins:
The three-dimensional conformation (the primary, secondary, tertiary and even in some cases, quaternary structure) is characteristic of a native protein. This conformation can be upset and disorganized, without breakage of any peptide linkage, only by the rupture of linkages which enabled the structure to maintain its conformation in space; this is called denaturation.
It can be triggered by diverse physical or chemical agents:
1. Heat (coagulation of ovalbumin of egg white is a well-known example of denaturation),
2. Ultraviolet and ionizing radiations,
3. pH variations. Certain acids (nitric, trichloroacetic, perchloric etc.) and certain acid solutions of heavy metals (Hg, Pb) are used in biological analysis to eliminate the proteins of the medium (blood for example); they are deproteinizing agents,
4. Detergents,
5. Organic solvents (except at a temperature less than 0°),
6. Urea or guanidine solutions,
7. Simple dilution or simple agitation can also cause the denaturation of proteins; this causes great inconvenience when purifying a protein.
Denaturation is sometimes irreversible (for example, in the coagulation of ovalbumin) and sometimes reversible. This process needs to be studied; we have here the passage from a highly ordered state to a less ordered state; there is an increase of entropy (entropy is a measure of the probability of existence of a state, and the least ordered states are the most probable).
In the native state, the protein has the most stable conformation — from the energetic point of view — in intra-cellular conditions; if these conditions are changed, the conformation of the protein will be altered; there will be rupture of the hydrogen bonds and other secondary bonds studied earlier, disorganization of secondary and tertiary structures resulting in increased sensitivity to proteolytic enzymes, increase of reactivity of certain groups which were either involved in secondary bonds or inaccessible and, above all, a loss of biological properties (especially in the case of enzymes).
Determinism of the Three-Dimensional Conformation:
The study of certain cases of reversible denaturation suggests that conformation is determined by the sequence of amino acids. We will take the example of ribonuclease. It is an enzyme. Its sequence is known (124 amino acids). Its three-dimensional conformation is maintained especially by 4 disulphide bridges resulting from the association in pairs of 8 cysteine residues of the chain.
If the enzyme is reduced, the four S-S bridges are broken (generating 8 thiol groups), the conformation collapses and we have a denatured, inactive enzyme.
But this denaturation is reversible, the four S-S bridges can form again by oxidation, the ribonuclease resumes a three-dimensional conformation and we have again an active enzyme. But the remarkable fact is that the same SH groups interact to form again the same four S-S bridges, although there is a large number of possible combinations (28).
It therefore appears that the sequence of amino acids contains the information required to impose a given spatial structure in defined intra-cellular conditions. We will see however, that small molecules (substrates, activators, inhibitors) are capable of producing slight modifications of the three-dimensional conformation in the case of allosteric enzymes.
Important Properties of Proteins:
1. Solubility:
Water solubility of proteins is so variable that it was proposed to classify them according to their solubility. Certain proteins are soluble in pure water (albumins), others — especially the globulins — dissolve only in presence of neutral salts or when the medium is slightly acidic or weakly alkaline, and certain others (scleroproteins) are insoluble even in these diverse conditions.
A. Effect of Electrolytes:
The action of neutral salts depends on their concentration and the charge of ions, i.e. the ionic strength. When ionic strength is low a dissolving effect is observed. On the contrary when the ionic strength is high, salting out takes place, i.e. the precipitation of protein molecules.
These two phases are distinctly observed for example, while studying the solubility variation of hemoglobin when ionic strength is increased by addition of ammonium sulphate (see fig. 1-20).
All proteins are not equally sensitive to this salting out effect by addition of a neutral salt; thus, certain proteins precipitate in a solution half saturated with (NH4)2 SO4, others only when the solution is 55% or 60% saturated etc. By gradual addition of a neutral salt (and centrifuging after each addition) it is therefore possible to fractionate a mixture of proteins, if not completely, at least partially.
B. Effect of pH:
Several methods of fractionation of proteins are based on the fact that under constant ionic strength and constant temperature, the solubility of a protein is minimal near the isoelectric pH (see fig. 1-21). In certain cases, crystallization of a protein may be achieved by increasing salt concentration in a solution maintained at a pH near the isoelectric point.
