NIT

ROGE

NO

US

COM

POU

ND

S-Ch

.15,p

.237

A-M

etho

ds f

or Q

uant

ifying

Prot

eins

I. M

etho

ds b

ased

on

elem

enta

ry a

naly

sis

of

nitr

ogen

(Kje

ldah

l, D

umas

)II

. M

etho

ds B

ased

on

Dir

ect

Det

erm

inat

ion

of

Prot

eins

(Biu

ret,

Low

ry, D

ye-b

indi

ng)

III.

Ind

irec

t M

etho

ds (T

urbi

dim

etry

, NIR

, T.D

)

B-M

etho

ds f

or A

sses

smen

t of

Pro

tein

Qua

lity

I. I

n vi

vo t

ests

(BV,

PER

, NPU

etc

.)II

. In

vi

tro

test

s (n

on-p

rote

in

nitr

ogen

de

term

inat

ion,

amin

o ac

id

com

posi

tion

, A

A.

avai

labi

lity)

.

C -M

etho

ds f

or e

luci

dati

ng p

rote

in str

uctu

reI.

The

seq

uenc

e of

am

ino

acid

s II

. Con

form

atio

nal

stre

oche

mis

try

of p

rote

ins.

Prot

eins

are

poly

mer

sof a

min

o ac

ids l

inke

d by

“am

ide”

linka

ges.

They

have

sign

ifica

nce

in fo

odsf

rom

:-n

utrit

iona

l, -r

heol

ogic

al(f

unct

iona

l)-o

rgan

olep

ticas

pect

s,Pr

otei

nsar

eim

porta

ntfo

rbio

logi

calf

unct

ions

and

cell

stru

ctur

e; th

eyin

clud

e“e

ssen

tiala

min

oac

ids”

whi

chca

nnot

be p

rodu

ced

in th

ehu

man

body

; foo

dte

xtur

ean

dfla

vour

are

also

very

muc

hin

fluen

ced

bypr

otei

n co

nten

ts.

Ther

efor

e, p

rote

in a

naly

sisi

s im

porta

ntfo

rbio

logi

cal

activ

ityde

term

inat

ions

, fun

ctio

nalp

rope

rtyin

vest

igat

ions

and

of c

ours

efo

rfoo

dla

belin

g.

MW

of p

rote

ins:

5000

-106

dalto

nsom

pose

dof

H,C

,N,O

,S.

Prot

eins

oc

cur

in

food

s of

ten

in

com

bina

tion

wi

th

othe

r m

acro

food

com

pone

nts:

asLi

popr

otei

ns

(as

in

bloo

d pr

otei

ns);a

s Gl

ycop

rote

ins(

asin

eg

g wh

ite)

;

as

met

allo

prot

eins

( as

min

eral

com

plex

es li

ke F

e in

hem

oglo

bin,

I

in t

hyro

glob

ulin

).A

llpr

otei

nsar

em

ade

upof

≅24

am

ino

acid

s,

eigh

tof

whi

char

e“e

ssen

tial

”. Ev

ery

a.a.

h

as a

t le

ast

one

amin

oan

dat

leas

ton

eca

rbox

ylic

grou

pin

its

mol

ecul

e.

Am

ino

acid

s co

me

toge

ther

firs

tfo

rmin

gpe

ptid

e ch

ains

(fir

stol

igop

epti

des;

then

the

poly

pept

ides

) an

dfi

nally

the

poly

mer

icpr

otei

n m

olec

ule.

In

all

prot

ein

mol

ecul

es,

ther

eis

~1

6% N

pr

esen

t(%

13-1

9).

COOH

CH

NH

R I−

−−

−−

−−

−

−

OO

R

2N

H

IIII

I

2I

CN

HC

CH

NH

CH

R

Exac

t N

itro

gen

rati

o in

pro

tein

s ra

nges

fro

m13

.4to

19.

1%:

Whe

at p

rote

in f

acto

r : 1

7.55

% N

; 10

0/17

.55=

5.7

Milk

pro

tein

fac

tor:

15.

67%

N; 1

00/1

5.67

=6.3

8

Oils

eeds

pro

tein

fac

tor:

18

.87%

N; 1

00/1

8.87

=5.3

Prot

ein

cont

ents

of

food

s:

Beef

:18%

asi

s;80

-90%

d.m

.; Fi

sh:7

0-80

%(d

.m.)

Milk

: 3.5

%; S

kim

med

Milk

pow

der:

36%

; Che

ese:

16-2

5%

Brea

d:8%

; Flo

ur: 9

-13%

Frui

ts:0

.2-1

.5%

Veg

etab

les:

1-2%

factor

protein

→≅

25.6

16100

PRO

TE

IN A

NA

LY

SIS

Why

are

we

inte

rest

ed in

the

over

all p

rote

in

cont

ent o

f a fo

od?

