92 .

SOHE GENERAL PHYSICAL AND CHENICAL PROPERTIES OF

The large class of compounds now classified as "pmteins" repre- sent a polycomplexity of individual species, yet, all have many common characteristics, constructed from similar chemical units and exhibit similar physical andare chemical properties. Proteins are biosynthesized from a variety of chemical molecules which are broadly classified as a-amino . As the synthesis proceeds and the chain is formed, the a-amino acids are caref'ully selected and one added to another is a specific manner until the growth process is terminated. Nature is specific in the selection of the correct amino at the time it is needed in the forma- tion of the polymer chain. The chemical characteristics of the proteins depend upon the indi- vidual amino acids units present in the complete protein molecule and the sequence in the units as they are placed in the polymer chain. That is, the chemical properties will not only be influenced by which has en- tered the chain but also upon its nearest neighbors. However, some of the physical properties will be relatively independent of the chemical constmc- tion and will result mostly from the fact that proteins just as other syn- thetic high molecular weight compounds, are in essence, giant molecules whose physical behavior is memly a reflection of their enormous size.

The chemical units of constmction, the a-amino acids, have many distinguishing features and are schematically represented by the following formula;

N OH H' 'H vhere the starred carbon is asymmetric and thus exists in two, optical isomeric forms, the right (d) and left (1) hand molecules. Since nature produces and utilizes only a-srrcino acids in the l-form the joining of d and 1 units does not become a complication in protein structure.

The R group attached to the asymmetric carbon atom is responsible for the distinction of one pamino acid from another. R can be either pure hydrocarbon in chemical nature or may contain other elements such as oxygen and nitrogen or amino groups.

However, the simple molecule described above has other more in- teresting features since it contains both a basic substituent (-NHz) and an 93. acidic substituent (-CCOH) making the amino acids rather unique molecules In basic solution the carboxylic acid portion would be more completely Ionized wheE the molecule would take a structure indicated by 11, thus becoming an anion, and H 0

in acidic solution a structure as shown in I11 results where the amino acid be come 8 0

a cation. Consequently in an electrolytic cell the migration of an amino acid to the negative electrode (cathode) or the positive electrode (anode) will depend not dnly on the individual amino acid but the pH of the media in which it Is dissolved.

The ability of 8 molecule to behave both as a and a6 an acid is termed amphoterism and the molecules ampholytes. This phenomena is not limited to amino-acids but is also of property of many simple metal hy- droxides.

The of an amino acid in aqueous solution will depend principally on the substituent R. Depending on the structure and composition of R sow amino acids in aqueous solution will be acidic in nature (pH < 7), Amino acids of this type are weak acids and a portion of the carboxylic acid groups interact wIth and dissociate as indicated below (IV).

Other amino acids will interact with water at the site a8 shown in (V), H 0 0 1 I, HI

and thus give basic aqueous solutions. Still others react equivalently with water as in IV and V SO that no net change in structure results and the amino acid is essentially neutral in nature. 94.

By proper adjustment of the pH of an aqueous solution of amino acids LL point is reached where the net electrical charge on the amino acid is zero. specific conditions the molecule would not migrate to Under these The pH where either electrode under the influence of an electric field. this phenomena is exhibited is the and is a char- termed IWy other physical prop- acteristic physical property of all amino acids. erties such as membrane potential, optical rotation, solubility, diffusion, stability and resistance to denaturation will show a maxima or minima at this unique pH.

When proteins a= formed the amino acids act as difunctional monomers and polycondense by eliminating water to form amide linkages and a polypeptide chain of the nature illustrated by(V1)

Although the amine and carboxylic acid groups are destroyed in the peptide fomation, the protein so formed may retain free amino or acid groups as dangling side groups (pendant groups as embodied in R) or as terminal un- reacted end groups. However, even though the peptide linkage longer will no Thus take part in pH titrations all residual amine and acids groups will. proteins are Ellso mpholytes and exhibit an isoelectric point where proper- ties such as Solubility, solvent swelling snd molecular size in solution will be drastically affected by slight alteration of the pH. In fact, the isoelectric point can be more sharply defined for proteins than for many of its contributing a-amino acids when the fomr is measured by the Tiselius electrophoresis method.

In this method a dissolved aqueous solution of protein is placed in an electric field and the pH of the solution is changed by titration with an acid or base. The moving boundary of can be followed by suit- able instrumental methods and when the boundary ceases to move in the field the molecule is in the isoelectric state. Many of the physical characteristics of proteins depend upon Structure wise, their structuE and the fact that they am giant molecules. the proteins as a class generally have regions of high crystallinity. How- ever, as in all long chain molecules it is impossible for proteins of high molecular weight to completely crystallize. This results from the compli- cations involved in making a molecule composed of many segments rearrange each segment in a specific within the and with respect to its order chain Thus proteins nearest neighbor molecules in a reasonable period of time. which crystallize contain both crystalline regions where high local order persists, andwill amorphous regions where very little order exists. It is these latter regions which are the most vulnerable to the action of solvents.

