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

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undertaken on a number of different levels, i.e., total

protein content, defining and quantifying the individ-

ual protein components, and characterization of pro-

tein biological activity (direct or indirect) and

functional properties.

Food Matrix

0002 Food manifests itself in a wide variety of guises from

the basic, raw material such as fruit through to highly

refined processed foods. Food contains a large

number of other components, as well as proteins,

such as carbohydrates, fats, and minerals/salts.

These components all interact to form the food

matrix. It is usually necessary to separate the protein

fraction from the other nonprotein material before

any meaningful analysis of the individual proteins

can be undertaken. The effect of the other compon-

ents in the food matrix must be considered, however,

when characterizing the protein biological activity

and functional properties.

Total Protein Content

0003 The gross analysis of protein in food is often required

prior to the characterization of the individual

protein components. The amount of protein is

also necessary during the purification of the individ-

ual proteins to determine their relative purity. The

three principal protein analysis methods are total

nitrogen, UV adsorption, and chemical or dye-

binding reactions.

0004 The Kjeldahl method, in which the sample is

digested with acid, a boiling aid, and a catalyst to

produce an ammonium salt that is then reacted with

alkali, and the resulting free ammonia is determined

either titrimetrically or photometrically, results in an

approximate protein level. Interference by nonpro-

tein nitrogen-containing material and inaccuracies

caused by differences in the nitrogen content of vari-

ous proteins must be considered.

0005 UV adsorption at either 280 nm (aromatic band of

tyrosine and tryptophan) or 205–220 nm (peptide

band) provides a rapid, nondestructive, and sensitive

estimate of protein content. This is subject to interfer-

ence from nucleic acids and other compounds and can

be used to quantify purified proteins if their extinc-

tion coefficients are known.

0006 A number of chromophore or dye-binding assays

are available, including the Biuret, Lowry, and

bicinchoninic acid reagents and the Coomassie Blue

dye. The methods differ in their sensitivity and re-

quire a prepurified calibrating protein for quantita-

tive results.

Individual Protein Characterization

Electrophoresis

0007Electrophoresis encompasses a wide range of tech-

niques used for the separation and analysis of complex

mixtures of proteins and peptides, and for the purifi-

cation of small amounts of these different compounds.

Separation is based on the differential movement of

charged particles under the influence of an electric field

where the overall motion is the product of the charge

on the particles and the field strength, and where this

movement is balanced by the frictional resistance of

the medium. Four parameters, viz. differences in mo-

lecular size, charge or hydrophobicity, and specific

interactions with other molecules, can be used to opti-

mize the separation of molecules from each other, thus

making electrophoresis a very powerful analytical

technique. In the separation of proteins and peptides,

the charged amino acids and carboxyl and amino ter-

minal groups contribute to the overall charge on the

protein and result in migration to either the anode or

the cathode when an electric field is applied.

0008There are presently two methods commonly used

for the electrophoretic separation of proteins and

peptides. Gel electrophoresis utilizes a ‘solid,‘ immo-

bilized matrix to retard the movement of the sample

through the aqueous phase. Initially, cellulose acetate

strips and starch gels provided this matrix, although

they have generally been superseded by polyacryla-

mide. Capillary electrophoresis (CE) occurs in free

solution in very narrow bore capillary tubes and

with high separation voltages, and is the electrophor-

etic analog of high-performance liquid chromatog-

raphy (HPLC). Narrow capillary tubes allow rapid

separation through their excellent heat dissipation,

thus minimizing convective mixing and diffusion

spreading of the separated zones.

0009Polyacrylamide gel electrophoresis Polyacrylamide

gels are three-dimensional networks of acrylamide

reacted with the bifunctional reagent N,N

-methyl-

ene-bis-acrylamide (abbreviated as Bis) via a free-

radical initiated vinyl polymerization mechanism.

The pore size of the gel is very reproducible and is

directly related to the ratio of acrylamide to Bis. The

resulting gels are described in terms of %T, the con-

centration (w/v) of acrylamide and Bis, and %C, the

weight percentage of the cross-linker in T. For pro-

teins, %T values of 5–10% result in gels with relative

molecular mass (M

) ranges of 20 000–200 000 Da.

Separation of proteins in complex samples based on

size, net charge, and hydrophobicity is possible using

different polyacrylamide gel electrophoresis (PAGE)

formats.

PROTEIN/Determination and Characterization 4825

0010 Sodium dodecyl sulfate–polyacrylamide gel electro-

phoresis This technique separates proteins acco-

rding to their subunit size. The natural charge of the

protein is masked by saturation with the anionic de-

tergent sodium dodecyl sulfate (SDS), which also de-

natures the protein degrading the secondary, tertiary,

and quaternary protein structure and aiding solubil-

ization. The inclusion of a reducing agent such as

2-mercaptoethanol breaks any disulfide bonds,

resulting in rod-like protein monomers containing

one SDS molecule for every two amino acid residues.

In an electric field, the protein–SDS complexes there-

fore migrate towards the anode at a rate dependent

on the number of SDS molecules (or the M

of the

protein) and the pore size of the gel.

0011 The M

of the sample proteins can be determined

by direct correlation of their migration distance with

proteins of known M

. Resolution of the protein

bands in SDS–polyacrylamide gel electrophoresis

(SDS–PAGE) can be improved by using a discontinu-

ous gel system, where a stacking gel is used to concen-

trate the protein sample into sharp bands before the

main separation gel. Differences in pH and ionic

strength between the stacking gel and the electrophor-

esis buffer (isotachophoresis) are used to achieve this.