For the two values of ionic strength used, solubility is minimal near the isoelectric pH, but it is observed that whatever the pH, solubility is greater when µ = 0.01. This is therefore the range of dissolving effect as far as ionic strength is concerned. To obtain the precipitation of the protein, it is advantageous to act on both factors.
C. Effect of Organic Solvents:
Ethanol, methanol, acetone, can be used to precipitate the proteins, provided temperature is maintained around -5° C to avoid their denaturation by these solvents. A fractionation of plasma proteins can be achieved by increasing especially alcohol concentration.
2. Molecular Weight:
Cryoscopy is not applicable because the lowering of the freezing point is too small to be measured accurately.
The measurement of osmotic pressure is not very convenient, but it has been used to determine the molecular weight of some proteins through the relation:
∏ (osmotic pressure) = RT C/M (C is the concentration by weight and M, the molecular weight).
Three methods are commonly used:
A. Filtration through Dextran Gel:
The dextran gels used (Sephadex type) are polyacids containing a varying number of cross-linkages which produce a certain degree of porosity; macromolecules penetrate these gels with varying ease depending on their size, hence the name “molecular sieve” given sometimes to these dextran gel columns: the largest molecules excluded from the gel will be the first to come out of the column, whereas the smallest molecules which can penetrate into the gel, will be delayed and will come out last.
These dextran gel columns can be “calibrated” by passing proteins of known molecular weight; a protein of unknown molecular weight is then passed, and its molecular weight determined on the basis of the time it takes to come out of the column (see fig. 1-22). Columns of acrylainide gel (Biogel) can be used for the same purpose.
B. Light Scattering:
When a beam of light passes through a protein solution a part of the light is scattered (Tyndall effect); the proportion of scattered light increases (while the proportion of transmitted light obviously decreases) when the number and size of molecule in solution increase. The molecular weight of a protein can therefore be calculated from the ratio: incident light/transmitted light.
C. Ultracentrifugation:
We owe to Svedberg this high speed centrifugation method, carried out in devices rotating at 60 000 revolutions/minute, which corresponds to a gravitation field of 500 000 X g or even more (depending on the geometry of the rotor used).
The protein molecules in solution, subjected to this ultracentrifugation, sediment according to their density (which is greater than that of the solvent) and concentration variations resulting from the sedimentation are observed by measuring the refractive index of the solution during the centrifugation and without interfering with the centrifugation operation (measurements are made with the help of an optical recording device).
a) Sedimentation Velocity:
When a homogeneous, monodisperse solution (containing only one protein) is subjected to ultracentrifugation, all molecules migrate with the same velocity and one observes only one boundary which moves in course of time and corresponds to the zone where protein concentration passes from 0 (pure solvent) to a certain value C. (see fig. 1-23 B).
For a given angular velocity (ω), C is a function of the distance between the molecule and the rotation axis of the rotor (r). In fact, what one follows through the optical system, is the displacement of a peak corresponding to the variations of dc/dr (see fig. 1-23 C). The displacement velocity of this peak, or sedimentation velocity, is r2-r1/t2— t1, or, at the limit dr/dt; it depends therefore on the gravitation field.
This determines a sedimentation constant, characteristic of a given protein, S = (dr/dt)ω2r, referred to defined conditions (sedimentation at 20°, in water) and expressed in Svedberg Units (S). One Svedberg unit is equal to 10-13 cm/s/field unit. For most proteins S is between 1 and 200.
From S, we can estimate the molecular weight, with Svedberg’s equation: M = S X f X N/1 – V̅ρ, where N is Avogadro’s number, ρ the density of the centrifugation medium, V̅ the partial specific volume of the protein and f the friction coefficient of the protein which is a complicated function of its conformation (shape, dimensions, rigidity, degree of preferential solvation).
If we subject to ultracentrifugation a heterogeneous, poly-disperse solution (containing a mixture of proteins, or different oligomers of a same sub-unit) we obtain several peaks corresponding to diverse particles sedimenting at different velocities. We can thus estimate the molecular weights of diverse proteins or diverse oligomers.