These types of protein molecules behave somewhat as synthetic net- work molecules such as Vulcanized rubber and will reach an equilibrium 95 . degree of swelling providing the imbibed solvent-polymer interactions are too weak to overcome the forces of crystallinity. The crystalline content of proteins can be both enhanced and destroyed by suitable changes in pH or metal strength of imbibed water. Some proteins whose crystallinity has been destroyed by dissolution will fail to recrystallize after removel from the solvent and are thus considered denatured or altered in an irre- versible fashion.

While some protein molecules are joined together by a mutual sharing of some of their segments in a crystallite, others are cross linked intemittently by covalent chemical bonds. This type of restric- tion tends to reduce the symmetry of a protein chain thus resulting in low degrees of crystallinity. Often these cross links can be selectively broken and the resulting chains solubilized. However, as cross links they are network junction points and greatly influence certain physical proper- ties of the proteins (such as equilibrium swelling, modulue of elasticity, etc.).

One of the properties affected is the degree to which a network may be swollen by imbibing solvent. With the cross links acting as restrictions the polymer will swell with solvent until the elastic retrac- tive energy of the chain reaches a state of equilibrium with energy re- sulting from solvent-protein interactions. The equilibrium degree of swelling should be reproducible once the conditions for equilibrium are reached and providing no degradation of protein takes place. Several factors affect the degree to which a polymer network will swell before reaching equilibrium, The first is the number and distribu- tion of cross links. The more cross links in a network, the lower the de- gree of swelling. Second is the molecular weight of the individual chains. The longer the chains the fewer cmss links needed to bring about a three dimensional system. Finally a third important factor is the chemical nature of the protein, that is, the type and distribution of R groups along the backbone of the chain.

A surfeit of hydrophobic R groups will decrease the equilibrium swelling of the protein by water whereas a large collection of ionizable (basic or acidic) groups in R will cause the protein to swell considerably. When pendant ionizable groups are present, the equilibrium swelling will incmase at both high and low pH, but will reach a minimum at the iso- electric point just aa solubility does for soluble proteins,

In summary, the chemical properties of proteins, in particular their behavior in acid, base and neutral aqueous solution are dependent up- on the constituent amino acids which form the polypeptide chain. The principal chemical features of the peptide chain will be embodied in the structure and chemical nature of the pendant R groups. The physical nature of the peptide chain will depend upon the repetition of the R groups along the chain. Regularity will encourage the crystalline state to form thus requiring greater interactions between the protein and the solvent to over- come the crystalline forces and cause solubilization. Irregular sequences of R will encourage amorphous arrangements and will considerably reduce the amount of interaction necessary for dissolution. 96.

Whcn the R groups are joined by chemical cross links, crystallinity is diminished but the protein will remain insoluble unless degraded, The polymer will swell however, because of the interaction between the chain and the solvent until the network has stored sufficient energy to counteract the solvent-polymer interactions through its own elastic force. The degree of swelling will be highly dependent on the chemical nature of the R gmups, the pH of tile solvent and the number of cross links. mrther changes in cross linking can occur by the introduction of multivalent ions which would tend to fora inter-molecular cross links. Finally, protein structure is complex in nature. It has been shown that some proteins can arrange into mare than one crystalline form (allotropy) and transitions from one to the other cam be readily obtained by subtle changes in pH or temperature, In addition proteins are subject to degradation (denaturization) either through cleaving of the main peptide chain (low probability), irreversible rearrangement of crystalline structure, disarrangement to a random structure, non-reversible dissolution, or altera- tion of pendant chemical groups by heat, ions, and pH changes. The fundamental causes of changes which take place in animal muscle tissue will be snore thoroughly understood once the chemistry and physics of large molecules has advanced to a stage where more detailed knowledge of the charscter of chemical bonding can be detennined more precisely at the mole- cular level*

MR. PEARSON: We will hold the questions for the time being, when we come to the final discussion we will allow time for the questions from the speakers.

Next topic this afternoon is the Physical Characteristics of Muscle Tissues as Related to Imbibed Water. George Wilson, American Meat Institute has consented to discuss this,

MR, GM)RGE WILSON: Thank you, Al. I think at this point I feel myself in a position that we Dr. Ibty has, Dr. Schweigert has on occasion at the Foundation, of explaining to some of our contributors that we do have a basic f'undamental program on whether it is meat hydration or meat pigments, or whatever it might be, to answer their question as to has this got anything to do with the price of hot dogs, or the price of mutton. I think perhaps it is the sssignment, some of the rest of us on the program, I hope others will be able to come through to explain a little bit what effects properties of colloids do have on the price of fra.nkCurters or some other commodity that we put on the market.