When a sample contains proteins with a wide range

of M

values, a gradient polyacrylamide gel (e.g.,

5–20% acrylamide) can be used.

0012 The determination of M

of glycoproteins and lipo-

proteins by SDS–PAGE can often result in erroneous

estimates due to the different binding characteris-

tics of SDS to carbohydrate and lipid moieties.

0013 Native gel electrophoresis This electrophoretic

technique is generally used when the biological activ-

ity of the sample to be detected is required. It is

generally considered to be a ‘gentle‘ method with a

lower resolving power than SDS–PAGE. Separation is

based on the native charge, size, and shape of the

sample molecules and on the properties of the separ-

ating gel. Homogeneous or gradient gels can be used,

and the sample must be soluble, as any aggregated or

precipitated material will not enter the gel matrix.

Identification of individual proteins within these

gels can be difficult due to the complex separation

mechanisms.

0014 Urea gel electrophoresis Urea gel electrophoresis is

similar to SDS–PAGE in that it uses a detergent,

6–8 M urea, and a reducing agent, usually 2-

mercaptoethanol, to ensure sample solubilization

and disulfide bond disruption. In this method, how-

ever, the urea does not impart any charge on to the

individual proteins, and separation is due to both

charge and subunit size. In food analysis, the method

is particularly useful for the separation of the individ-

ual casein proteins from the casein micelles in milk.

0015Isoelectric focusing Isoelectric focusing (IEF) is a

high-resolution technique where proteins are separ-

ated according to their isoelectric points within a

continuous pH gradient. The high resolving power

allows the separation of compounds differing by

only 0.01 pH units in pI. Separation is achieved by

initially establishing a pH gradient along the gel using

a mixture of low-M

carrier ampholytes such as

amino acids or small peptides. The sample compon-

ents then travel through the pH gradient under an

electric field until their individual charges reach

zero, i.e., when they attain the pH corresponding to

their isoelectric point. For this technique, a low poly-

acrylamide or agarose gel is used as it is only a stabil-

izing medium and does not participate in the sample

separation. As urea carries no charge, samples can be

run under either native or denaturing conditions (8 M

urea).

0016Two-dimensional electrophoresis With the advent

of proteomics, two-dimensional gel electrophoresis

has become an indispensable tool with which to

tease apart the individual proteins in cell systems.

Proteins in a sample are initially separated according

to their pI by isoelectric focusing (often in 8 M urea),

and then a sample strip is applied at right angles along

the edge of a SDS gel. Electrophoresis in the second

direction is therefore according to subunit size, and

the resulting pattern provides a unique ‘fingerprint‘ of

the sample.

0017Protein/peptide fixing and detection Proteins are

immobilized in the gels after separation by fixing,

usually with trichloroacetic, methanolic acetic or sul-

fosalicylic acid. Dyes such as Coomassie brilliant blue

R-250, silver nitrate or Sypro Orange or Red are then

used to visualize the protein bands.

Capillary Electrophoresis

0018Capillary electrophoresis (CE) includes a range of

methods where samples are analyzed electrophoreti-

cally in capillary tubes. The capillaries may contain

buffers, sieving gels, or more classic HPLC porous

beads. As in HPLC, the numerous separation mech-

anisms available contribute to the versatility of CE

with the different modes generally accessible simply

by altering the buffer composition. Separation is

based on differences in component migration in an

electric field where the migration is a sum of the

electric force on the component and the frictional

drag through the medium. This is often highly de-

pendent on the pH and composition of the running

4826 PROTEIN/Determination and Characterization

buffer and is also affected by the electroosmotic flow

(or bulk flow) of liquid in the capillary.

0019 Capillary zone electrophoresis Fundamentally, ca-

pillary zone electrophoresis (CZE or free zone elec-

trophoresis (FZE)) is the simplest CE format,

principally because the capillary is only filled with

buffer. Separation of both anionic and cationic com-

ponents in a sample occurs when they migrate in

discrete zones at different velocities due to electroos-

motic flow. The selectivity of the separation can be

altered using additives or coatings that alter the

charge and hydrophobicity of the capillary wall.

0020 CZE has a diverse range of applications for protein

analysis such as purity validation, screening protein

variants, and contaminants, and in conformational

studies. It is also well suited for analysis of posttran-

slational modifications such as N- or C-terminal

modifications, phosphorylations, carboxylations or

N-glycosylations, and qualitative peptide mapping

to detect subtle differences in proteins.

0021 Micellar electrokinetic chromatography Micellar

electrokinetic chromatography (MEKC) is a hybrid

of electrophoresis and chromatography which uses

surfactants in the running buffer, thus making the

separation of neutral species possible. The surfactant

molecules form micelles above their critical micelle

concentration that can then interact with the neutral

components. MEKC can be used for charged, un-

charged, hydrophilic, and hydrophobic compounds

such as peptides and amino acids.

0022 Capillary gel electrophoresis The size-based separ-

ation of macromolecules such as proteins and nucleic

acids is affected in capillary gel electrophoresis (CGE)

by the electrophoresis of the compounds through a

polymer network that acts as ‘molecular sieve.‘ It is

directly comparable to PAGE and uses polymer

matrices such as acrylamide, dextran, and agarose.

0023 For protein chemistry applications, the separation

of native and SDS-complexed proteins in either the

reduced (by 2-mercaptoethanol) or nonreduced state

has been achieved using either cross-linked poly-

acrylamide or linear dextran polymers.