Func

tiona

lity

(hyd

ratio

n, g

ella

tion,

su

rface

prop

ertie

s..)

Dis

ulfid

e bo

nds

(whe

at g

lute

n)

Prot

ein

Ana

lyse

s

Tota

lpro

tein

con

tent

Amin

oac

idco

mpo

sitio

nC

onte

ntof

a p

artic

ular

prot

ein

oraa

Prot

ein

isol

atio

nsan

did

entif

icat

ions

Non

prot

ein

nitro

gen

Nut

ritiv

eva

lue

(per

,nitr

ogen

bala

nce

etc.

)

Met

hods

base

don

Ele

men

tary

Ana

lysi

sof

Nit

roge

n:A

. Kje

ldah

lMet

hod

for

Crud

ePr

otei

nPr

inci

ple

step

s:1-

Dig

esti

on in

con

cent

rate

d H

2SO

4 +

cata

lyst

mix

ture

:

N is

con

vert

edto

amm

oniu

mio

n.2-

Neu

tral

isat

ion:

Conc

entr

ated

al

kalin

e so

luti

on

is

adde

d,

follo

wed

by s

team

dis

tilla

tion

of

liber

ated

NH

3, co

llect

ing

into

kno

wn a

mou

nt o

f ac

id:

3-Ti

trat

ion

of u

nrea

cted

acid

wit

h st

anda

rdiz

ed s

olut

ion:

At

turn

ing

poin

t,m

oles

HCl

=mol

esN

H3

1ml o

f 0.

1N H

Cl=1

.400

8 m

gN

or1m

l 0.12

53 N

HCl

=10

mg

Prot

ein

[]

↑↑

+→

+−

−2

24

44

22

2)(

SOCO

SONH

SOmH

NH

Cn

↑→

+−

34

4)

(NH

OH

SONH

H2S

O4

Cata

lyst

(Hg,

Cu,

Se)

Boil

(NH4

)2.SO

4

incr

ease

bp

with

pota

ssiu

m s

ulfa

te

NaO

H

NH3

NH3

NH3

NH4

+ H

2BO

3-Boric

aci

d

H2BO

3-

Titr

ate

Bora

teW

ith

AcidStan

dard

HCl

1

32

Kje

ldah

lMet

hod

Cal

cula

tion:

Gra

m n

itrog

en/ g

ram

of s

ampl

e =

*(m

l of s

ampl

e -m

l of b

lank

) ×N

stan

dard

aci

d ×

0.01

4g/m

eq

wei

ght o

f sam

ple

* m

l of h

ydro

chlo

ric a

cid

requ

ired

to ti

trate

sam

ple

solu

tion.

KJEL

TEC:

Aut

omat

ed

devi

ce

usin

g M

icro

Kj

elda

hlpr

inci

ples

(20

sam

ples

/hou

r)

Dis

adva

ntag

es:

not a

ll N

is p

rote

in.

Purin

e

Pyrim

idin

eD

NA

, RN

A, e

tc.

Ure

a

Man

y pl

ant t

issu

es h

ave

> 50

% n

on-p

rote

in N

.

% N

×6.

25 =

% P

rote

in

B. D

UM

AS

Met

hod(

1830

’s):

3 St

eps:

1. Py

roly

sis:

Nit

roge

n is

fre

ed f

rom

org

anic

mat

ter

by

pyro

lysi

s(70

0-90

0ºC)

, und

era

stre

amof

O2

toyi

eld

a ga

sm

ixtu

re(N

2, N

Ox,

, CO

, CO

2,CH

4) a

ndas

h.

2. R

educ

tion

:N

itro

gen

oxid

es i

n th

e ga

s st

ream

are

re

duce

d to

N g

as b

y he

ated

met

allic

cop

per.

NO

x→N

2 3.

Qua

ntif

icat

ion:

The

inte

rfer

ing

gase

s (C

O2,

CH4,

CO)

are

rem

oved

by

spec

ialc

hem

ical

ads

orba

nts

and

N g

as

is

dete

rmin

ed e

ithe

rvo

lum

etri

cally

orno

wby

Gas

Ch

rom

atog

raph

y us

ing

TCD

th

erm

al

cond

ucti

vity

de

tect

or.

Smal

lsam

ple

size

s(0.

1g)

and

take

s2

min

utes

; how

ever

hom

ogen

izat

ion

is n

ot e

asy.

