Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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PROTEIN

Contents

Chemistry

Food Sources

Determination and Characterization

Requirements

Functional Properties

Interactions and Reactions Involved in Food Processing

Quality

Digestion and Absorption of Protein and Nitrogen Balance

Synthesis and Turnover

Deficiency

Heat Treatment for Food Proteins

Sources of Food-grade Protein

Chemistry

J K P Weder and H-D Belitz

,TechnischeUniversita

nchen, Garching, Germany

Background

0001 Like peptides, proteins are formed from amino acids

through amide linkages. Covalently bound hetero

constituents can also be incorporated into proteins.

For example, phosphoproteins such as milk casein or

phosvitin of egg yolk contain phosphoric acid esters

of serine and threonine residues. Glycoproteins, such

as k-casein, various components of egg white and egg

yolk, collagen from connective tissue, and serum pro-

teins of some species of fish contain one or more

monosaccharide or oligosaccharide units bound

O-glycosidically to serine, threonine, or d-hydroxy-

lysine or N-glycosidically to asparagine.

0002 The structure of a protein is dependent on the

amino acid sequence (the primary structure) which

determines the molecular conformation (secondary

and tertiary structures). Proteins sometimes occur

as molecular aggregates which are arranged in an

orderly geometric fashion (quaternary structure).

Amino Acid Sequence

Amino Acid Composition and Subunits

0003 Sequence analysis can only be conducted on a pure

protein. First, the amino acid composition is deter-

mined after acid hydrolysis. The procedure (separ-

ation on a single cation exchange resin column and

color development with ninhydrin reagent) has been

standardized and automated (amino acid analyzers).

More recently, derivatization of amino acids to form

fluorescent derivatives, e.g., with o-phthaldialdehyde

(OPA) and 2-mercaptoethanol, followed by reversed-

phase high-performance liquid chromatography

(RP-HPLC) has been introduced (precolumn deriva-

tization). The use of chiral thiols in this procedure

allows the separation of amino acid enantiomers.

0004Selenocysteine has been detected as a relatively new

protein amino acid in glutathione peroxidase (EC

1.11.1.9) in 1973, and later also in other oxidoreduc-

tases and other proteins (e.g., in selenoprotein P, 10

residues per molecule). The incorporation of seleno-

cysteine occurs during ribosomal protein synthesis

cotranslationally by a complex mechanism in which

a specific tRNA

(ser)sec

is first loaded with serine, then

transformed into selenocysteyl-tRNA

(ser)sec

with sele-

nophosphate as the selenium donor and bound with

its anticodon region to a UGA codon of the mRNA.

Some selenocysteine, however, is also formed by

posttranslational selenium–sulfur exchange, e.g., in

selenoprotein P.

0005It is also necessary to know the molecular weight of

the protein. This is determined by gel column chro-

matography, ultracentrifugation, sodium dodecyl

sulfate–polyacrylamide gel electrophoresis (SDS-

PAGE) or mass spectrometry (matrix-assisted laser

desorption/ionization time-of-flight mass spectrom-

etry, MALDI-TOF-MS, or electrospray ionization

mass spectrometry, ESI-MS). Furthermore, it is neces-

sary to determine whether the protein is a single

molecule or consists of a number of identical or

different polypeptide chains (subunits) associated

through disulfide bonds or noncovalent bonding.

Deceased.

PROTEIN/Chemistry 4805

Dissociation into subunits can be accomplished by a

change in pH, by chemical modification of the pro-

tein, such as by succinylation, or with denaturing

agents (urea, guanidine hydrochloride, sodium dode-

cyl sulfate). Disulfide bonds, which are also found in

proteins consisting of only one peptide chain, can be

cleaved by oxidation of cystine to cysteic acid or by

reduction to cysteine with subsequent alkylation of

the thiol group to prevent reoxidation. Separation of

subunits is achieved by chromatographic or electro-

phoretic methods.

Terminal Groups

0006 N-Terminal amino acids can be determined by

treating a protein with 2,4-dinitrofluorobenzene

(DNFB, Sanger’s reagent) or 5-

(dimethylamino)naphthalene-1-sulfonyl chloride

(dansyl chloride). Another possibility is the reaction

with cyanate, followed by elimination of the N-ter-

minal amino acid in the form of hydantoin, and sep-

aration and recovery of the amino acid by cleavage of

hydantoin. The N-terminal amino acid (and the

amino acid sequence close to the N-terminus) is ac-

cessible by hydrolysis with aminopeptidase M (EC

3.4.11.2), in which case it should be remembered

that the hydrolysis rate is dependent on amino acid

side chains and that proline residues are not cleaved.

A special procedure is required when the N-terminal

residue is acylated (N-formyl- or N-acetyl-amino

acids, or pyroglutamic acid).

0007 Determination of C-terminal amino acids is

possible via the hydrazinolysis procedure. The C-ter-

minal amino acid is separated from the amino acid

hydrazides, e.g., by a cation exchange resin, and iden-

tified. The C-terminal amino acids can be removed

enzymatically by carboxypeptidase A (EC 3.4.17.1),

which preferentially cleaves amino acids with aro-

matic and large aliphatic side chains; carboxypepti-

dase B (EC 3.4.17.2), which preferentially cleaves

lysine, arginine, and amino acids with neutral side

chains; carboxypeptidase C (EC 3.4.16.1), which

cleaves with less specificity and cleaves proline; car-

boxypeptidase P (EC 3.4. 16.1), hydrolyzing almost

all amino acids (including proline, aspartic acid,

and glutamic acid; release of serine and glycine is

retarded); or carboxypeptidase Y (EC 3.4. 16.1),

which shows high catalysis rate if the penultimate

and/or terminal residue is an aromatic or aliphatic

amino acid including proline (release of glycine and

aspartic acid is considerably retarded).