0024 Capillary isoelectric focusing Capillary isoelectric

focusing (CIEF) is similar to IEF–PAGE and separates

proteins and peptides according to their pI values. It

is a ‘high-resolution‘ technique with a resolution of

0.005 pI units and less. Ampholytes are used to form

a pH gradient within the capillary, and the proteins to

be separated migrate (or are focused) through the

ampholyte medium until they become uncharged at

their pI values.

0025The pI values of proteins can be determined using

protein standards with known pI values. Protein iso-

forms, proteins whose separation by other methods

can be problematic, such as immunoglobulins and

hemoglobins, and dilute biological solutions have all

been successfully analyzed by CIEF.

0026Capillary isotachophoresis This is a ‘moving bound-

ary‘ electrophoretic technique where the separated

zones move at the same velocity sandwiched between

two buffer systems (called the leading and terminat-

ing electrolytes). The method creates very sharp

boundaries between the zones and is mainly used to

separate anions or cations.

0027Whilst it has not been used extensively for proteins,

it has great potential for separating components of

very similar mobility such as removing small amounts

of impurity in recombinant proteins.

0028Capillary electrochromatography Capillary electro-

chromatography (CEC) is a true hybrid of electro-

phoresis and chromatography. It uses capillaries

packed with chromatographic material, and the sep-

aration is based on electrophoresis and the chromato-

graphic distribution of the various components

between the stationary solid phase and the liquid

mobile phase. It can also be compounded by inter-

actions between the sample components and the

capillary walls.

0029In theory, CEC has similar applications to CZE and

MEKC. In practice, however, macromolecules such as

proteins have an increased surface adsorption to both

the capillary wall and the chromatographic matrix

that restricts its usefulness. The problems should not

be as severe with peptides.

Liquid Chromatography

0030Liquid chromatography (LC) consists of a family of

methods in which two phases are used to separate

structurally different analytes in a mixture. For the

separation and purification of proteins, a variant of

LC, HPLC, is now routinely used. This method uses

small-diameter, wide-pore, solid-phase packings, and

several techniques are available based on differences

in the protein properties of size, charge, and hydro-

phobicity. The methods all use a stationary solid

phase immobilized in a column and a liquid mobile

phase. The protein mixture is introduced on to the

column via the mobile phase, separation occurs via

interaction with the stationary phase, and the individ-

ual components are detected usually at 214 or 280 nm

using the UV-absorptive properties of proteins.

0031Reversed-phase chromatography Separation of pro-

teins by reversed-phase chromatography (RPC)

PROTEIN/Determination and Characterization 4827

depends on the reversible adsorption/desorption of

the proteins, which have varying degrees of hydro-

phobicity, to a hydrophobic stationary matrix. The

nature of the hydrophobic binding interaction is

thought to be the result of a favorable entropy effect

in areas adjacent to the hydrophobic regions where

there is a higher degree of organized water structure,

although the actual mechanism has yet to be fully

elucidated. The matrices are generally silica-based or

synthetic organic polymers and contain covalently

bound alkyl chains of different lengths such as n-

octadecyl (C18), n-butyl (C4), or phenyl groups.

The strength of the hydrophobic interaction increases

as the alkyl chain size increases.

0032 Proteins are initially introduced on to the chroma-

tographic matrix in conditions that promote binding,

e.g., 5% organic modifier (acetonitrile) containing a

weak hydrophobic ion-pairing reagent such as tri-

fluoroacetic acid (0.1%, pH * 2), and then eluted

with an aqueous gradient of the organic modifier

(5–60% v/v). The resulting separations are generally

quick, and the proteins are usually highly resolved.

0033 RPC has only recently become the method of

choice for the separation and characterization of

proteins/peptides. This is because of concerns that

the organic solvents, e.g., acetonitrile, routinely used

for the mobile phase can lead to protein denaturation

and loss of biological activity. Strategies such as using

short columns of short alkyl chain length, e.g., C4,

organic modifiers such as ethanol or isopropanol, and

omitting trifluoroacetic acid have been developed to

allay these apprehensions.

0034 Hydrophobic interaction chromatography Separa-

tion of proteins by hydrophobic interaction chroma-

tography (HIC) is similar to RPC and utilizes

hydrophobic interactions between the proteins in a

mixture and the hydrophobic ligands (usually butyl,

octyl, or phenyl groups) attached to an immobile

matrix. HIC is different to RPC, however, in its use

of aqueous mobile phases of high ionic strength and

neutral pH that do not denature proteins. The

ligands in HIC are also less hydrophobic and less

densely bound to the solid matrix than in RPC

matrices.

0035 Proteins are bound to the column in a mobile phase

that contains a high salt concentration, e.g., 1–3M

ammonium sulfate, and then eluted with a descending

salt gradient that resolvates the proteins and makes

binding thermodynamically unfavorable. This differs

from RPC, where an organic solvent gradient is used

to neutralize hydrophobic binding interactions. The

protein-binding selectivity and strength can be ma-

nipulated by the mobile-phase pH and temperature,

and elution of strongly adsorbed hydrophobic

proteins can be achieved using ethylene glycol to

lower the polarity of the mobile phase.

0036Affinity chromatography Affinity chromatography

uses unique aspects of the biological or individual

chemical structure of a protein to affect its purifica-

tion. By selecting an interacting ligand which has a

high natural specificity to the target protein, highly

selective separations can be achieved. The protein to

be purified or quantified is specifically and reversibly

adsorbed by the complementary ligand immobilized

on an insoluble matrix. Purification factors of up to

several thousand can be achieved, together with fur-

ther concentration during the elution step and high

recoveries of the initial active protein.