Ultracentrifugation can also be used as a criterion of purity but the fact that a protein gives only one peak during ultracentrifugation does not mean that it is pure (it may very well be a mixture of two proteins or even more, as revealed by another technique like electrophoresis on polyacrylamide gel for example).
b) Sedimentation Equilibrium:
A pure protein solution may be subjected to ultracentrifugation until the distribution of the protein in the liquid column reaches a state of stable equilibrium i.e. until displacement of the protein is no longer observed (equilibrium between sedimentation and diffusion phenomena).
If we determine protein concentration at different distances from the centre of rotation, we can deduce the molecular weight of the protein. This method does not necessitate the determination of the sedimentation constant; on the other hand, a comparatively small centrifugal force is sufficient, but the disadvantage of the method is that centrifugation must be carried out for a rather long time to reach equilibrium.
D. Results:
The molecular weights of proteins can vary from about 10 000 (ribonuclcase) to one million or even more (hemocyanins).
A few examples are given below:
3. Amphoteric Character:
Most amino and carboxylic groups of amino acids are — in proteins — involved in peptide linkages. But we have seen that the side chains contain ionizable groups which confer on the proteins an amphoteric character.
Depending on the pH of the solution, these groups are more or less ionized according to their pK, so that when pH is raised the protein may exist in the following 3 states:
The isotonic point is reached when the number of positively charged groups is equal to the number of those negatively charged; the number of protons combined with the basic groups is then equal to the number of protons liberated by the dissociation of acid groups.
As in the case of amino acids, this isoionic point (characteristic of a pure protein in solution in water) is near the isoelectric point where the total charge of the protein is nil (taking into consideration the other ions in solution, which may fix themselves to the protein molecule).
The pH, can vary widely in different proteins:
Due to the large number of groups capable of reacting reversibly with protons, and in a rather wide range of pH values, proteins have considerable buffer capacity in biological media.
From the practical point of view, these properties of proteins lead to — as in the case of amino acids — a number of interesting applications:
i. The Solubility of a Protein is Minimal Near its Isoelectric Point:
It can therefore be precipitated during its separation and purification,
ii. Proteins can be Fractionated by Ion Exchange Chromatography:
Modified celluloses are often used for this purpose; these substances are either anion exchangers like diethylaminoethyl-cellulose (DEAE-cellulose), or cation exchangers like carboxymethyl-cellulose (CM-Cellulose). The principle of this method was explained in connection with the fractionation of amino acids,
iii. Except at the Isoelectric Point, Proteins Migrate when placed in an Electric Field:
If the pH is higher than the isoelectric pH, the protein is charged negatively and migrates towards the anode, but if the pH is lower than the isoelectric pH, the protein is charged positively and migrates towards the cathode. One can therefore fractionate proteins by electrophoresis, either in a liquid medium in a Tiselius type apparatus (U-tube), or on a support like cellulose acetate paper, starch gel or polyacrylamide gel (zone electrophoresis).
This second technique is being increasingly used because it is more convenient; with the Tiselius apparatus, an optical system must be used to follow the displacement of the boundary between the protein and the buffer by measuring the refractive index; in zone electrophoresis, the position of different zones containing the proteins which migrated differently is visualised by a specific coloration of proteins (by amido-schwartz for example); this method also permits a quantitative analysis of the different proteins of a mixture, as one can either titrate by photodensitometry the coloration of different zones, or cut out these zones and elute the proteins, or even collect the different fractions at the end of the gel which they reach at different times on account of differences in their electrophoretic mobility.
In a given electric field, this mobility mainly depends on the charge and size of the protein molecules. For example, the electrophoresis of serum proteins has very great importance in medicine because it permits the diagnosis of certain diseases which are reflected by anomalies of the electrophoregram like the increase or decrease of a protein fraction. Figure 1-24 shows the electrophoretic profile of proteins of a normal serum.
Immuno-electrophoresis is an application of electrophoresis as well as of the specific precipitation produced by the mixture of a protein and the corresponding antibody.
Proteins have an antigenic capacity, i.e. when introduced in a foreign organism they induce the production of specific γ-globulins or antibodies which are means of defence of the organism against the protein (or antigen) introduced (for example, vaccination induces the formation of specific antibodies).
The protein mixture to be analysed is therefore subjected to a zone electrophoresis and a serum containing a given antibody is allowed to diffuse. If the antibody meets the corresponding protein (i.e. its antigen) we observe an arc-shaped precipitation zone which reveals the presence of this protein (see fig. 1-25).