Partial Hydrolysis

0008 Longer peptide chains are usually fragmented. The

fragments are then separated and analyzed individu-

ally for amino acid sequences. Selective enzymatic

cleavage of peptide bonds is accomplished with tryp-

sin (EC 3.4.21.4), which only cleaves Lys-X and Arg-

X bonds; chymotrypsin (EC 3.4.21.1), which cleaves

peptide bonds with less specificity (Tyr-X, Phe-X,

Trp-X and Leu-X); and, more recently, with endopro-

teinase Arg-C (EC 3.4.22.8), hydrolyzing only Arg-X

bonds; endoproteinase Asp-N (EC 3.4.24.33), hydro-

lyzing X-Asp and X-CySO

H bonds; endoproteinase

Glu-C (EC 3.4.21.19), hydrolyzing Glu-X or Glu-X

and Asp-X bonds depending on pH and buffer;

or endoproteinase Lys-C (EC 3.4.21.50), hydrolyzing

only Lys-X bonds. The enzymatic attack can be

influenced by modification of the protein.

0009The most important chemical method for selective

cleavage uses cyanogen bromide (BrCN) to attack

Met-X linkages. Separation of peptide fragments is

achieved by gel and ion exchange column chromatog-

raphy using a volatile buffer as the eluent (e.g., pyri-

dine, morpholine acetate) which can be removed by

freeze-drying of the fractions collected. Recently the

separation of peptides and proteins by RP-HPLC has

gained great importance, using volatile buffers mixed

with organic, water-soluble solvents (e.g., aceto-

nitrile) as the mobile phase.

0010The fragmentation of the protein is performed by

different enzymatic and/or chemical techniques, at

least by two enzymes of different specificity. The

arrangement of the peptides obtained, in the same

order as they occur in the intact protein, is accom-

plished with the aid of overlapping sequences.

Sequence Analysis

0011The Edman degradation is by far the most important

method in sequence analysis. It involves stepwise

degradation of peptides with phenyl isothiocyanate,

starting at the N-terminus of the polypeptide. The

resultant phenylthiohydantoin is either identified

directly or the amino acid is recovered. The stepwise

reactions are performed in solution or on peptide

bound to a carrier, i.e., to a solid phase. Both ap-

proaches have been automated (sequencer). Carriers

used include resins containing amino groups (e.g.,

aminopolystyrene) or glass beads treated with

aminoalkylsiloxane. The peptides are then attached

to the carrier by carboxyl groups (activation with

carbodiimide or carbonyl diimidazole, as in peptide

synthesis) or by amino groups. Nowadays, Edman

degradation has been miniaturized and is performed

on gas-phase or pulsed-liquid-phase sequencers, in

which the peptide is adsorbed on a polybrene-treated

glass-fiber disk and the reagents are applied either as

vapors in a carrier gas stream or in small liquid doses

separated by carrier gas bubbles.

4806 PROTEIN/Chemistry

0012 Methods for C-terminal sequencing have also

been reported. After activating the polypeptide at

the a -carboxyl group with acetic anhydride, the

activated polypeptide is reacted with thiocyanic

acid (formed from ammonium thiocyanate with an

acid) and the resulting thiohydantoin is cleaved with

a base to give the thiohydantoin of the C-terminal

amino acid and the polypeptide reduced by one

amino acid residue. Another procedure uses diphenyl

phosphoroisothiocyanatidate (DPP-ITC), pyridine

for reaction and cyclization, and sodium trimethylsi-

lanoate for cleavage. A modification of the first

procedure has been automated including S-alkyla-

tion of the thiohydantoin with 2-(bromomethyl)-

naphthalene and cleavage of the C-terminal amino

acid derivative simultaneously with the formation of

the next thiohydantoin by addition of ammonium

thiocyanate plus trifluoroacetic acid. It should be

mentioned that C-terminal sequencing procedures

are by far less sensitive than Edman degradation

and that only a few steps can be interpreted without

doubt.

0013 Methods other than chemical degradations can

provide additional information. These include deter-

mination of terminal residues with amino- and

carboxypeptidases, as already discussed, or mass

spectrometry. Mass spectrometric sequencing can be

done by MALDI-TOF-MS (peptide ladder sequencing

and postsource decay (PSD)-MALDI) or ESI-MS

(atmospheric pressure ionization collision-induced

decay, API-CID; single or multiple fragmentation of

selected ions, MS-MS or MS

). Peptide ladder sequen-

cing combines a modified Edman degradation (in-

complete by the addition of 5% phenyl isocyanate)

with the determination of the molecular masses of the

resulting mixture consisting of phenylcarbamoyl de-

rivatives of peptides stepwise reduced by one amino

acid. Because of the small difference between the

molecular mass of aspartic and glutamic acid and

their amides, results can only be interpreted up to

5 kDa. The other two methods include fragmentation

of the peptide chain, and unknown sequences can

only be interpreted up to 2 kDa without doubts. The

main area of mass spectrometric sequencing is the

confirmation of already known sequences. None of

the methods can differentiate between leucine and

isoleucine.

0014 Recently, amino acid sequences of proteins have

increasingly been deduced from the nucleotide

sequences of the corresponding genes. Examples of

food proteins are gliadins and glutenins from wheat.

If the gene responsible for the expression of a protein

is known, the sequence analysis of the nucleic acid

can be performed much easier than that of the protein

itself.