0037Whilst the method is now routinely used for the

purification of monoclonal antibodies and their cor-

responding antigens, receptor proteins, DNA-binding

proteins, and ‘tagged‘ recombinant proteins, its use in

protein analysis is less prevalent.

0038Immobilized metal affinity chromatography Immo-

bilized metal affinity chromatography (IMAC) is a

specialized variant of affinity chromatography

where the proteins or peptides are separated

according to their affinity for metal ions that have

been immobilized by chelation to an insoluble matrix.

At pH values around neutral, the amino acids histi-

dine, tryptophan, and cysteine form complexes with

the chelated metal ions (e.g., Zn

2þ

,Cu

2þ

,Cd

2þ

,Co

2þ

,Ni

2þ

, and Fe

2þ

). They can then be eluted

by reducing the pH, increasing the mobile phase ionic

strength, or adding ethylenediaminetetraacetic acid

(0.05 M) to the mobile phase. This technique is espe-

cially suited for purifying recombinant proteins as

poly-histidine fusions and for membrane proteins

and protein aggregates where detergents or high-

ionic-strength buffers are required.

0039Size-exclusion chromatography Size exclusion (or

gel filtration) chromatography (SEC or GFC) separ-

ates proteins in solution according to differences in

their size and shape as they travel through a solid

phase (gel) matrix. As most proteins are spherical,

their shape, or radius of gyration, will approximate

to their M

. The gel matrices are usually cross-

linked three-dimensional polymers of dextran, agar-

ose, or silica that contain pores of controlled size

ranges to allow free transfer of the liquid phase and

limited diffusion of the proteins to be separated. By

using matrices with pores of carefully controlled

sizes, different M

ranges of proteins can be separ-

ated.

0040Separation of a mixture of proteins occurs when

the passage of the smaller proteins, peptides, and

4828 PROTEIN/Determination and Characterization

other molecules through the column is retarded by

their diffusion into the pores of the gel beads. In

comparison, the large proteins do not diffuse into

the pores, effectively only ‘see‘ a smaller volume

of the mobile phase, and are said to elute with

the ‘solvent front.‘ Hence, the proteins are eluted

from the column according to their size, with the

large proteins eluting earlier. The M

of the eluting

proteins can be calculated by using suitable reference

proteins.

0041 The gel matrix and buffer systems are designed to

minimize any adsorption of the protein to the gel

matrix (e.g., 20 mM sodium phosphate, pH 7 or 50–

100 mM NaCl for silica matrices). Denaturants such

as 0.1% SDS or 6 M guanidine hydrochloride can

also be added to the mobile phase to disrupt aggre-

gates, promote the formation of uniform, rod-like

conformations, and reduce interactions between the

sample and the column matrix.

0042 Ion-exchange chromatography Separation of pro-

teins by ion-exchange chromatography (IEXC) is

based on the interaction between proteins containing

different charged groups and the oppositely charged

moieties covalently linked to a chromatographic

matrix. Thus, negatively charged proteins (i.e., in a

mobile phase with a pH greater than the isoelectric

point of the proteins) will separate on a positively

charged solid phase (such as diethylaminoethyl

groups) using anion exchange. The converse situation

occurs for positively charged proteins usually using a

negatively charged carboxymethyl ion exchanger and

apH5–6 buffer. The technique is very powerful and

can separate two proteins differing by only one

charged amino acid.

0043 Proteins are initially introduced on to the column

under conditions that encourage binding. They are

then sequentially eluted from the matrix usually by

increasing the ionic strength of the eluting buffer by

including a salt gradient or by changing the pH of the

eluting buffer. Separation is due to different proteins

having different degrees of interaction with the ion

exchanger because of differences in their charges,

charge densities, and distribution of charges on their

surfaces.

Immunoassays

0044 Immunochemical techniques require an antibody (or

immunoglobulin) to recognize and bind to a three-

dimensional site on an antigen giving rise to a meas-

urable secondary effect. This reaction is of necessity

very specific for the given antigen and can be moni-

tored by a number of detection systems, including

precipitation of the antibody–antigen complex,

agglutination, or the use of labels, e.g., radioactive,

enzymatic, or fluorescent.

0045Marker-free immunoassays These techniques rely

on the ability to measure the antigen–antibody com-

plex ‘physically‘ and include turbidity, nephelometry,

and immunodiffusion. The first two methods rely on

differences in the light-scattering properties of the

antigen–antibody complex and are not generally con-

sidered to be as sensitive or accurate as other im-

munoassays. This may change with the introduction

of surface plasmon resonance that detects changes in

the refractive index of the reaction solution close to a

sensor surface and is sensitive down to the picomole

level.

0046Immunodiffusion covers a number of formats

where antigen and an excess of antibody are allowed

to react as they diffuse through a permeable gel. The

end product of the reaction is an immunoprecipitate

that is ‘trapped‘ in the gel.

0047In single radial immunodiffusion (RID), the anti-

gen is added to a well in the gel and diffuses radially

through the gel that contains the antibody. Initially,

the antigen is in excess of the antibody, resulting in

soluble complexes. The antigen diffuses further until

the antibody concentration is greater than the anti-

gen, and a precipitation ring forms.

