92 . SOHE GENERAL PHYSICAL A N D CHENICAL

PROPERTIES OF PROTEINS

The large c lass of compounds now c lass i f ied as "pmteins" repre- sent a polycomplexity of individual species, yet, a l l have many common character is t ics , are constructed f r o m similar chemical un i t s and exhibit similar physical and chemical properties. a var ie ty of chemical molecules which a re broadly c lass i f ied as a-amino acids. a-amino acids a re caref'ully selected and one added t o another is a specif ic manner u n t i l the growth process i s terminated. Nature is specif ic i n the select ion of the correct amino acid at the t i m e it is needed i n the forma- t i on of the polymer chain.

Proteins are biosynthesized f r o m

As the synthesis proceeds and the protein chain i s formed, the

The chemical character is t ics of the proteins depend upon the indi-

That is, the vidual amino acids un i t s present i n the complete protein molecule and the sequence i n the un i t s as they are placed i n the polymer chain. chemical properties w i l l not only be influenced by which amino acid has en- te red the chain but a l so upon i ts nearest neighbors. However, some of t h e physical properties w i l l be re la t ive ly independent of the chemical constmc- t i o n and will resu l t mostly from the fact that proteins j u s t as other syn- t h e t i c high molecular weight compounds, are i n essence, giant molecules whose physical behavior i s memly a ref lect ion of their enormous s ize .

The chemical un i t s 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 i s asymmetric and thus exists i n two, op t ica l isomeric forms, the right (d) and l e f t (1) hand molecules. Since nature produces and u t i l i z e s only a-srrcino acids i n the l-form the joining of d and 1 uni t s does not become a complication i n protein structure.

The R group attached t o the asymmetric carbon atom is responsible R can be either pure f o r the d is t inc t ion of one pamino acid from another.

hydrocarbon i n chemical nature o r may contain other elements such as oxygen and nitrogen carboxylic acid o r amino groups.

However, the simple molecule described above has other more in- te res t ing features since it contains both a basic substi tuent (-NHz) and an

93.

acidic substi tuent (-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 s t ructure as shown i n I11 resu l t s where the amino acid be come 8

0

a cation. acid t o the negative electrode (cathode) o r the posit ive electrode (anode) w i l l depend not dnly on the individual amino acid but the pH of the media i n which it I s dissolved.

Consequently i n an e lec t ro ly t ic c e l l the migration of an amino

The abil i ty of 8 molecule t o behave both as a base and a6 an acid is termed amphoterism and the molecules ampholytes. l imited t o amino-acids but is also of property of many simple metal hy- droxides.

This phenomena is not

The acid strength of an amino acid i n aqueous solution w i l l depend pr incipal ly on the substi tuent R. of R sow amino acids i n aqueous solution w i l l be acidic in nature (pH < 7), Amino acids of this type are weak acids and a portion of the carboxylic acid groups interact wIth water and dissociate as indicated below (IV).

Depending on the s t ructure and composition

Other amino acids w i l l in te rac t with water at the amine s i te a8 shown in (V),

0 I ,

H 1

H 0 I

and thus give basic aqueous solutions. water as i n IV and V SO that no net change i n s t ructure resu l t s and the amino acid is essent ia l ly neutral i n nature.

S t i l l others react equivalently with

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By proper adjustment of the pH of an aqueous solution of amino acids LL point is reached where the net e l e c t r i c a l charge on the amino acid is zero. Under these specific conditions the molecule would not migrate t o either electrode under the influence of an e lec t r i c field. The pH where t h i s phenomena is exhibited is termed the i soe lec t r ic point and is a char- a c t e r i s t i c physical property of a l l amino acids. IWy other physical prop- e r t i e s such as membrane potential , op t ica l rotation, solubi l i ty , diffusion, s t a b i l i t y and resistance t o denaturation w i l l show a maxima o r minima at t h i s unique pH.

When proteins a= formed the amino acids a c t as difunctional monomers and polycondense by eliminating water t o form amide linkages and a polypeptide chain of the nature i l l u s t r a t ed by(V1)

Although the amine and carboxylic acid groups are destroyed i n the peptide fomation, the protein so formed may retain free amino o r acid groups as dangling side groups (pendant groups as embodied in R) o r as terminal un- reacted end groups. take part i n pH t i t r a t i o n s a l l residual amine and acids groups w i l l . proteins are Ellso mpholytes and exhibit an isoelectr ic point where proper- ties such as Solubility, solvent swelling snd molecular size i n solution w i l l be dras t ica l ly affected by slight a l te ra t ion of the pH. In f ac t , the i soe lec t r ic point can be more sharply defined f o r proteins than f o r many of i t s contributing a-amino acids when the f o m r is measured by the Tisel ius electrophoresis method.

However, even though the peptide linkage will no longer Thus

In t h i s method a dissolved aqueous solution of protein is placed i n an e lec t r i c f ie ld and the pH of the solution is changed by t i t r a t i o n with an acid o r base. The moving boundary of ions can be followed by su i t - able instrumental methods and when the boundary ceases t o move in the field the molecule is i n the isoelectr ic s t a t e .

Many of the physical character is t ics of proteins depend upon the i r s t r u c t u E and the f a c t t ha t they a m giant molecules. the proteins as a class generally have regions of high c rys ta l l in i ty . ever, as i n a l l long chain molecules it is impossible f o r proteins of high molecular weight t o completely crystal l ize . This resul ts f r o m the compli- cations involved i n making a molecule composed of many segments rearrange each segment i n a specific order within the chain and with respect t o its nearest neighbor molecules i n a reasonable period of time. Thus proteins which c rys ta l l ize will contain both crystalline regions where high loca l order persists, and amorphous regions where very l i t t l e order exis ts . It is these la t ter regions which are the most vulnerable t o the action of solvents.