4. Osmotic Pressure:
Let us consider two compartments separated by a membrane permeable to ions but impermeable to proteins. The presence of a protein in one of the compartments causes an unequal distribution of diffusible ions on the two sides of the membrane (whereas in the absence of protein there is equal distribution, at equilibrium).
Figure 1-26 shows that in the initial state, the compartment contains negatively charged protein molecules denoted P (anions). There is a disequilibrium in the distribution of CI– and the CI– ion will therefore diffuse from B to A; but in order to maintain electrical neutrality, an equivalent quantity (x) of Na+ ions will also move from B to A.
A final state is therefore reached where the distribution of Na+ and CI– is unequal on the two sides of the membrane; this is the DONNAN equilibrium; its corresponding equation is:
In other words, the ratio of NaCl concentrations on the two sides of the membrane is equal to 1 + the ratio of protein concentration in A to the initial NaCl concentration in B. This means that the higher the protein concentration in A, the more unequal the final distribution of diffusible ions, and therefore, the greater the osmotic pressure.
To the osmotic pressure of protein (proportional to its concentration, but inversely proportional to its molecular weight, because, ∏ = RT C/M), is therefore added in A, the osmotic pressure due to the excess of diffusible ions: the sum represents the oncotic pressure.
It is clear that this excess of ions will be minimum at the isoelectric point of the protein and will increase for values diverging from this pH, because it depends on the degree of ionization of the protein.
In blood plasma, albumin contributes to osmotic pressure to the extent of 75 to 80% (although it represents no more than half the plasma proteins), because its molecular weight is lower and on the other hand, at the blood pH, it is further away from its pH, value than the globulins. The oncotic pressure due to plasma proteins plays an important role in the passage of extravascular water of the interstitial liquid towards the intravascular compartment.
It should be noted that if, in the example of figure 1-26, we replace the diffusible cation Na+ by H+, we will have on the two sides of the membrane an unequal distribution in H+ ions, i.e. a difference of pH, and compartment B will be less acid than compartment A.
On the other hand, if in the initial state, the pH and the isoelectric point of the protein are such that we have cations P+ (in presence of an equivalent concentration in Cl– ions), compartment B will be more acid in the final state.
Isolation, Fractionation and Purification of Proteins:
The isolation of a pure protein from a homogenate of cells is generally a long and delicate task, because in most cases we face several hundreds of proteins having somewhat similar chemical properties. Besides, care must be taken not to denature the protein to be isolated.
The methods most frequently used were mentioned while studying the properties of proteins; they are mainly:
1. Dialysis, to eliminate the small molecules,
2. Precipitation by neutral salts (or salting out); ammonium sulphate is very often used because it is highly soluble, even at the low working temperature adopted to avoid denaturation,
3. Precipitation at the isoelectric point,
4. Precipitation by organic solvents at low temperature,
5. Adsorption chromatography on gel (alumina, calcium phosphate or hydroxyapatite etc.) which allows to retain certain proteins and then elute them by gradually varying the pH or saline concentration,
6. Ion exchange chromatography,
7. Chromatography by filtration on dextran gels (or molecular sieves),
8. Affinity chromatography which allows to purify an enzyme protein for example. The method consists in bonding covalently on a solid support, a steric analogue of the enzyme substrate (competitive inhibitor or modified substrate) to form with the enzyme a complex of the enzyme-substrate type.
Therefore, during the passage on the column, the enzyme will be delayed with respect to the other proteins which, not recognizing the analogue, will come out of the column more rapidly than the enzyme,
9. Immuno-adsorption is very similar in its principle, to affinity chromatography: the protein is first purified by the previous methods, specific antibodies against this protein are prepared by injecting it to a rabbit. These antibodies are then isolated and chemically fixed covalently on a solid support.