0048In the double diffusion (Ouchterlony) immuno-

assay, the antigen and antibody are applied to separ-

ate wells and diffuse towards each other until a

precipitation line is formed. This method can be

used to compare the specificity of an antibody for

different antigens.

0049Immunoelectrophoresis combines electrophoresis,

initially to separate the antigens (proteins), and then

gel immunodiffusion, to visualize them by precipita-

tion with specific antibodies. A variation of this

method, rocket immunoelectrophoresis, permits

quantification of the antigen by performing the elec-

trophoesis in a gel that contains antibody. A precipi-

tation line in the shape of a ‘rocket‘ is formed whose

peak height depends on the concentration of the

antigen.

0050Labeled-reagent immunoassays Immunoassay sen-

sitivity can be increased by several orders of magni-

tude by using labeled reagents instead of marker-free

immunoassays. The label can be an enzyme, a radio-

active antigen or antibody, or a fluorescent chromo-

gen. There are a large number of methods available

based on either competitive or noncompetitive for-

mats. In competitive assays, free antigen competes

with labeled or solid-phase bound antigen for a

limited amount of antibody. In noncompetitive

methods, the amount of antigen is limited and is

PROTEIN/Determination and Characterization 4829

bound by specific labeled antibodies. Various formats

such as microtiter plates, dipsticks, immunodots, and

immunofiltration devices can be used.

Other Technologies

0051 Protein bioassays When proteins or peptides have

specific functions, e.g., enzymes, folate-binding pro-

tein, or biologically active peptides, individual bio-

assays are often used to determine their bioactivity.

These assays are often more informative to the re-

searcher than the more physical data. For proteins

(and enzymes), this activity is usually also indicative

of the conformational state of the protein with pro-

tein denaturation correlating to loss of activity.

0052 Mass spectrometry Recent developments in mass

spectrometry, such as electrospray ionization and

matrix-assisted laser desorption ionization, have

made it possible to determine the M

of proteins

and peptides that weigh in excess of 100 000 Da

and to analyze minute amounts of these compounds

down to the femtomole level. This has resulted in

mass spectrometry becoming a common adjunct to

the UV adsorption detector for the detection and

characterization of proteins and peptides by HPLC

and CE.

Further Reading

Andrews AT (1986) Electrophoresis: Theory, Techniques,

and Biochemical and Clinical Applications, 2nd edn.

Oxford: Oxford University Press.

Ashurst PR and Dennis MJ (eds) (1998) Analytical Methods

of Food Authentication. London: Blackie Academic &

Professional.

Hames BD and Rickwood D (eds) (1990) Gel Electrophor-

esis of Proteins: A Practical Approach, 2nd edn. Oxford:

IRL Press.

Oliver RWA (ed.) (1998) HPLC of Macromolecules: A

Practical Approach, 2nd edn. Oxford: IRL Press.

Pomeranz Y and Meloan CE (1994) Food Analysis: Theory

and Practice. New York: Chapman & Hall.

Scopes RG (1993) Protein Purification: Principles and

Practice, 3rd edn. New York: Springer-Verlag.

Sorensen H, Sorensen S, Bjergegaard C and Michaelsen S

(1999) Chromatography and Capillary Electrophoresis

in Food Analysis. Cambridge: The Royal Society of

Chemistry.

Requirements

A E Bender

, Fetcham, Leatherhead, Surrey, UK

This article is reproduced from Encyclopaedia of Food Science,

Introduction

0001The protein requirement is defined as the lowest level

of dietary protein that will balance the losses of nitro-

gen (N) in persons maintaining energy balance. The

body proteins are constantly undergoing breakdown

and resynthesis at rates that vary from tissue to

tissue. The amino acids released by tissue breakdown

are mostly reused for synthesis but there is some

loss – the obligatory nitrogen loss – by oxidative

catabolism.

0002It is necessary to specify that the subject is in energy

balance because utilization of dietary protein is influ-

enced by energy intake. It has been shown that at any

given level of dietary protein, the addition of energy

improves nitrogen balance until it reaches a plateau,

which represents the limitation imposed by protein.

Any change above or below the energy needs of the

subject is likely to influence nitrogen balance. This

is why the current international recommendations

consider energy and protein requirements together.

(See Energy: Measurement of Food Energy; Energy

Expenditure and Energy Balance.)

0003If more protein is consumed than is physiologically

required, the excess is metabolized as a source of

energy. However, there has been some concern that

excessive protein intakes may contribute to deminer-

alization of bone and deterioration of renal function

in patients with renal disease; it is therefore suggested

that the upper limit should be 1.5 g of protein per kg

of body weight per day.

Earlier Recommendations

0004According to the perceived wisdom of the day, figures

for requirements for protein have varied enormously

(Table 1). In the early days such figures were effect-

ively pronouncements by leading physiologists and it

was not until the discussions of the Food and Agricul-

ture Organization (FAO) of the United Nations that

figures were based on scientific evidence. At intervals

of about 10 years, namely 1957, 1965, 1973 and

1985, revised FAO recommendations have been

made in the light of increasing knowledge.

0005The first of these reports, in 1957, emphasized the

proportions of the various amino acids required and

Deceased.

4830 PROTEIN/Requirements

they quantified dietary protein requirements in terms

of a theoretical reference protein of perfect amino

acid composition, i.e., satisfying the experimentally

determined needs for each amino acid. The reason for

terming this a theoretical protein is that dietary pro-

tein is most effectively assimilated only at low levels

of intake (as used in the laboratory assessment of

protein quality) and efficiency falls off as the level

rises. The efficiency of utilization of the theoretical

protein is constant at all levels of intake.