Structure wise, How-

These types of protein molecules behave somewhat as synthetic net- work molecules such as Vulcanized rubber and w i l l reach an equilibrium

95 . degree of swelling providing the imbibed solvent-polymer interactions a re too weak t o overcome the forces of c rys ta l l in i ty . The crys ta l l ine content of proteins can be both enhanced and destroyed by sui table changes i n pH o r metal ion strength of imbibed water. Some proteins whose c rys t a l l i n i ty has been destroyed by dissolution w i l l f a i l t o recrys ta l l ize after removel f r o m the solvent and are thus considered denatured o r a l te red i n an irre- versible fashion.

While some protein molecules are joined together by a mutual sharing of some of their segments i n a c rys t a l l i t e , others are cross linked i n t e m i t t e n t l y by covalent chemical bonds. t i o n tends t o reduce the symmetry of a protein chain thus resul t ing i n low degrees of c rys t a l l i n i ty . broken and the resul t ing chains solubilized. However, as cross l inks they are network junction points and great ly influence cer ta in physical proper- t ies of the proteins (such as equilibrium swelling, modulue of e l a s t i c i ty , e tc . ) .

This type of r e s t r i c -

Often these cross l inks can be select ively

One of the properties affected i s the degree t o which a network may be swollen by imbibing solvent. res t r ic t ions the polymer w i l l swell with solvent u n t i l the elastic retrac- t i v e energy of the chain reaches a state of equilibrium with energy re- su l t ing from solvent-protein interactions. swelling should be reproducible once the conditions f o r equilibrium are reached and providing no degradation of protein takes place.

With the cross l inks act ing as

The equilibrium degree of

Several fac tors a f f ec t the degree t o which a polymer network w i l l swell before reaching equilibrium, t i o n of cross links. gree of swelling. The longer the chains the fewer cmss l inks needed t o bring about a three dimensional system. nature of the protein, that is, the type and dis t r ibut ion of R groups along the backbone of the chain.

The f irst i s the number and d is t r ibu- The more cross links i n a network, the lower the de-

Second is the molecular weight of the individual chains.

Finally a third important f ac to r is the chemical

A s u r f e i t of hydrophobic R groups w i l l decrease the equilibrium swelling of the protein by water whereas a large col lect ion of ionizable (basic o r ac id ic ) groups i n R w i l l cause the protein t o swell considerably. When pendant ionizable groups are present, the equilibrium swelling w i l l incmase a t both high and low pH, but will reach a minimum a t the iso- e l e c t r i c point just aa so lub i l i t y does f o r soluble proteins,

In summary, the chemical properties of proteins, i n par t icu lar their behavior i n acid, base and neutral aqueous solution are dependent up- on the constituent amino acids which form the polypeptide chain. pr incipal chemical features of the peptide chain w i l l be embodied i n the s t ructure and chemical nature of the pendant R groups. of the peptide chain w i l l depend upon the repet i t ion of the R groups along the chain. requiring greater interactions between the protein and the solvent t o over- come the c rys ta l l ine forces and cause solubilization. of R will encourage amorphous arrangements and w i l l considerably reduce the amount of interact ion necessary f o r dissolution.

The

The physical nature

Regularity w i l l encourage the crys ta l l ine state t o form thus

Irregular sequences

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Whcn the R groups a re joined by chemical cross l inks, c rys t a l l i n i ty is diminished bu t the protein w i l l remain insoluble unless degraded, The polymer will swell however, because of the interaction between the chain and the solvent u n t i l the network has stored suf f ic ien t energy t o counteract the solvent-polymer interactions through i ts own e l a s t i c force. swelling w i l l be highly dependent on the chemical nature of the R gmups, the pH of tile solvent and the number of cross links. mrther changes i n cross l inking can occur by the introduction of multivalent ions which would tend t o fo ra inter-molecular cross l inks.

The degree of

Finally, protein s t ructure is complex i n nature. It has been shown that some proteins can arrange in to mare than one c rys ta l l ine form (allotropy) and t rans i t ions f r o m one t o the other cam be readily obtained by subt le changes i n p H o r temperature, degradation (denaturization) e i t h e r through cleaving of the main peptide chain (low probabili ty), i r revers ib le rearrangement of crystal l ine structure, disarrangement t o a random structure , non-reversible dissolution, o r a l te ra - t i o n of pendant chemical groups by heat, ions, and pH changes.

In addition proteins are subject t o

The fundamental causes of changes which take place i n animal muscle tissue will be snore thoroughly understood once the chemistry and physics of large molecules has advanced t o a stage where more detai led knowledge of the charscter of chemical bonding can be detennined more precisely a t the mole- cu lar l eve l *

MR. PEARSON: We w i l l hold the questions f o r the time being, when we come t o the final discussion we w i l l a l low time f o r the questions from the speakers.

Next topic t h i s afternoon is the Physical Characterist ics of Muscle Tissues as Related t o Imbibed Water. In s t i t u t e has consented t o discuss th i s ,

George Wilson, American Meat

MR, GM)RGE WILSON: Thank you, Al. I think a t t h i s point I f e e l myself i n a posit ion that we Dr. Ibty has, Dr. Schweigert has on occasion at the Foundation, of explaining t o some of our contributors that we do have a basic f'undamental program on whether it i s meat hydration o r meat pigments, o r whatever it might be, t o answer their question as t o has t h i s got anything t o do with the price of hot dogs, o r the pr ice of mutton. think perhaps it is the sssignment, some of the rest of us on the program, I hope others w i l l be able t o come through t o explain a l i t t l e b i t w h a t e f f ec t s properties of colloids do have on the price of fra.nkCurters o r some other commodity that we put on the market.

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