2. D

ye B

indi

ng M

etho

ds

Prin

cipl

e:A

t low

pH

, bas

ic g

roup

s of p

rote

in a

re (+

) cha

rged

. Th

ese

will

qua

ntita

tivel

y bi

nd a

(-) c

harg

ed d

ye.

Exam

ples

of th

ese

basi

c gr

oups

are

show

nbe

low

:

NH

3+

CHCH

2

CH

2

CH

2

CH

2

NN

H

HC O

C CH

2

CN

H+

CH

CH

NH

C

H

CO

N H

CH

2

CH

2

CH

2

NHC N

H2N

H2+

Lysi

ne

Arg

inin

e

His

tidin

e

“DYE

BIN

DIN

G M

ETH

OD

”s, c

ont.

Und

er

spec

ified

co

nditi

ons,

th

e to

tal

acid

ic o

r ba

sic

grou

ps o

f pr

otei

ns b

ind

quan

titat

ivel

y w

ith c

erta

in o

rgan

ic d

yes,

fo

rmin

gin

solu

ble

com

plex

es.

Afte

rse

para

tion

of

this

com

plex

, by

dete

rmin

ing

amou

ntof

of u

nbou

nd d

yein

so

lutio

n,

we

can

dedu

ce

prot

ein

cont

ents

. B

ut

al

lsp

ectr

opho

tom

etric

eval

uatio

ns n

eed

to b

e co

rrel

ated

with

st

anda

rdis

edm

etho

dsan

dsp

ecifi

cca

libra

tion

grap

hs.

Bin

ding

sele

ctiv

ity-E

xam

ple

Cati

onic

gro

ups

Cati

onic

gro

ups

of t

he a

min

o ac

ids

reac

t wi

th a

n of

the

am

ino

acid

s re

act

with

an

anio

nic

anio

nic

sulf

onic

sulf

onic

acid

dye

acid

dye

(i.e

(i.e

Am

ido

Am

ido

Blac

k)Bl

ack)

The

mor

e dy

e th

at w

as b

ound

, the

mor

e pr

otei

n Th

e m

ore

dye

that

was

bou

nd, t

he m

ore

prot

ein

pres

ent

in t

he s

ampl

e.pr

esen

t in

the

sam

ple.

Aci

d O

rang

e 12

:

N =

NHO

SO3-

Proc

edur

e:

1.M

ix p

rote

in, d

ye, b

uffe

r pH

= 2

.

2.Fi

lter o

r cen

trifu

ge.

3.M

easu

re o

ptic

al d

ensi

ty (O

.D.)

of fi

ltrat

e.

Exam

ple

forD

ye B

indi

ng M

etho

d

Abs

orba

nce

of d

ye b

ound

by

prot

ein

=(A

dye

initi

al -

A fi

ltrat

e)

A. at 470 nm

% P

rote

in (K

jeld

ahl)

68

1012

1416

Skim

milk

Dye

Bin

ding

Met

hod

Fact

ors I

nflu

enci

ng D

ye B

indi

ng d

eter

min

atio

n:

1.Te

mpe

ratu

re

2.N

on-p

rote

ins.

3.B

uffe

rs sy

stem

s.

4.Pr

otei

n qu

ality

.

Dye

Bin

ding

Met

hod

II.M

etho

ds M

easu

ring

Pro

tein

s D

irec

tly

Thes

ede

pend

on

char

acte

rist

icre

acti

ons

of

prot

eins

.

Exam

ple

1:

BIU

RET

MET

HO

D:

Colo

rim

etri

c (1

914)

m

etho

d

depe

ndin

g on

the

pri

ncip

le t

hat

prot

eins

(or

any

m

olec

ule

with

mor

e th

an 2

pep

tide

bon

ds)

form

a

purp

le-c

olou

red

com

plex

wi

thCu

pric

salt

s(i.e

. Cu

S04)

in a

lkal

ine

solu

tion

s. I

t is

use

d m

ainl

y fo

r

prot

eins

in

so

luti

ons

and

is

sim

ple,

ra

pid

and

inex

pens

ive.

urea

urea

“biu

ret”

22

NH

O ıı CN

H

O ıı CH

−−

−−

Cup

ric io

ns re

act w

ith p

eptid

e bo

nds

unde

r al

kalin

e co

nditi

ons

(cop

per s

ulfa

te +

K-N

a-ta

rtrat

e +

alka

li)M

easu

re c

olor

in S

PEC

at 5

40 n

m

The

Biu

ret M

etho

d:

N N

OH

RH O

H

Cu2+

OH

-

ON N

HH

H

R O

N N

OH

RH O

HCu

2+

Purp

le b

iure

t com

plex

Biu

retM

etho

d(c

ont.)