National Recommended Dietary

Allowances (RDAs)

0006 While the FAO reports represent the conclusions of

expert committees, each national authority has com-

piled its own figures based on the opinions of the

committee involved and taking national habits into

account. This has led to different figures in different

countries. Thus, the RDA for protein for adult males

is 81 g day

1

in France, 64 g day

1

in Germany, 70 g

day

1

in the Netherlands, and 54 g day

1

in Spain. In

practical food terms, the amount of protein one

would recommend in the diet must depend on the

total food (energy) intake; otherwise, heavy work

would apparently call for provision of the extra

energy from pure carbohydrate and fat. Two

examples of the practical food approach that obviate

this difficulty are the figure of 10–15% of the total

energy intake as protein recommended by the Scandi-

navian countries, and 10% (69 g of protein on aver-

age) in the 1979 UK tables.

0007 The 1991 UK revision (Table 2) adopted the FAO/

World Health Organization (WHO)/United Nations

University (UNU) figures. For adult males aged 19–50

years this is listed as 42.6 g of protein for males

weighing 74 kg as the estimated average requirement,

with 55.5 g as the reference nutrient intake – the

term introduced in the 1991 revision and which is

equivalent to RDA. For women weighing 60 kg, the

corresponding figures are 36 and 45 g day

1

. The

figures are based on milk or egg protein which are

taken as completely digestible. For diets based on

unrefined cereal grains and vegetables, a correction

factor for digestibility of 85% is applied; for diets

based on refined cereals, the correction factor is

95%. (See Dietary Reference Values.)

Methods of Assessment of Needs

0008There are two methods of determining nitrogen re-

quirements. The one used until the 1985 FAO report

was the factorial method in which the losses of nitro-

gen from the body are measured to establish the

amount that is required. The second method, adopted

in the 1985 report, measures the intake of nitrogen

needed to maintain nitrogen equilibrium.

Factorial Method

0009On a diet free from protein the body loses nitrogen in

the urine as urea, creatinine, uric acid, and ammo-

nium salts, together with small amounts of other

compounds; this is termed the endogenous loss.

There are also losses in the feces from bacteria, muco-

sal cells shed from the lining of the intestine, and

residues of digestive juices that have not been re-

absorbed; this is termed metabolic loss.

tbl0001 Table 1 History of recommended protein intakes (adult man)

Author Recommendedintake

Playfair (1865) 57–184 g (observed)

Voit (1881) 118 g (recommended)

Chittenden (1905) 50–55 g (observed and recommended)

Sherman (1920) 33–40 g at 70 kg body weight

(0.59 g per kg)

League of Nations (1936) 1 g per kg body weight

FAO (1957) 0.35 g kg

1

FAO (1965) 0.71 g kg

1

FAO (1973) 0.57 g kg

1

FAO (1985) 0.86 g kg

1

FAO, Food and Agriculture Organization.

tbl0002Table 2 UK dietary reference values for protein

Age Weight

(kg)

Estimated

average

requirement

(gday

1

)

Reference

intake

(gday

1

)

0–3 months 5.9 12.5

4–6 months 7.7 10.6 12.7

7–9 months 8.8 11.0 13.7

10–12 months 9.7 11.2 14.9

1–3 years 12.5 11.7 14.5

4–6 years 17.8 14.8 19.7

7–10 years 28.3 22.8 28.3

Males

11–14 years 43.0 33.8 42.1

15–18 years 64.5 46.1 55.2

19–50 years 74.0 44.4 55.5

50þ years 71.0 42.6 53.3

Females

11–14 years 43.8 33.1 41.2

15–18 years 55.5 37.1 45.4

19–50 years 60.0 36.0 45.0

50þ years 62.0 37.2 46.5

Pregnancy þ6

Lactation

0–6 months þ11

6þ months þ8

Reproduced from Department of Health (1991) Dietary Reference Values

for Food Energy and Nutrients for the United Kingdom. Report on Health

and Social Subjects 41. London: Her Majesty’s Stationery Office.

PROTEIN/Requirements 4831

0010 In addition, there are small losses from skin cells,

hair, finger nails, and body fluids.

0011 These obligatory losses are measured on subjects

fed a diet free from protein. On such a diet the urinary

output of nitrogen does not attain a constant level but

falls sharply for 4–6 days and then very slowly. The

levels reached are 2–3 g day

1

or 30–45 mg per kg of

body weight. Forty-five milligrams of nitrogen ap-

proximates to 2 mg of nitrogen per basal kcal. The

two phases of this decline are taken to indicate two

processes. The first rapid fall is loss from organs with

a rapid protein turnover and is called labile body

protein. The second is largely from muscles and

skin. The obligatory nitrogen loss is taken as the

figure at the end of the rapid fall. This relative in-

exactitude is one of the disadvantages of the factorial

method.

0012 The 1965 report took the figures of 46 mg nitrogen

per kg (2 mg per basal kcal) for urine loss, 20 mg

1

for fecal loss, and 20 mg for other losses. In

the light of better evidence, the 1973 report changed

these findings to 37, 12, and 5 mg respectively. This

accounts for the marked reduction in protein recom-

mendations between 1965 (0.71 g kg

1

) and 1973

(0.57 g kg

1

). At first consideration, obligatory losses

should be balanced by an intake of the same amount.