0015Examples for amino acid sequences of food pro-

teins are summarized in Tables 1–3 (legume 11S glo-

bulins, legume 7S globulins, wheat proteins). Further

examples (milk proteins, collagen, monellin, and

thaumatin) are presented in the first edition of this

Encyclopedia.

Conformation

0016Information about conformation is available through

X-ray crystallographic analysis of protein crystals

and through the determination of the H–H distances

by proton nuclear magnetic resonance (NMR) spec-

troscopy in solution. In principle, the conformation of

the protein in crystalline form can be assumed to be

similar to that of the protein in solution. In 1960 the

structure of myoglobin (17.8 kDa) was elucidated

with a resolution of 0.2 nm. Individual atoms are

well revealed at 0.11 nm. Such a resolution has only

been achieved with few proteins. Reliable localization

of the C

atoms of the peptide chain requires a reso-

lution of less than 0.3 nm. Most protein conform-

ations have been studied with a resolution between

0.2 and 0.3 nm and, more recently, between 0.15 and

0.2 nm.

Extended Peptide Chains

0017X-ray structural analysis and other physical measure-

ments of a fully extended peptide chain reveal the

lengths and angles of bonds. The peptide bonds have

partial (40%) double-bond character with p-electrons

shared between the C

–OandC

–N bonds. The res-

onance energy is about 83.6 kJ mol

1

. Normally the

bond has a trans configuration, i.e., the oxygen of the

carbonyl group and the hydrogen of the NH group

are in the trans position; a cis configuration only

occurs in exceptional cases (e.g., in small cyclic

peptides or in proteins before proline residues). Be-

cause of the partial double-bond character, six atoms

of the peptide bonds, C

iþ1

and C

iþ1

lie in one plane. For a trans peptide bond, o

is 180



The position of two neighboring planes is determined

by the numerical value of the angles c

(rotational

bond between a carbonyl carbon and an a-carbon)

and f

(rotational bond between an amide nitrogen

and an a-carbon). For an extended peptide chain c

180



and f

¼ 180



. The position of side chains can

also be described by a series of angles w

1n

Secondary Structure (Regular Structural Elements)

0018The primary structure gives the sequence of amino

acids in a protein chain whereas the secondary and

tertiary structure reveal the arrangement of the chain

in space. The peptide chains are not in an extended or

PROTEIN/Chemistry 4807

unfolded form (c

, f

6¼ 180



). It can be shown with

models that c

and f

, at a permissible minimum

distance between atoms can assume only particular

angles. It has been shown for many proteins empiric-

ally that they have values of c

, f

pairs within the

permissible range. When a multitude of equal c

, f

pairs occurs consecutively in a peptide chain, the

chain acquires regular repeating structural elements

(secondary structure). Examples of some important

secondary structures are shown in Figures 1–3.

0019 b-Sheet Three regular structural elements (pleated-

sheet structures) have values in the range of f ¼

120



and c ¼þ120



. The peptide chain is always

lightly folded on the C

atom, thus the R side chains

extend perpendicularly to the extension axis of the

chain, i.e., the side chains change their projections

alternately from þ z to z. Such a pleated structure

is stabilized when more chains interact along the x

axis by hydrogen bonding, thus providing the cross-

linking required for stability. When adjacent chains

run in the same direction, the peptide chains are

parallel. This provides a stabilized, planar, parallel

sheet structure (Figure 1). When the chains run in

opposite directions, a planar, antiparallel sheet struc-

ture is stabilized (Figure 2). The lower free-energy,

twisted sheet structures, in which the main axes of the

neighboring chains are arranged at an angle of 25



are more common than planar sheet structures.

0020The b-structures can also be regarded as special

helical structures with a continuation of two residues

per turn. With proline, the formation of a b-structure

is not possible.