Prin

cipl

es: C

u++

in a

lkal

ine

solu

tion

form

com

plex

ity w

ith

pept

ide

bond

s -gi

ving

pink

ish-

purp

le c

olor

.

Mea

sure

the

inte

nsity

of c

olor

at 5

40 n

m.

A at 540 nm

% P

rote

in (K

jeld

ahl)

Ex2:

LOW

RY

MET

HO

DPr

inci

ple:

Prot

eins

in

tera

ct

with

ph

enol

re

agen

t*an

d co

pper

und

er a

lkal

ine

cond

ition

s, o

xidi

zing

the

arom

atic

amin

oac

ids.

*Fol

in-C

ioca

lteau

Rea

gent

:M

ixtu

reof

Pho

spho

tung

ustic

and

phos

phom

olyb

dic

acid

sol

utio

ns,

yiel

ding

a bl

ue

colo

urfo

llow

ing

copp

er-c

atal

yzed

oxi

datio

n of

a.a

.,whi

chis

re

adsp

ectr

opho

tom

etric

ally

.

4.Lo

wry

Met

hod

•C

u++in

alk

alin

e so

lutio

n to

form

com

plex

ity w

ith p

rote

in.

•C

u++

cata

lyse

s oxi

datio

n of

phe

nol g

roup

of t

yros

ine

with

ph

osph

omol

ybdi

c-ph

osph

otun

gstic

acid

.A at 750 nm

gof

pro

tein

(Kje

ldah

l)µ

III.

IN

DIR

ECT

Met

hods

1.Nep

helo

met

ryan

d Tu

rbid

imet

ry:

Turb

idit

y re

sult

ing

from

pre

cipi

tati

ng p

rote

ins

with

a

prec

ipit

atin

gag

ent(

Ex:3

-10%

Tric

hlor

oace

tic

acid

, su

lfos

alys

ilic

acid

, po

tass

ium

ferr

icya

nide

)wi

llbe

pr

opor

tion

al

to

prot

ein

co

nten

t an

dis

to

be

mea

sure

dby

redu

ctio

nin

tra

nsm

itta

nce

valu

esat

60

0nm

or

dire

cttu

rbid

omet

erre

adin

gsM

ost

suit

able

for

nitr

ogen

com

poun

dsin

sol

utio

n.

2.Th

erm

al

Ana

lysi

s Te

chni

ques

:Re

lyin

gon

m

easu

ring

the

heat

chan

ges

in

ther

mal

deco

mpo

siti

on

of

prot

eins

unde

rco

ntro

lled

cond

itio

ns.

N

eutr

onac

tiva

tion

an

d pr

oton

acti

vati

on a

naly

ses

are

also

bei

ngus

ed.

Ultr

a-vi

olet

Abs

orpt

ion

(UV

) at 2

80 n

m

1.C

hrom

opho

ricsi

de c

hain

s of a

rom

atic

am

ino

acid

s (T

rosi

ne, T

rypt

opha

n).

2.A

bsor

ptio

n at

280

nm

. “N

on-d

estru

ctiv

e m

eans

to

dete

rmin

e pr

otei

n”.

3.C

alcu

latio

n pr

otei

n co

nc. b

ased

upo

n ab

sorp

tion

valu

e.

Fluo

resc

ence

Met

hod

Tyro

sine

is a

fluo

resc

ent c

ompo

und.

Tryp

toph

ane

is a

fluo

resc

ent c

ompo

und.

Exci

te th

ese

amin

o ac

ids a

t 280

nm

.

Mea

sure

em

issi

on a

t 348

nm

.

Adv

anta

ge:

mor

e se

nsiti

ve th

an U

V a

bsor

ptio

n.

mg

of p

rote

in/m

l of s

olut

ion

Emitted Fluorescence at 348 nmFluo

resc

ence

Met

hod

Prin

cipl

eof

Flu

ores

cenc

e M

etho

ds

Wha

t is f

luor

esce

nce

and

how

to m

easu

re it

?

Gro

und

Stat

e

Exci

ted

Stat

eEm

its ra

diat

ion

(flu

ores

cenc

e)

Dec

ay y

ield

s flu

ores

cenc

e at

lo

nger

wav

elen

gth

By

usin

g sp

ecifi

c λ

(wav

elen

gth)

to e

xcite

and

mea

sure

out

put a

t a

spec

ific λ.

It i

s rat

her s

peci

fic.