This was assumed in the 1965 report but it was later

realized that when an individual is supplied with the

same amount of nitrogen as his or her obligatory loss,

he or she remains in negative nitrogen balance. It

was found necessary to increase that figure by

30% to achieve nitrogen equilibrium. An additional

30% was added to allow for individual variation.

All measurements are made as nitrogen and then

converted into protein by multiplying by the

factor 6.25.

0013 While the basic data show a small difference for

nitrogen losses between males and females, figures

are not sufficiently firm to allow any distinction.

The difference between final protein figures is due to

the different standard body weights used for males

and females; in the 1973 report this was 0.71 g of

protein per kg of body weight per day, which is 39 g

for females and 46 g for males. This was called the

practical allowance and based on full utilization of

the protein in the diet, i.e., high-quality protein,

termed reference protein.

0014 An extra allowance is made for protein of lower

quality, i.e., protein with net protein utilization

(NPU) values of 70–80 in developed countries and

60–70 in less developed countries (with values as

low as 50–60 in special conditions); this is stated in

the 1973 report.

0015 Apart from the difficulty of deciding the level of the

endogenous loss (since there is no clear sharp break in

the curve), most workers did not measure the cutane-

ous losses because of the difficulty of doing so, and

they accepted published values which were derived

from only a limited number of experiments.

Nitrogen Balance Method

0016Nitrogen balance is the difference between nitrogen

excreted from the body and nitrogen ingested in the

diet (of which the greater part by far is protein).

During growth, pregnancy, lactation, and recovery

from convalescence, the body is in positive nitrogen

balance since it is retaining nitrogen for the purpose

of synthesizing new protein tissues. During dietary

deprivation, most illnesses, and certain types of stress,

the body loses nitrogen and is in negative balance.

The healthy adult is in nitrogen equilibrium. The

basis of this method of determining nitrogen require-

ments is to feed the subjects a series of diets with

different levels of protein while measuring nitrogen

excretion, then to interpolate to nitrogen equilibrium

(zero nitrogen balance).

0017In early studies the diets included very low levels of

protein and a zero level but since the nitrogen balance

response is not linear throughout the entire submain-

tenance range, recent studies use levels around the

expected range of requirements. An allowance has

to be made for the variable losses of nitrogen in

sweat, which are considerable in heavy work in a

hot climate.

0018It takes some time at a given level of dietary protein

to achieve a steady state, i.e., adjustments in urine

output do not immediately follow changes in nitrogen

intake, so that diets are fed for periods of 1–3 weeks

at each intake level. These short-term nitrogen bal-

ance determinations do not take account of adapta-

tion to low levels of dietary protein as experienced in

developing countries. Long-term studies require sev-

eral months, but these are usually limited to a single

level of protein intake which would provide evidence

that a particular diet is adequate.

0019It must be borne in mind that in these detailed

consultations about protein (and energy) require-

ments and recommendations, the FAO is largely con-

cerned with the adequacy of diets in poorly fed

populations of developing countries. In the well-fed

western world there is never a problem of protein

shortage in healthy people as long as enough food is

consumed to satisfy hunger. The fundamental physio-

logical considerations, of course, still apply.

0020Direct nitrogen balance studies are the currently

accepted method of determining protein require-

ments but they suffer from the lack of long-term

balance studies, the absence of independent valid-

ation of an optimal state of protein nutrition, and

lack of knowledge of the functional significance of

4832 PROTEIN/Requirements

the size of the total nitrogen pool and rate of turnover

of tissue proteins. There are no functional indicators

of protein inadequacy at a stage before clinically de-

tectable changes occur.

0021 The limitations of the data are indicated by the

small numbers of studies at the time of the 1985

report – nine short-term studies of single protein

sources on a total of 93 subjects, eight short-term

studies on the typical mixed diets of eight countries

on a total of 73 subjects, and six long-term studies,

24–89 days, five on egg and one on milk, on a total of

34 subjects.

0022 The short-term studies provide an estimate of a

mean daily requirement of 0.63 g of highly digestible

good-quality protein – a figure slightly higher than

the 1973 ‘safe level.‘ The long-term studies suggest

that 0.58 g kg

1

is a reasonable estimate.

0023 From all the available results it is suggested that

0.6 g per kg of body weight per day is the average

requirement for good-quality proteins such as meat,

milk, egg, and fish.

0024 The coefficient of variation was 12.5%, i.e., 2 sd

equals 25%. This provides the currently accepted

figure of 0.75 g of protein per kg of body weight per

day as the safe level of intake for an adult.

Quality and Digestibility

0025Earlier recommendations for protein requirements

were based on ‘good-quality‘ protein or reference

proteins such as eggs or milk, i.e., proteins with

NPU close to 1.0 (100 in the older nonmenclature).