0021Helical structures There are three regular structural

elements in the range f ¼60



and c ¼60



,in

which the peptide chains are coiled like a threaded

screw. These structures are stabilized by intrachain

hydrogen bridges which extend almost parallel to the

helix axis, cross-linking the CO and NH groups,

specifically the CO group of amino acid residue i

with the NH group of residue iþ3(3

-helix), iþ4

(a-helix) or iþ5(p-helix). The a-helix, and for poly-

peptides from l-amino acids exclusively the right-

handed a-helix, is the most common (Figure 3). The

-helix is only observed at the ends of a-helices,

whereas the existence of a p-helix is hypothetical. A

helix is characterized by the angles f and c, or by the

tbl0001 Table 1 Amino acid sequences of 11S globulins from legumes

Glycinin A

from soya bean, a polypeptide

505050 5050

1 L REQAQQNE CQI QKL N AL KPDNRI ESEGGF I ET WNPN NKPFQC AGVAL SR

51 CTLNRNALRRPSYT NGPQEI YI QQGNGI F GMI FP GCP STYQEP QESQQRG

101 RSQRPQDRHQKVHRFREGDLI AV PTGVAWWMYNNEDT PVVAVS I I DTNSL

151 ENQL DQMPR RF YL A GN QE QEF L K Y QQQQQ GGSQS QKG KQQE E E NE GSN I L

201 SGFAPEF LKEAFGV NMQI VRNLQGENEEE DSGAI VTVKGGLRVTAPAMRK

251 PQQEEDDDDEEEQP QCVETDKGCQRQSKRS RN

Glycinin A

from soya bean, a polypeptide

505050 5050

1 GI DETI CTMRL RQN I GQNSSPDI YNPQAGS I T T ATSL DF PALWL L KLS AQ

51 YGSL RKN AMFVPHY TL NANSI I Y AL NGRA L V QVV NCN GERVF D GELQE GG

101 VL I VPQNFAVAAKS QS DNF EYVS FKTNDR P S I GNL AGANSL L N ALPEE VI

151 QHT F NLKSQQARQV KNNNPFSF L VPPQES QR RAV A

Legumin J from pea, b polypeptide

505050 5050

1 L ATSSEF DR L NQCQL DSI NAL EP DHRVVS E A GL T ETWNPNHPE L KCAGVS

51 LI RRTI DPNGLHLP SF SPSPQLI FI I QGKGVLGL SFPGCPETYEEPRSSQ

101 SRQESRQQQGDSHQKV RRFRKGDI I AI PS GI PYWT YN HGDEPL VAI SL L D

151 TSNI ANQLDST PRV FY L GGNPET EFPET QE E QQGRHR QKHSYP VGRRS GH

201 HQQEEESEQQNEGNSVLSGFSSEFLAQTF NT EEDTAKRLRSPRDERSQI V

251 RVEGGLRI I KGRTE EE KEQSHSHSHREEKE E EEE EEE DEEEKQRSEERKN

Legumin J from pea, b polypeptide

505050 5050

1 GLEETI C SAKI RENI ADAARADL YNPRAGR I STA NSL T L PVLR YLRLS AE

51 YVRLYRNGI YAPHWNI NANSL L Y VI RGEGRV RI RSCE LPT NTMF DNKL RK

101 GHLVVVPQNFVVAE QAGEEEGLEYVVFKT NDRAAVSHVQQVFRATPSEVL

151 ANAFGL R QR QVTEL KL SGNRGPMVHPRSQS QSH

Amino acids are given in the International Union of Pure and Applied Chemistry one-letter notation. The two polypeptides of glycinin and legumin

are connected by a disulfide bond Cys(a84)-Cys(b7) and Cys(a89)-Cys(b7), respectively. The two remaining cysteine residues of the a subunits (10/43 and

13/46, respectively) probably form an intrachain disulfide bridge.

Data from Wright DJ (1987) The seed globulins. In: Hudson BJF (ed.) Developments in Food Proteins, vol. 5, pp. 81–157. London: Elsevier Applied Science.

4808 PROTEIN/Chemistry

parameters derived from these angles: n, the number

of amino acid residues per turn; d, the rise along

the main axis per amino acid residue; and r, the radius

of the helix. Thus, the equation for the pitch, p,is

p ¼nd.

0022 Reverse turns An important conformational feature

of globular proteins is the reverse turns, b-turns, or

b-bends. They occur at ‘hairpin‘ corners, where the

peptide chain changes direction abruptly. Such

corners involve four amino acids residues, among

them frequently proline. Glycine is favored in the

third position of b-bends on the basis of energy con-

siderations. Different types of b-turns are known, for

which different amino acids are allowed.

0023 Super secondary structures Analysis of known

structures has demonstrated that regular elements

can exist in combined forms. Examples are the

coiled-coil a-helix, chain segments with antiparallel

b-structures (b-meander structure) and combinations

of a-helix and b-structure (e.g., baba).

Tertiary and Quaternary Structure

0024Proteins can be divided into two large groups on the

basis of conformation: (1) fibrillar (fibrous) or sclero-

proteins, and (2) folded or globular proteins.

0025Fibrous proteins The entire peptide chain is packed

or arranged within a single regular structure for a

variety of fibrous proteins. Examples are wool

keratin (a-helix), silk fibroin (b-sheet), and collagen

(a triple helix). Stabilization of these structures is

achieved by intermolecular binding (electrostatic

interaction and disulfide linkages, but primarily

hydrogen bonds and hydrophobic interactions).