Prob

lem

s: T

urbi

dity

/Que

nchi

ng (s

elf o

r oth

ers)

/Exp

ensi

ve/

Qua

ntita

tion

is d

iffic

ult.

Infr

ared

SPEC

TRO

SCO

PY

IR s

pect

rosc

opy

mea

sure

sth

eab

sorp

tion

of ra

diat

ion

in n

earo

rmid

IR re

gion

sby

mol

ecul

esin

fo

ods.

Diff

eren

tfun

ctio

nalg

roup

sab

sorb

diffe

rent

frequ

ernc

ies

of ra

diat

ion:

forp

rote

ins,

6.47

µm,1

560-

1670

µm

and

3300

-350

0 µm

are

char

acte

ristic

for

the

pept

ide

bond

. The

refo

re, i

rradi

atin

ga

sam

ple

with

the

char

acte

ristic

wav

elen

gth

and

mea

surin

gth

een

ergy

that

is re

flect

edor

trans

mitt

edby

sam

ple

(whi

chin

turn

is in

vers

ely

prop

ortio

nalt

oth

een

ergy

abso

rbed

and

prot

ein

cont

ent)

can

lead

toqu

qntit

ativ

ees

timat

ions

of p

rote

in c

onte

nt.

B-M

etho

ds f

or a

sses

smen

tof

pro

tein

qua

lity

B.I.

In

vi

vo

test

:Ra

tio

of

the

regr

essi

onco

effi

cien

tof

gr

owth

of

Prot

oza

of

genu

s Te

trah

ymen

a,

Bact

eria

lik

e St

rept

ococ

ci,

Leuc

onos

toc,

Fu

ngi

like

A.

Flav

us(i.

e.

Dry

myc

elia

lwe

ight

afte

r72

hou

rs)

on N

con

tent

of t

est

mat

eria

lto

the

regr

essi

onco

effi

cien

tof

the

grow

thof

sam

eor

gani

smon

the

N

cont

ent

of c

asei

ngi

ves

“nut

riti

onal

inde

xof

pr

otei

n qu

alit

y”.

(Stu

dypp

.268

-276

in

yo

urbo

ok)

Esse

ntia

lam

ino

acid

s?

Am

ino

acid

s whi

ch th

e bo

dy c

anno

t mak

e (o

r mak

e en

ough

of)

for p

rote

in sy

nthe

sis d

ue to

lack

of e

nzym

es.

Esse

ntia

l Am

ino

Aci

ds:

His

tidin

e,

Isol

euci

ne,

Leuc

ine

Lysi

ne,

Met

hion

ine,

Phe

nyla

lani

ne

Thre

onin

e,V

alin

e

Lim

iting

am

ino

acid

is th

e es

sent

ial a

min

o ac

id w

hich

is

lack

ing

in th

e pr

otei

n to

hav

e a

bala

nced

pro

tein

.

Cor

nLy

sine

Oat

sLy

sine

Ric

eLy

sine

Whe

at

Lysi

neSe

sam

e Se

edLy

sine

Cow

’s M

ilkM

ethi

onin

ePo

tato

Met

hion

ine

Chi

ck P

eaM

ethi

onin

eG

reen

Pea

Met

hion

ine

Cot

ton

Seed

Isol

euci

neB

eef

Val

ine

Prod

uct

Lim

iting

Am

ino

Aci

d

Chi

cken

Tryp

toph

an

PRO

TEIN

QU

ALI

TY D

ETER

MIN

ATI

ON

1.Pr

otei

n Ef

ficie

ncy

Rat

io.

2.B

iolo

gica

l Val

ue.

3.N

et P

rote

in U

tiliz

atio

n.

Wha

t are

the

mea

sure

men

ts o

f pro

tein

qua

lity?

For l

abel

ing

purp

oses

, one

nee

ds to

kno

w th

e pr

otei

n ef

ficie

ncy

ratio

.

1.If

PER

= c

asei

n (2

.5),

the

RD

A =

45

g/da

y.

2.If

0.5

< P

ER <

2.5

, the

n R

DA

= 6

5 g/

day.

3.If

PER

< 0

.5 (2

0% o

f cas

ein)

, the

n “n

ot a

si

gnifi

cant

sour

ce o

f pro

tein

”.

How

doe

s one

det

erm

ine

PER

?1.

Mal

e la

b ra

ts ≥

21 d

ays, ≤

28 d

ays o

f age

, at l

east

10

rats

/gro

up.

2.Fe

ed a

stan

dard

ized

die

t con

tain

ing

salt

mix

, vita

min

s, co

tton

seed

oil,

cel

lulo

se, s

tarc

h or

sucr

ose

+ w

ater

for 2

8 da

ys.

