What is the experiment of Protein coagulation?

Answer 1

The term denaturation is used more frequently than coagulation by scientific investigators at the present time to denote certain changes in proteins. Definite characteristics of the proteins are changed when they are coagulated, among which is loss of solubility in water and dilute salt solutions. In some instances and under certain conditions the coagulation process may be reversible.

Manner in which denaturation may be brought about. Coagulation of proteins may be brought about by a variety of processes. In cookery one of the principal means of coagulation is heat. But in addition to heat the action of acids, alkalies, salts, alcohol, mechanical agitation, radiation, and ultra-sonic vibrations may denature the protein and convert it from a soluble into an insoluble form.

Some changes in the proteins during denaturation. All investigators agree that denaturation is brought about in two steps. The first step is a preliminary alteration of the protein or denaturation. The second is a physical change which leads to coagulation or aggregation. Clayton in discussing "Foods as Colloid Systems" reviews some of the theories of protein denaturation. "Hydrolysis has been frequently reported as the cause of denaturation, but present views incline to the idea of some structural rearrangement within the molecule. Thus, the refractive index increases during heat denaturation, whilst X-ray diffraction patterns lead to the view that coagulation is accompanied by the elimination of water between NH1 and COOH groups. . . . Cubin holds that denaturation is the distortion or opening up of the protein unit, whilst flocculation is the process following this and rendered possible by it. Interaction of NH1 and COOH groups situated on contiguous colloid units leads to aggregation and, hence, coagulation."

No matter how denaturation is brought about, the denatured product has sulfur atoms, the combination of which differs from those in the native protein. Mirsky and Anson have shown that in native egg albumin no sulfhydryl (SH) and disulfide (S-S) groups are detectable by certain methods. But in completely coagulated protein the number of SH and S-S groups detectable is the same as in hydrolyzed protein. These workers have also shown that in partially coagulated protein when the soluble and insoluble fractions are separated the soluble portion contains no detectable SH or S-S groups, but the insoluble fraction has the number of reactive SH and S-S groups characteristic of the completely denatured protein. In the interfacial coagulation of a protein, i.e., when a film of insoluble protein forms at the surface of a protein solution, SH and S-S groups appear, the number being the same as that found in the hydrolyzed protein. Also when the proteins are denatured by ultra-violet light, by acids, or by other means the SH and S-S groups appear. From these results they conclude that the formation of insoluble proteins and increase in detectable SH and S-S groups are closely linked phenomena; that denaturation is a definite chemical reaction; and that a given protein molecule is either completely native or completely denaturated.

In a later paper Mirsky and Anson report that the number of detectable SH and S-S groups in different proteins varies with the pH and the temperature. To illustrate, native hemoglobin had no detectable SH groups at pH 6.8. But with increase of pH the SH groups become detectable in increasing numbers up to pH 9.6. But native egg albumin showed no detectable group at pH 6.8 or pH 9.6. However, denatured hemoglobin had detectable groups at pH 6.8 and still more at pH 9.6. They found that intact, unhydrolyzed proteins possess in addition to SH groups other reducing groups which can be oxidized by ferricyanide. The number and activity of these groups vary from protein to protein. They are probably contained in the tyrosine and tryptophane component of proteins. "It can now be seen that the activation of SH and S-S groups in protein denatura-tion is part of a more general process."

Heat coagulation. As has been indicated heat coagulation of proteins is used in preparation of food products, and, fortunately for the mental equilibrium of the cook, heat coagulation of proteins is ordinarily not reversible. Otherwise, many cooked dishes would, with certain treatment, revert to their original uncooked consistency.

Some of the changes occurring during heat coagulation of the proteins have been indicated. But these are not the only factors playing a role in the process. Electrolytes have some role in heat coagulation of proteins. This is shown in the work with distilled water egg custards. It has been shown that, if the mineral content of egg white is lowered through dialysis, coagulation does not occur on heating. The effect of electrolytes in heat coagulation may be brought about either by chemical reaction or by adsorption. If the effect of salts is brought about by adsorption, the salts must be very strongly adsorbed and almost impossible to remove from the aggregated protein by washing the protein, for the process is usually irreversible. Any theory of heat coagulation of the proteins must not only explain how the proteins are rendered insoluble by heat but the effect of other factors. That the heat coagulation of proteins is influenced by electrolytes, sugar, temperature, time, the reaction of the solution, and the presence of water and other factors is evident when the cooking of eggs, custards, salad dressings, cheese and egg dishes, baked products, and meat is observed. The effect of some of these factors can be determined in the laboratory; but the understanding of the manner of their action is lacking in many instances and awaits explanation by the colloid chemist or biochemist.

Bancroft and Rutzler have reported that heat-coagulated egg white may be peptized by dextrose and certain salts. They showed that the coagulated and repeptized egg-white sols are identical with the original solution by immuno-biological tests for species specificity and isoelectric point measurements. Because of the similarity of the reversed protein to the original protein they believe that coagulation is a colloidal reaction which is due to a physical rather than a chemical change.

Interfacial denaturation. Proteins are also denatured at interfaces, typical examples being the insoluble portion of beaten egg white, and froth or foam on milk. When egg white or milk foams are allowed to remain undisturbed, they gradually collapse and the wrinkled membranes, skin, or films may be observed through the microscope. Mention has already been made that protein can be recovered from a solution by removing the foam. Denaturation of protein solution occurs by shaking and in some instances spontaneously, an example being the membrane formed at the interface of an air/protein solution when no agitation has occurred.

Neurath and Bull state that both heat and surface denaturation processes involve an unfolding of the peptide chains which in the natural state are curled up in the interior of the molecule and become stretched out when the molecule comes in contact with the surface of the bulk of the solution. The polar groups of the protein molecule, the amino, carboxyl, the OH groups of the hydroxy acids, the sulfur-containing groups, and the peptide linkages, have an affinity for water; whereas the non-polar or lyophobic groups, the hydrocarbon residues, tend to be repelled by water. Thinking that an interaction between the amino and carboxyl groups during heat denaturation might diminish the lyophobic or polar properties of natural protein, whereas an unfolding of the peptide chains by surface denaturation might expose lyophobic groups to the surface, which in the native state are buried in the interior, Neurath and Bull measured the volume contraction of native, heat-denatured, and surface-denatured proteins. They found that the native protein had the lowest density, heat-denatured ones were intermediate, and the surface-denatured protein had the highest density.

This membrane-forming property of protein through denaturation is important in food preparation, in all products in which beaten egg white is used, in emulsions, and wherever interfacial reactions occur.

Membranes form readily on the surface of protoplasm and play important parts in cell functions. The presence of calcium has been shown to stiffen the surface membranes in some instances, whereas sodium and potassium in the absence of calcium tend to soften and dissolve the membrane.

This suggests that salts may also have some influence in surface denatura-tion and that the salts of flour, egg, and milk used in cooked products may modify the denaturation at surfaces.

Clayton states that high concentrations of sugar in egg white will prevent surface denaturation, which of course has application in making angel cakes, meringues, and sweetened souffles.