tbl0002 Table 2 Amino acid sequences of 7S globulins from legumes

b-Conglycinin from soya bean, a subunit

50 5 0 5 05 050

1 VEE EEECEEGQI P RPRP QHPERE RQQHGE KEEDEGEQP RPF PFPRPRQPH

51 QEEEHEQKEEHEWHRKE EKHGGKGSEEEQDEREHPRP HQPHQKEEEKHEW

101 QHK QEKHQGKESE EEEE DQDEDE EQDKES QESEGSES QREPRRHKNKNPF

151 HF NSKRFQTL F KN QYGHVRVL QR F NKRSQQLQNLRDY R I L EFNSKPNTLL

201 L PH HADADYL I VI L NGT AI L T L V NNDDRDSYNL QSGD A LRVPAGTTFYVV

251 NPDNDENL RMI AGTTFY VVNPDN DENL RMI T LAI PVNK PERF ESFFL SST

301 QAQQSYL QGF SKN I LEA SYDT KF EEI NKV LF GREEGQQQGEERLQESVI V

351 EI S KKQI REL SKHAKSS SRKTI S SEDKPF NL GSRDPI Y SNKL GKL F EI TQ

401 RNP QLRDL DV FLS VVDMNEGAL F L PHF NS KAI VVLVI N EGEANI ELVGI K

451 EQQQRQQQEE QPL EVRKYRAELSEQDI FVI PAGYPVMVNATSDLNFFAFG

501 I NA ENNQRNF L AGSKDNVI SQI P SQVQEL AF PRSAKDI ENL I KSQSESYF

551 VDAQPQQKEE GNK GRKGPL SSI L RAF Y

Vicilin from pea

50 5 0 5 05 050

1 RSDPQNPFI F KSNKFQTLFENENGHI RLLQKFDQRSKI FENLQNYRLLEY

51 KSK PHTI F LP QHT DADY I LVVL S GKAI LT VL KPDDRNS FNERGDTI KL PA

101 GTI AYLVNRDDNEELRVLDLAI PVNRPGQLQSFLLSGNQNQQNYLSGFSK

151 NI L EASF NT D YEE I EKV L L EEHE KET QHRRSL KDKRQQSQEENVI VKL SR

201 GQI EEL SKNAKST SKKS VSSESE PFNL RS RGPI YSNE P GKFF EI T PEKNP

251 QLQDLDI F VNSVE I KEGSL L L PH YNSRAI VI VTVNEGK GDFEL VGQRNEN

301 QQE QRKEDDE EEE QGEE EI NKQV QNYKAKL SSGDVF V I PAGHPVAL KASS

351 NL DLLGF GI NAEN NQRNFL AGDE DNVI SQVQRPVKEL AFPGSAQEVDRI L

401 ENQKQSHF AD AQP QQRE RGSRET RDRL SS V

Phaseolin from garden bean, b subunit

50 5 0 5 05 050

1 ATS LREEEES QDN PF YF NSDNSWNTLF KN QYGHI RVL QRFDQQSKRL QNL

51 EDY RLVEF RS KPE TLLL PQQADA ELLL VV RSGSAI L V L VKPDDRREYFFL

101 TQGDNPI FSDNQKI PAGTI FYLVNPDPKEDLRI I QLAMPVNNPQI HEFFL

151 SSTEAQQSYL QEFSKHI LEASFNSKFEEI NRVLFEEEGQQEEGQQEGVI V

201 NI D SEQI EEL SKH AKSS SRKSHS KQDNTI GNEFGNL T E RTDNSL NVL I SS

251 I EMKEGALFV PHYYSKAI VI LVVNEGEAHVELVGPKGNKETLEFESYRAE

301 L SKDDVFVI P AAY PVAI KATSNV NFTGF GI NANNNNRNLLAGKTDNVI SS

351 I GR AL DGKDV L GL T F S G S GE E V MKL I NKQSGS Y F VDGH HHQQE QQKGSHQ

401 Q E Q Q K G R K G A F V Y

Data from Wright DJ (1988) The seed globulins – part II. In: Hudson BJF (ed.) Developments in Food Proteins, vol. 6, pp. 119–178. London: Elsevier Applied

Science, and references therein.

PROTEIN/Chemistry 4809

tbl0003 Table 3 Amino acid sequences of gluten proteins from wheat

HMW subunit of glutenin (x-type), 1Bx7 (Cheyenne)

5050505050

1EGE AS GQL QC E H EL EA C QQV V DQQ L RD VS P G CR P I T V SPGT R QYE Q QP V V

51PSKAGSFYPSETTPSQQLQQMI FWGI PALLRRYYPSVTSSQQGSYYPGQA

101SP Q QS GQ G Q Q P G QE QQP GQGQ QHQ Q P GQR QQ GY Y P T S PQQ P GQGQQL GQG

151QP G Y Y P T S Q Q PG QKQQA GQGQQS G Q GQQGYY P T S P Q Q SGQGQQP GQ GQP G

201YY P T S P QQ S GQWQQP G Q G QQP GQG Q QS GQ GQ QGQQP G QGQRP GQGQ QG Y Y

251P I S P QQP G Q G QQ S GQGQP GY Y P T S L RQ P GQWQQ P GQG QQP GQ GQQG QQ P G

301QG Q QS GQ G QQ GY Y P T S L Q QP G QGQ Q L GQG QP GY Y P T S QQS E Q GQQP GQ GK

351QP GQGQQGYY PT SPQQSGQGQQLGQGQPGYY PT SP QQSGQGQQSGQGQQG

401YY P T S P QQS GQG QQP GQGQSGYF P T S R QQSG QG QQ P GQGQQS GQGQ QG Q Q

451P G Q GQQA Y Y P T S S QQS R QRQQ AGQ WQR PGQG QP GY Y P T S P QQP GQE QQ S G

501QAQQS GQWQL VYYPTSPQQPGQLQQPAQGQQPAQGQQSAQEQQPGQAQQS

551GQWQWQL V Y Y PT SPQQP GQLQQPAQGQQGYYPT SP QQSGQGQQGYYPT SP

601QQ S GQ GQ Q GY Y P T S PQQ S GQGQQP G QGQQP R QGQQGY YP I S P QQS G QGQQ

651PGQGQQGYYPTS PQQS GQGQQPGHEQQPGQWLQPGQGQQGYYPTSS QQSG

701QGH QS GQGQQGY YPTS L WQPGQGQQGY AS PY HV SAE Y QAA RL KVAKAQQL

751AA QLP AMC R L EGSDAL S T RQ

HMW subunit of glutenin (y-type), 1Dy10 (Cheyenne)