3.M

easu

re w

eigh

t gai

n an

d fo

od in

take

at r

egul

ar in

terv

als,

not >

7

days

.

4.PE

R =

Wei

ght G

ain/

Gra

m o

f Pro

tein

in D

iet.

5.U

sual

ly n

orm

aliz

ed fo

r cas

ein

= 2.

5.

6.D

eter

min

e pr

otei

n qu

ality

of s

ampl

e as

ratio

of s

ampl

e PE

R to

re

fere

nce

case

in P

ER.

Prot

ein

Effic

ienc

y R

atio

= G

ain

in w

eigh

t per

gra

m p

rote

in ta

ken.

Prot

ein

Effic

ienc

y R

atio

for D

iffer

ent F

oods

Prod

uct

PER

Ric

e10

0%2.

30R

ice

70%

Bla

ck B

eans

30%

2.70

50%

50%

2.60

20%

80%

1.30

100%

NIL

Cor

n+

0.4%

Lys

ine

+ 0.

07%

Try

ptop

han

2.14

Cor

n (5

0%) +

Bla

ck B

eans

(50%

)2.

05

.

Prot

ein

Effic

ienc

y R

atio

for F

oods

Prod

uct

PER

Soyb

ean

2.32

Cot

ton

Seed

Mea

l2.

25

Egg

3.90

Chi

ck P

eas

1.68

Pean

uts (

grou

nd n

uts)

1.65

Kid

ney

Bea

ns0.

88

Bio

logi

cal V

alue

(BV

)

Net

Pro

tein

Util

izat

ion

(NPU

)

BV

=R

etai

ned

Nitr

ogen

(nitr

ogen

inta

ke -

feca

l &

urin

ary

nitro

gen)

/Abs

orbe

d N

itrog

en

(nitr

ogen

inta

ke -

feca

l nitr

ogen

)

NPU

= R

etai

ned

Nitr

ogen

/Inta

ke N

itrog

en =

BV

×

Dig

estib

ility

OTH

ER P

RO

TEIN

QU

ALI

TY D

ETER

MIN

ATI

ON

MET

In-v

ivo

Indi

ces

of P

rote

in Q

ualit

y: I

ndex

→in

dice

s

diet

)

nonp

rote

in

fed

an

imal

s

by N

excr

eted

-an

imal

ste

st

by

(Nex

cret

ed-

Nin

take

A=

==

=

di

et,

no

npro

tein

a fe

d

anim

als

for

B

Ban

imal

s,by

test

Nex

cret

ed-

Nin

take

B**

A-B

*B

100

valu

eB

iolo

gica

l

0

0

inta

kepr

otei

nw

eigh

tbo

dyin

gain

ratio

)ef

ficie

ncy

(Pro

tein

PER

=

anim

alte

stof

inta

keN

diet

free

Non

anim

alof

cote

ntN

Bod

ypr

otei

nte

stw

ithfe

dan

imal

of

cont

ent

NB

ody

n)ut

iliza

tiopr

otei

nN

PU(N

et−

=

NPR

atio

=[G

ain

in b

.w.w

ithte

st p

rote

in+l

ossi

n b.

w. W

ithN

-fre

edi

et]/P

rote

in in

take

PRO

TEIN

QU

ALIT

Y TE

STIN

G(N

utrit

iona

l Val

ue)

Tes

ts•

In v

ivo

test

s–

mon

itor

anim

al g

rowt

h,

nitr

ogen

bal

ance

–m

easu

re h

ow w

ell a

pro

tein

is

met

abol

ized

and

use

d by

bod

y. T

hese

tend

to

be e

xpen

sive

and

tim

e co

nsum

ing.

Adu

lt -0

.75g

/kg

body

wei

ght (

ca. 5

2.5g

/70

kg) m

ust b

e eq

uiva

lent

to

egg

or m

ilk p

rote

ins –

stan

dard

s of q

ualit

y.

Bio

logi

cal T

ests

Prot

ein

Effi

cien

cy R

atio

(PER

) –ol

d m

etho

d –

1919

.M

easu

res

the

grow

th r

ate

of y

oung

rap

idly

gro

wing

rat

s re

lati

ve t

o ca

sein

(not

a g

reat

pro

tein

as

refe

renc

e)O

ver

esti

mat

es t

he v

alue

of

som

e an

imal

pro

tein

s re

lati

ve t

o hu

man

s.U

nder

est

imat

es t

he v

alue

of

som

e pl

ant

prot

eins

. Pr

oble

m w

ith

an a

nim

al m

odel

: rat

s du

ring

rap

id g

rowt

h ne

ed h

ighe

r le

vels

of

cert

ain

amin

o ac

ids

than

hu

man

s.Co

stly

and

tim

e co

nsum

ing

B.II

. In

vitr

o te

sts

for

prot

ein

qual

ity

:

A.