The support thus prepared is used to make an immuno-absorbent column which will specifically retain the antigen protein, while all other proteins will pass through such a column without being retained. The antibody-antigen complex thus formed will then be dissociated on the column to recover the purified protein,
10. Electrophoresis
11. Electro-focusing. In this technique, a pH gradient is produced in a liquid column with the help of appropriate electrolytes. After introducing the protein mixture in the column, an electrophoretic migration is effected. A given protein will migrate, depending on its initial charge, towards one of the electrodes, until it reaches the zone corresponding to its isoelectric pH; in that zone its charge becomes zero and its migration stops. By this method, proteins are therefore fractionated according to their pHi,
12. Preparatory ultracentrifugation,
13. Crystallization,
Purification may be followed either by physico-chemical methods such as electrophoresis or analytical ultracentrifugation, or by biological methods such as determination of specific activity in the case of enzymes (catalytic activity per unit of protein mass, which of course, will increase as purification proceeds).
Classification of Proteins:
Different types of classifications have been proposed:
1. Classification Based on Molecular Shape:
As mentioned while studying the three-dimensional conformation of proteins we distinguish:
A. Fibrous Proteins:
Also called scleroproteins, they consist — as indicated by their name — of fibers or fibrils; they are practically insoluble. This group includes silk fibroin, collagens found in conjunctive tissues, cartilages, tendons (and which on heating with water in the autoclave give gelatins) and keratins present in the skin and superficial body growths (hair, fur, nails, horns, feathers etc).
B. Globular Proteins:
They are also called spheroproteins on account of their spherical or ovoid shape; they are generally more easily soluble. This group includes mainly albumins and globulins which will be described in the following.
2. Classification Based on Solubility:
The following are generally distinguished:
1. Albumins, which are soluble even in distilled water. They precipitate on addition of ammonium sulphate between 70 and 100% saturation. Their isoelectric point is in general, distinctly less than 7 (serumalbumin, 5.3; ovalbumin 4.7); they are therefore acid in character;
2. Globulins, are insoluble in pure water but soluble in dilute saline solutions (e.g., 5% NaCl). They precipitate on addition of ammonium sulphate at 50% saturation. They are often glycoproteins or lipoproteins;
3. Protamins and histones are soluble proteins of comparatively small size (especially the protamins with their molecular weight varying between 2 000 and 5 000; in fact they are polypeptides rather than proteins); they have a very distinct basic character due to the presence of a large proportion of lysine and arginine (up to 90% in certain cases) giving them a high isoelectric point (around 11).
On account of this basic character, they can combine with various acid compounds (nucleic acids, acidic proteins) and they are found combined with deoxyribonucleic acids in cell nuclei;
4. Globins differ from the above proteins by a comparatively high histidine content (up to 10%). They constitute the protein part of hemoglobins and myoglobins, chromoproteins whose prosthetic group is heme.
5. Prolamins and glutelins are plant proteins, insoluble in water but soluble in acids and dilute bases;
6. Scleroproteins, insoluble in water and dilute saline, acid or alkaline solutions;
7. Soluble fibrilar proteins: these proteins constitute the fibrils in muscle cells (where they are very abundant) or microtubules of the cytoskeleton.
Fibrils of muscle cells contain four types of proteins called contractile proteins:
1. Myosin, molar weight 460 000, organized in two chains in coiled double helix (130 nm length) ending with a globular head which can link with a second protein, the actin.
2. Actin, a globular protein of molar weight 42 000, which polymerizes in long filaments containing about 400 monomers; it forms a double helix of 2µm length.
Two other proteins are found along these filaments:
1. Troponin (molar weight 80,000) a globular protein and tropomyosin (molar weight 70,000) a fibrilar protein.
Within other cells are found similar organizations constituting what is called the cycloskeleton thanks, in particular, to a protein called tubulin which can polymerize into microtubules.
3. Classification Based on Composition:
There are two broad groups:
1. The holoproteins, consisting exclusively of amino acids.
2. The heteroproteins, which contain, on one hand, one or several polypeptide chains and on the other hand, a non-protein part called prosthetic group which is bonded covalently.
Heteroproteins form an extremely heterogeneous family, not only by the variety of prosthetic groups, but also the number of groups present per molecule of protein and the nature — more or less labile — of linkages between the prosthetic groups and the polypeptide chains. Heteroproteins include phosphoproteins, glycoproteins and chromoproteins.
The proteins can also be associated in vivo with very diverse compounds and if these associations are sufficiently stable to withstand the extraction and purification processes, lipoproteins and nucleoproteins may be isolated. There are even ternary associations like the phosphoglycoproteins or phospholipoproteins.