5050505050

1EGE AS RQL QCER EL QE S S L EACRQV VD QQL A GR L P WS TGL QMRCCQQL RD

51VSAKCRSVAVSQVARQYEQTVVPPKGGSFYPGETTPLQQLQQGI FWGTSS

101QT V QGYY P GV T S P RQGS Y YP G QAS P QQ PGQG QQ P GK WQEP GQGQQWYY P T

151SL QQP GQ G Q QI G KGQQ GY Y P T SL QQ P GQGQQ GY YP T S L QH T GQRQQ P V Q G

201QQ P E Q GQ Q P GQWQQGY Y P T SP QQL GQGQQ P R QWQQ S G QGQQGHYP T SL QQ

251P G Q GQQGH Y L AS QQQP G Q GQQGHY P AS QQ QP GQGQQ G HYP AS QQQP GQGQ

301QGH YP AS Q Q E P G QGQQ G Q I P A SQQQ PGQGQQ GH YP A S L QQPGQGQQ GH Y P

351T S L QQ L GQ GQ QT GQP GQ K QQP GQGQ QT GQGQQP E Q E Q QP GQGQQGY YP T S

401L QQ P G QG Q Q Q GQ GQQG Y Y P T S L QQ P GQ GQ QG HY P A S L QQP GQ GQPG QR QQ

451PGQGQHP E QGKQPGQGQQGYYPTS P QQPGQGQQLGQGQQGYYPTSP QQP G

501QGQQP GQGQQGHCPTS PQQSGQAQQPGQGQQI GQVQQPGQGQQGYYPT SV

551QQ P GQ GQQS G QGQQSGQGHQP GQG Q QS GQ E Q QG Y D S P Y HV SA E QQA AS P M

601VAKAQQPATQLPTVCRMEGGDALSASQ

LMW subunit of glutenin, clone LMWG-1D1 (Chinese spring)

5050505050

1RCI PGLERPWQQQPLPPQQTFPQQPLFSQQQQQQLFPQQPSFSQQQPPFW

51QQ QP P F S Q Q Q PI L PQQP P F SQ QQQ L V L P Q QP P F S Q QQ QP V L P PQQS P F P Q

101QQ Q QH QQ L V Q QQ I P VV Q P SI L QQL N PC KV F L QQ QC S P VAMPQ RL AR SQML

151QQ S S C HV MQQ QC CQQL P Q I PQ QS R Y E A I R AI I Y S I I L QEQQQ V QGS I Q S Q

201QQQPQQL GQCVS QPQQQS QQQLGQQPQQQQL AQGT F L QPHQI AQLEVMT S

251I AL RI LPT MCSVNVPL YRTTT SVP F GV GT GV GAY

a-Gliadin clone A 1235 (Cheyenne)

5050505050

1VRV PVPQL QP QNPSQQQP QEQVPL MQQQQQF PGQQE QFPPQQPYPHQQPF

51P S Q QP Y P Q P Q PF P P QL P Y P QT QP F P P Q QP YP QP QP QY P QP QQ PI S Q QQA Q

101QQ Q QQQQ T L Q QI L QQQ L I P CR DVV L QQHN I A HA SS Q V L QQSS YQQL QQL C

151CQQLF QI P E QSR CQAI H NVVHAI I L HHHQQQQQQP S S QVS YQQPQE QY P S

201GQV SF QSSQQNPQAQGSVQPQQLP QFQEI RNLALQT L PAMCNVYI P PY CS

251TTI APFGI F GTN

g-Gliadin clone gene A (Yamhill)

5050505050

1NMQ V D P S G Q V QWP QQQP V L L P QQP F SQQP QQ T F P QP Q QT F P H QP QQ QF P Q

51PQQPQQQF L QPQQPFPQQPQQPYP QQP QQPF PQTQQP QQL FP QSQQPQQP

101YP Q QP QQP F P QT QQPQ Q Q F PQSQQ P QP F P QP QQ P QQ S F PQ QQ P SF I QP S L

151QQ Q L N P C K N L L L QQCR P V S L V S S L WS MI WP Q SA CQV MRQQ CC QQL A QI P Q

201QL QCA AI H S V V H SI S MQ E QQQQQQQ QQQQQQ QQGMR I L L P L Y QQQQ VGQG

251T L V QGQGI I QPQQPAQL E AI RSL V L QT L P TMCN VY V P PECSI I KAP F A S I

301VT GI GGQ

The high-molecular-weight (HMW) subunit (x-type) contains one intramolecular disulfide bond (Cys (10)-Cys (17)), whereas the remaining two cysteines

(Cys (32) and Cys (758)) form intermolecular disulfide bonds probably with other HMW subunits. In HMW subunits (y-type), the two adjacent cysteines

(Cys(43) and Cys(44)) form two intermolecular disulfide bonds with two adjacent cysteines of another y-type HMW subunit, Cys(513) forms an

intermolecular disulfide bond with low-molecular-weight (LMW) subunits, whereas the linkage of the remaining four cysteines is still unknown (probably

one intermolecular disulfide bond, Cys(10)-Cys(22)). The LMW subunit contains three intramolecular disulfide bonds (Cys(127)-Cys(162), Cys(135)-

Cys(155) and Cys(163)-Cys(260)), whereas the remaining two cysteines (Cys(2) and Cys(210)) form intermolecular disulfide bonds with other LMW

4810 PROTEIN/Chemistry

0026 Globular proteins Regular structural elements are

mixed with randomly extended chain segments

(random-coiled structures) in globular proteins.

The proportion of regular structural elements is

highly variable: 20–30% in casein, 45% in lyso-

zyme, and 75% in myoglobin. Five structural sub-

groups are known in this group of proteins: (1)

a-helices occur only; (2) b-structures occur only;

(3) a-helical and b-structural portions occur in sep-

arate segments on the peptide chain; (4) a-helices

and b-structures alternate along the peptide chain;

and (5) a-helices and b-structures do not exist. The

process of peptide chain folding is not yet fully

understood. It occurs spontaneously, probably

arising from one center or from several centers of

high stability in larger proteins. Folding of the pep-

tide chain packs it densely, by formation of a large

number of intramolecular noncovalent bonds. Data

about the nature of the bonds involved are provided

in Table 4.

subunits(headtoheadorheadtotail),y-typeHMWsubunitsorglutenin-bound g-gliadins. In a-gliadins,threeintramoleculardisulfidebondsoccur

(Cys(120)-Cys(150),Cys(151)-Cys(241)andCys(163)-Cys(249)). In g-gliadins,fourintramoleculardisulfidebondsoccur(Cys(157)-Cys(191),Cys(165)-

Cys(184),Cys(192)-Cys(282)andCys(204)-Cys(290)).