Qua

ntif

icat

ion

of in

divi

dual

esse

ntia

l a.a

. is

done

m

ainl

y by

HPL

C or

Aut

omat

ed a

min

oaci

dan

alyz

er.

Sam

ple

prep

arat

ion

invo

lves

3 st

ages

:1.

Hyd

roly

sis

of p

rote

in(b

reak

ing

down

the

pe

ptid

e lin

kage

s)wh

ich

is d

one

eith

erwi

th6N

HCl

at 1

10°C

orwi

then

zym

es.

2.

Sepa

rati

onby

ion-

exch

ange

chro

mat

ogra

phy:

The

hy

drol

ysed

amin

o ac

ids

are

elut

ed

from

an

io

n-ex

chan

gere

sin

colu

mn,

byst

epwi

sech

angi

ngth

epH

and

ioni

cst

reng

hth

of

elue

ntan

dea

ch a

.a. b

eing

elu

ted

at a

spe

cifi

c ra

te

corr

elat

ed w

ith

its

mol

ecul

ar s

ize

and

char

ge.

3. Q

uant

ific

atio

nwi

thni

nhyd

rin:

The

Aut

omat

ed

a.a.

an

alyz

er

uses

a

Nin

hydr

inre

agen

t,wh

ich

toge

ther

wit

h ea

chel

utin

ga.

a.,f

orm

a

pink

ish

-vi

olet

col

our,

the

inte

nsit

y(or

Abs

orba

nce

valu

eat

57

0nm

)of

co

lour

bein

g co

rrel

ated

wi

th

a.a.

co

ncen

trat

ion.

In p

aral

lel,

a “s

tand

ard

a.a.

mix

ture

” (2

.5µm

oles

of e

ach

a.a.

/5 m

l) is

also

inj

ecte

d in

to t

he l

iqui

d co

lum

n.

Resu

lts

can

be ex

pres

sed

in e

ithe

rof

the

follo

wing

basis:

•[m

oles

a.a

./10

0 g

sam

ple]

•[m

g a.

a./1

00 g

sam

ple]

•[m

g a.

a./g

N in

sam

ple]

•[m

g a.

a./1

00 g

rN

in s

ampl

e]•[

mg

a.a.

/16

g N

in s

ampl

e]•[

mg

a.a.

/100

gdr

ysa

mpl

e-d.

m.b

asis

]

LYS

HIS

ASP

GLU

ALA

VA

L

LEU

pH 2

.25

pH 3

.25

pH4.

25

Mol

es/L

iter

CH

RO

MA

TOG

RA

M O

F A

MIN

O A

CID

S

Calc

ulat

ions

for

conv

erti

ng a

.a.

cont

ent

from

one

bas

is t

o an

othe

r:

Give

na

food

sam

ple

with

follo

wing

com

posi

tion

:N

%=

1.571

; Pro

tein

%=

1.5

71x6

.25=

9.82

%D

rym

atte

r%=

86.4

8;Gl

utam

icac

id=

4436

mg/

100g

rgr

of s

ampl

e(M

W o

f gl

utam

icac

id=

147.

13).

Expr

ess

resu

lts

of g

luta

mic

acid

anal

yses

in 4

dif

fere

ntba

ses:

2823

x 1

6= 4

5168

mg

glut

amic

acid

/16

g N

glu/

g.N

mg

2323

1.57

14.

436

=

sam

ple

grgl

u./1

00m

oles

3014

7.13

4436

=

mat

ter

dry

ggl

u./1

00m

g51

29x1

0086

.48

4436

=

B.

Bioa

vaila

bilit

yof

A

min

oaci

d:

Dur

ing

proc

essi

ng,

a.a.

sid

e-ch

ains

may

be

degr

aded

or

may

in

tera

ct

with

su

gars

to

fo

rm

typi

cal

arom

as.F

orex

ampl

e, in

bak

ing

orco

okin

gfo

rlo

ngdu

rati

ons,

lysi

neve

ry

easi

ly

bind

s to

gl

ucos

e,

beco

min

g un

avai

labl

enu

trit

iona

lly.

To

quan

tify

th

e bi

oava

ilabi

lity,

fo

llowi

ng

reag

ents

m

aybe

us

ed: 1.

DN

FB(d

init

rofl

uoro

benz

ene)

rea

gent

at

alka

line

pHre

acts

onl

y wi

th f

ree

lysi

ne,p

rodu

cing

yello

wco

mpo

unds

.2.