Thus, the recommendation of 0.75 g protein per kg of

body weight is for protein of this high quality and the

amount has to be increased proportionately to com-

pensate for lower quality. For example, 0.75 g of NPU

tbl0003 Table 3 Values for the digestibility of protein in humans

Protein source True

digestibility

(mean +

SD)

Digestibility

relative to

reference proteins

Egg 97 + 3

Milk, cheese 95 + 3 100

Meat, fish 94 + 3

Maize 85 + 689

Rice, polished 88 + 493

Wheat, whole 86 + 590

Wheat, refined 96 + 4 101

Oatmeal 86 + 790

Millet 79 83

Peas, mature 88 93

Peanut butter 95 100

Soya flour 86 + 790

Beans 78 82

Maize plus beans 78 82

Maize plus beans plus milk 84 88

Indian rice diet 77 81

Indian rice diet plus milk 87 92

Chinese mixed diet 96 98

Brazilian mixed diet 78 82

Filipino mixed diet 88 93

American mixed diet 96 101

Indian rice plus beans diet 78 82

Reproduced from WHO (1985) Energy and Protein Requirements. Report of

the Joint FAO/WHO/UNU Expert Consultation. WHO Technical Report

Series 724. Geneva: World Health Organization.

tbl0004Table 4 Safe level of protein intake (good-quality protein)

Age g perkgbody weight g perday

3–6 months 1.85 13

9–12 months 1.50 14

1–2 years 1.2 13.5

2–3 years 1.15 15.5

3–5 years 1.10 17.5

5–7 years 1.0 21

7–10 years 1.0 27

10–12 years

Male 1.0 34

Female 1.0 36

12–14 years

Male 1.0 43

Female 0.95 44

14–16 years

Male 0.95 52

Female 0.9 46

16–18 years

Male 0.9 56

Female 0.8 42

Adult

Male 0.75 49 (65 kg body weight)

Female 0.75 41 (55 kg body weight)

Pregnancy þ6

Lactation þ17.5

Reproduced from WHO (1985) Energy and Protein Requirements. Report of

the Joint FAO/WHO/UNU Expert Consultation. WHO Technical Report Series

724. Geneva: World Health Organization.

tbl0005Table 5 Protein losses related to surgery

Time of loss Protein loss (g)

Before surgery

Bleeding peptic ulcer 90 (over 5 days)

Long-bone fracture 140–190 (over 10 weeks)

Limb burn Up to 1000

During surgery (blood loss)

Gastric surgery 9–18

Pneumonectomy 20–60

Thyroidectomy 3–12

Catabolic response after surgery

Herniorrhaphy 18 (over 10 days)

Gastric resection 50–175 (5–10 days)

Appendectomy 50 (10 days)

Bed rest (inactivity), 3–4 weeks 300

PROTEIN/Requirements 4833

1.0 is equivalent to 1.07 g of quality 0.7, or 1.25 g of

NPU 0.6.

0026 In practice, the quality of the mixture of dietary

proteins in underdeveloped countries (which rely very

largely on a single staple food, usually a cereal), is

about 0.7, and the addition of protein-rich foods such

as meat and milk – cereals, meat, and milk are the

major sources of dietary protein in the developed

countries – increases this to only 0.8. Consequently,

the 1985 report lays less emphasis on the quality of

the protein because the figure recommended is higher

than the 1973 figure and digestibility correction

alone is adequate. Digestibility was not mentioned

at that time.

0027 Only four amino acids are likely to limit the protein

quality of mixed diets: they are lysine, the sulfur

amino acids, threonine, and tryptophan, and diets

in developing countries generally meet the adult

requirement for these amino acids. Quality can be

taken care of by calculating the proportion of the

most limiting amino acid compared with that in the

requirement pattern and then increasing the protein

figure in that ratio. (See Amino Acids: Metabolism.)

0028 Differences in digestibility arise from the nature of

the cell walls of the food, the presence of dietary fiber

and polyphenols, and from chemical reactions that

alter the release of amino acids during digestion. Ad-

justments are therefore made to the amount of food

protein to be ingested by comparing the digestibility

of the protein food with that of reference proteins

(Table 3).

0029 The digestibility of a complete diet can be calcu-

lated by using the values for individual sources of

protein and multiplying by the proportion of those

foods in the diet.

0030 Diets based on coarse, whole-grain cereals and

vegetables are generally about 85% digested, while

those based on refined cereals are 95% digested;

meat, milk, and eggs are 100% digested.

Extra Allowances

0031 Figures for protein requirements are based on meas-

urements made on young adults with corrections for

age, sex, and body weight. They apply to healthy adults

in nitrogen equilibrium. There are obviously greater

needs for growing children, pregnant women, and

lactating mothers, and these are shown in Table 4

(the UK figure for lactation, in Table 2, has been

modified). The extra needs for growth are small com-

pared with the nitrogen required to maintain the

tissues, even in young children, except for babies

under 1 year of age. For example, the average re-

quirement for growth of boys 4–5 years of age is

0.36 g of protein per day, while for maintenance it is

12.6 g. It is accepted that different amounts of protein

may be laid down from day to day as part of the

normal process of growth. The body has very limited

capacity for storing amino acids; it is therefore neces-

sary to provide enough protein every day for possible

extra demands of higher rates of growth. For this

reason the extra demands for growth were estimated

at 50% greater than the theoretical growth rate. (See

Children: Nutritional Requirements; Lactation:

Human Milk: Composition and Nutritional Value;

Physiology; Pregnancy: Safe Diet.)

Trauma

0032Protein requirements for healthy adults take into ac-

count the ‘normal stresses of everyday life‘ but there

are increased losses of nitrogen from the body in pain,

anxiety, and psychological stresses, with very large

losses in trauma, chronic disorders, and parasitic in-

fections. Clearly, these will vary enormously with the

severity and duration of the condition. Tables 5 and 6

show some examples of these losses which must be

made good during convalescence.

See also: Amino Acids: Metabolism; Children:

Nutritional Requirements; Dietary Reference Values;

Energy: Energy Expenditure and Energy Balance;

Measurement of Food Energy; Lactation: Human Milk:

Composition and Nutritional Value; Physiology;

Pregnancy: Safe Diet