DatafromM

llerSandWieserH(1997)Thelocationofdisulphidebondsinmonomeric a-typegliadins.JournalofCerealScience26:169–176,ShewryPR

andTathamAS(1997)Disulphidebondsinwheatglutenproteins.JournalofCerealScience25:207–223andreferencestherein.

(a)

(b)

fig00 01 Figure 1 Parallel b-sheet( f ¼119



, c ¼þ113



,n¼2.0,d¼0.32nm,r¼0.11nm;seetext).(a)Perspectiveviewoftwodecapeptide

chainsconsistingof10alanineresidueseach. TheN-terminusofbothchainsisontheleft;hydrogenatomsofthemethylgroupshave

beenomittedforclearness;hydrogenbridgesarepresentedasbrokenlines. (b)Viewinthedirectionofthechainaxis.

(a)

(b)

fig00 02 Figure 2 Antiparallel b-sheet( f ¼139



, c ¼þ135



,n¼2.0,d¼0.34nm,r¼0.09nm).(a),(b)Cf. Figure 1,buttheN-terminusofthe

upper chain is on the left and that of the lower chain is on the right.

PROTEIN/Chemistry 4811

0027 The hydrogen bonds formed between main chains,

main and side chains, and side-side chains are of

particular importance for folding. The portion of

polar groups involved in hydrogen bond build-up in

proteins of > 8.9 kDa appears to be fairly constant at

about 50%. The hydrophobic interaction of the non-

polar regions of the peptide chains also plays an

important role in protein folding. These interactions

are responsible for the fact that nonpolar groups are

folded to a great extent towards the interior of the

protein globule. The surface areas accessible to water

molecules have been calculated for both unfolded and

native folded forms for a number of monomeric pro-

teins with known conformations. The proportion of

the accessible surface of a stretched state which tends

to be fixed in the interior of the globule as a result of

folding is a simple linear function of the molecular

weight.

0028Proteins with disulfide bonds fold at a significantly

slower rate than those without disulfide bonds.

Folding is not limited by the reaction rate of disulfide

formation. Therefore the folding process of disulfide-

containing proteins seems to proceed in a different

way. The reverse process, protein unfolding, is very

much slowed down by the presence of disulfide

bridges which generally impart great stability to

globular proteins. This stability is particularly effect-

ive against denaturation. An example is the Bowman–

Birk inhibitor from soya beans, which inhibits the

activity of trypsin and chymotrypsin. Its tertiary

structure is stabilized by seven disulfide bridges. The

reactive sites of inhibition are Lys(16)-Ser(17) and

Leu(43)-Ser(44), i.e., both sites are located in rela-

tively small rings, each of which consists of nine

amino acid residues held in ring form by a disulfide

bridge. The thermal stability of this inhibitor is high

(Figure 4).

0029Quaternary structures In addition to the free energy

gain by folding of a single peptide chain, association

of more than one peptide chain (subunit) can provide

(a) (b)

(c)

fig0003 Figure 3 Right-handed a-helix (f ¼57



, c ¼47



, n ¼ 3.3, d ¼ 0.15 nm, r ¼ 0.23 nm). (a) Perspective view of a helical peptide chain

consistingof15alanineresidues;theN-terminusisatthebottom;inotherrespectsitisthesameas Figure 1. (b)Samerepresentation

as (a) with a lined band elucidating the right-handed helix running from bottom to top. (c) View in the direction of the helix axis.

tbl0004 Table 4 Bond-types in proteins

Type Examples Bond strength (kJmol

1

)

Covalent bonds -S-S- 230

Electrostatic bonds -COO



...H

-21

>C¼O...O¼C<þ1

a,b

Hydrogen bonds -O-H . . . O< 17

>N-H . . . O¼C< 13

Hydrophobic bonds -Ala . . . Ala- 3

-Val . . . Val- 8

-Leu . . . Leu- 9

-Phe . . . Phe- 13

-Trp . . . Trp- 19

For a dielectric constant of the surrounding medium e ¼ 4;

weak

repulsion due to the negative partial charge of the oxygens in a polarized

C–O double bond.

Data from Belitz H-D and Grosch W (1999) Food Chemistry, 2nd edn. Berlin:

Springer–Verlag.

4812 PROTEIN/Chemistry

further gains in free energy. For example, hemoglobin

(four associated peptide chains) DG



¼46 kJ mol

1

and the trypsin–trypsin inhibitor complex (associ-

ation of two peptide chains) DG



¼75.2 kJ mol

1

In principle, such associations correspond to the

folding of a larger peptide chain around several

structural domains without covalently binding the

subunits.

0030 Examples for protein conformations are given in

Figure 5, which shows the sweet proteins monellin

and thaumatin.

Denaturation

0031 The term ‘denaturation‘ denotes a reversible or

irreversible change of native conformation (tertiary

structure) without cleavage of covalent bonds (except

for disulfide bridges). Denaturation is possible with

any treatment that cleaves hydrogen bridges, or ionic

or hydrophobic bonds. This can be accomplished by

changing the temperature, adjusting the pH, increas-

ing the interfacial area, or adding organic solvents,

salts, urea, guanidine hydrochloride, or detergents

such as sodium dodecyl sulfate. Denaturation is gen-

erally reversible when the peptide chain is stabilized

in its unfolded state by the denaturing agent and

the native conformation can be reestablished after

removal of the agent. Irreversible denaturation occurs

when the unfolded peptide chain is stabilized by inter-

action with other chains (as occurs for instance with

egg proteins during boiling). During unfolding, react-

ive groups, such as thiol groups, that were buried or

blocked may be exposed. Their participation in the

formation of disulfide bonds may also cause an irre-

versible denaturation. Denaturation of biologically

active proteins is usually associated with loss of activ-

ity. The fact that denatured proteins are more readily

digested by proteolytic enzymes is also of interest.

