Physicochemical properties of proteins

Denaturation

A protein is denatured when its specific three-dimensional conformation is changed by rupture of certain connections without attack of its primary structure. It can be a question, for example, of the disorganization of zones in propeller α. The denaturation can be reversible or irreversible. It involves a loss total or partial of the biological activity. It very often produces a change of solubility of protein.

The agents of denaturation are numerous:

physical agents: heat, radiations, pH;

chemical agents: urea solution which forms new hydrogen bonds in protein, organic solvents, detergents…

Solubility

The relations of proteins with water are complex. The secondary structure of proteins depends on the whole on the interaction on the peptide connections with water via hydrogen bonds. Hydrogen bonds are also formed either between radicals of protein (structures alpha and beta) or between its free absorbent radicals and water. The proteins rich in static ball are thus a priori more soluble than the helicoid structures. On the level of the tertiary structure, water causes the orientation of the chains and radicals absorbent towards the outside of the molecule, whereas the chains and radical hydrophobic tend to react between them inside the molecule (cf hydrophobic effect). The solubility of proteins in an aqueous solution containing of salts depends on two antagonistic effects related on the one hand to the electrostatic interactions (" salting in"), and in addition with the hydrophobic interactions (" salting out").

With low ionic force, the solubility of proteins increases when the salt concentration believes, until a certain threshold. Beyond that, it decreases with the addition of salts. The increase in solubility (salting in) is related to the action of the electrostatic forces the reduction in solubility (" salting out") is allotted to various interactions gathered under the name " interactions hydrophobes" , without this term implying a precise mechanism.

Influence pH: solubility is minimal with isoelectric pH

Molecular weights and mass

It is a fundamental characteristic of each protein. It defined by two equivalent expressions which must be distinguished:

the " weight moléculaire" or " molecular mass relative" , Mr. is without dimension.

the " mass moléculaire" (symbol m), generally expressed let us daltons some (Da) for proteins.

For proteins, one often uses Mr. This characteristic is estimated by various methods, for example:

filtration on freezing, or steric chromatography of exclusion. Ball of polyosides with cross connections or an artificial polymer forms in water a gel functioning like a molecular sieve crossed quickly by the large molecules, slowly by the small ones which penetrate freezing. The principle is simple: after having calibrated the column with proteins of known molecular weight, there exists then a linear relation between the volume of elution and the logarithm of the molecular weight.

the electrophoresis on a polyacrylamide gel in gradient. Results close to those of filtration on freezing are obtained thanks to gel of electrophoresis of decreasing porosity in which the proteins are gradually " bloquées" according to their molecular weight.

The molecular weight of proteins varies few thousands with 1 million or more.

Electric properties

Amphoteric character

The peptide connection is not charged with pH being of physiological interest. Indeed with such pH the peptide formation starting from their amino-acids constitutive is accompanied by a net loss of a positive load and a negative charge by formed peptide connection. However, peptides are molecules carrying loads to the physiological pH because of the loads on the groupings C and NR final and on the functional groupings of carbons alpha of the polar amino-acids.

The load varies with pH: in acid medium, the proteins ionize like bases and are positively charged. In alkaline medium, they form anions.

There exists a point of pH for which the loads + and - are equivalent, the clear load being thus null; it is the isoelectric point, or phi, of the protein which then does not move any more in one electric field.

Electrophoretic mobility - Electrophoresis - Electrofocusing

Subjected to an electric field, the proteins move more or less according to their load: it is the electrophoretic mobility which will thus allow, if there is a mixture of proteins to separate them by electrophoresis. It is a very traditional process of fractionation of the proteins which can be either only analytical or also préparatif. It is generally carried out on supports: acetate, freezing of gélose, polyacrylamide gel. The electrophoresis of zone is very usually used in clinical biology to separate proteins from the blood serum.

The isoelectrofocalisation (IEF) rests on the property of the amphoteric molecules as proteins to be positively charged if pH is more acid than their phi, negatively if pH is basic than their phi. One can apply to the support carrying the gradient of pH an electric field such as the pole + or Anode is on the acid terminal and the pole - or cathode on the basic terminal. If the amphoteric molecule is neutral, it will remain motionless. If it at the beginning is charged negatively, it will be pushed back by cathode and will join under the effect of the electric field its phi. If it is positively charged, pushed back by the Anode, it will also join its phi. Thus, whatever was the starting point of protein in this gradient of pH subjected to an electric field, it will be focused, concentrated with its phi. These conditions are carried out by the use of a mixture of amphoteric molecules of low molar mass (or ampholytic) whose isoelectric pH cover the zone of selected pH.

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