Physical Properties

Dissociation

0032Proteins, like amino acids, are amphoteric. Depending

on pH, they can exist as polyvalent cations, anions, or

zwitterions. Since a-carboxyl and a-amino groups are

linked together by peptide bonds, the uptake or re-

lease of protons is limited to free terminal groups, and

mostly to side chains. In contrast to free amino acids,

the pK

values fluctuate greatly for proteins since the

dissociation is influenced by neighboring groups in

the macromolecule. For example, in lysozyme the

g-carboxyl group of Glu(35) has a pK

of 6–6.5,

SER

CYS

MET

ARG

PRO

THR

CYS

ARG

PRO

LYS

ASP

ALA

LEU

LYS

CYS

ASP

SER

PRO

SER

GLU

ASP

PRO

LEU

CYS

SER

ASN

CYS

SER

ASP

ASN

GLU

PHE

GLN

THR

TYR

HIS

PHE

ILE

GLU

TYR

PRO

LYS

ALA

CYS

ASP

LYS

CYS

ALA

ASP

VAL

ASP

LYS

ASN

GLU

fig0004 Figure 4 Bowman–Birk proteinase inhibitor from soya beans. Solid black lines, disulfide bridges between half-cystine residues;

asterisks, trypsin (Lys-Ser) and chymotrypsin (Leu-Ser) reactive sites. Adapted from Ikenaka T, Odani S, and Koide T (1974) Chemical

structure and inhibitory activities of soybean proteinase inhibitors. In: Fritz H, Tschesche H, Greene LJ and Truscheit E (eds) Bayer-

Symposium V Proteinase Inhibitors, pp. 325–343. Berlin: Springer-Verlag, with permission.

PROTEIN/Chemistry 4813

while the pK

of the b-carboxyl group of Asp(66)

is 1.5–2, of Asp(52) is 3–4.6, and of Asp(101) is

4.2–4.7. The total charge of a protein, which is the

absolute sum of all positive and negative charges, is

differentiated from the so-called net charge which,

depending on the pH, may be positive, zero, or nega-

tive. By definition the net charge is zero and the total

charge in general is maximal at the isoelectric point.

Lowering or raising the pH tends to increase the net

charge toward its maximum, whereas the total charge

becomes less than at the isolectric point.

0033 Since proteins interact not only with protons but

also with other ions, there is a further differentiation

between an isoionic and an isoelectric point. The

isoionic point is defined as the pH of a protein solu-

tion at infinite dilution, with no other ions present

except for H

and OH



. Such a protein solution can

be acquired by extensive dialysis (or, better, electro-

dialysis) against distilled water. The isoionic point is

constant for a given substance whereas the isoelectric

point is variable, depending on the ions present and

their concentration. In the presence of salts, e.g.,

when binding of anions is stronger than that of

cations, the isoelectric point is lower than the isoionic

point. The reverse is true when cation binding is

dominant. In most cases the shift in pH is consistently

positive, i.e., the protein binds more anions than

cations. At its isoelectric point a protein is the least

soluble and the most likely to precipitate (isoelectric

precipitation) and it is at its maximal crystallization

capacity. The viscosity of solubilized proteins and

the swelling power of insoluble proteins are at a

minimum at the isoelectric point.

Optical Activity

0034The optical activity of proteins is due not only to

asymmetry of amino acids but also to the chirality

resulting from the arrangement of the peptide chain.

Information on the conformation of proteins can be

obtained from recording the optical rotatory disper-

sion (ORD) or the circular dichroism (CD), especially

in the range of peptide bond absorption wavelengths

(190–200 nm). The Cotton effect occurs in this range

and reveals quantitative information on secondary

structure. An a-helix or a b-structure gives a negative

Cotton effect, with absorption maxima at 199 and

205 nm, respectively, whereas a random-coiled

conformation shifts the maximum to shorter wave-

lengths, i.e., results in a positive Cotton effect.

Solubility, Hydration, and Swelling Power

0035Protein solubility is variable and is influenced by the

number of polar and apolar groups and their arrange-

ment along the molecule. Generally, proteins are sol-

uble only in strongly polar solvents such as water,

glycerol, formamide, dimethylformamide, or formic

acid. In a less polar solvent such as ethanol, few

proteins have appreciable solubility (e.g., prolamins).

The solubility in water is dependent on pH and

on salt concentration. At low ionic strength, the

solubility rises with increase in ionic strength and

the solubility minimum (isoelectric point) is shifted

to a somewhat lower pH. This shift is due to prefer-

ential binding of anions to the protein. As a rule,

neutral salts have a twofold effect on protein

solubility. At low concentrations they increase the

solubility (‘salting-in‘ effect) by suppressing the elec-

trostatic protein–protein interaction (binding forces).

(b)

(a)

fig0005 Figure 5 Conformation of the peptide chains of the sweet pro-

teins (a) monellin and (b) thaumatin. b-sheet, |!; a-helix,

;

b-turn, ; N

, N

, C

denote the N- and C-termini of the A and

B chains. Adapted from Ogata C, Hatada M, Tomlinson G, Shin

WC and Kim S-H (1987) Crystal structure of the intensely sweet

protein monellin. Nature 328: 739–742, with permission.

4814 PROTEIN/Chemistry