Dan

syl

chlo

ride

in b

orat

ebu

ffer

at 4

0ºC

reac

tswi

thfr

eebu

t no

t wi

thbo

und

lysi

ne.T

hedi

dans

ylly

sine

peak

give

sin

dica

tion

of

the

amou

ntof

ava

ilabl

ely

sine

.

C. N

on-P

rote

in N

itro

gen

Det

erm

inat

ion

Thes

ein

clud

efr

eeam

ino

acid

s, n

itro

sam

ines

, ni

trit

esan

dni

trit

es,

amm

onia

and

amm

oniu

mco

mpo

unds

. The

seca

n be

se

para

ted

from

the

prot

eins

byei

ther

one

of t

hefo

llowi

ngpr

otei

n se

para

tion

met

hods

: D

ialy

sis,

ult

rafi

ltra

tion

with

spec

ial

mem

bran

es,

prec

ipit

atio

nwi

thhe

ator

bypr

otei

n-pr

ecip

itan

tslik

etr

ichl

oroa

ceti

cac

id,

picr

icac

id,

tann

icac

id, s

ulfo

saly

silic

acid

, pho

spho

tung

stic

acid

.1.A

mm

onia

and

am

mon

ium

com

poun

dsca

n be

det

ecte

d wi

th

“Nes

sler

Reac

tion

or

Nes

sler

izat

ion”

: N

essl

erre

agen

t[K

OH

+HgI

+KI]

rea

cts

with

NH

3yi

eldi

nga

red-

oran

geto

brow

nco

lore

dco

mpl

ex,

whic

hca

n be

quan

tifi

edsp

ecto

phot

omet

rica

llyat

440

nm(p

.754

-5).

NH

3+

K2H

gI4

+3KO

H→

7KI

+2H

2O+

Hg 2

OIN

H2

(red

-col

ored

com

plex

)

2.N

itra

tes,

nitr

ites

:Sp

ectr

opho

tom

etri

cde

term

inat

ion

afte

r re

duci

ng;

Grie

ss-I

losw

ayPr

oced

ure:

Int

erac

tion

wit

h pr

imar

y ar

omat

ic a

min

es, l

ike

Sul

fani

licac

id, i

n ac

idso

luti

on,

tofo

rm a

dia

zoni

umsa

lt,

whic

h is

the

nco

uple

d wi

th

an a

rom

atic

com

poun

d (i.

e.na

phty

lam

in)

to f

rom

an

azo-

colo

ur(p

.767

).

3.Fr

ee a

min

oaci

dsan

d a.

a. s

alts

(i.e

.MSG

):2-

dim

ensi

onal

TL

C.

Indi

vidi

ual

a.a.

ca

n be

qu

alit

ativ

ely

dete

rmin

ed

by

TLC

and

then

be

subj

ecte

d to

sp

ecif

ic

colo

urre

acti

ons

(Ex.

W

ith

ninh

ydri

nre

agen

t) F

ree

a.a.

can

als

obe

qua

ntif

ied

byFo

rmol

tit

rati

onwh

ere

the

amin

ogr

oup

is b

lock

edwi

thfo

rmal

dehy

de, a

ndth

enth

efr

eeCO

OH

gro

upis

ti

trat

edwi

thN

aOH

.4.

Nit

roso

amin

es

C-M

etho

ds f

or e

luci

dati

ng p

rote

in s

truc

ture

s:1.

A.a

. se

quen

ce d

ecip

heri

ngor

dete

rmin

atio

n;Th

is h

as

mad

e in

sulin

(51

a.a.

in

insu

linm

olec

ule)

synt

hesi

s po

ssib

le(1

950’

s-SA

NGE

R).

Th

e pr

inci

ple

behi

ndth

ese

met

hods

invo

lves

ste

pwis

e de

grad

atio

nof

pep

tide

s by

sp

ecif

ic c

hem

ical

rea

gent

s:a.

Sang

er m

etho

d:Th

eα-

amin

ogr

oup

form

sa

yello

wco

mpl

exwi

thFN

DB(

fluo

roni

trod

iben

zene

) re

agen

twh

ich

is

solu

ble

in n

onpo

lar

solv

ents

. Th

eD

init

roph

enyl

com

plex

form

edis

the

nel

uted

with

CHCl

3 an

dsu

bjec

ted

toa.

a.

anal

ysis

.b.

Edm

ande

grad

atio

n:

invo

lves

form

atio

nof

“a

.a.

Hyd

anto

ins”

wit

hph

enyl

isot

hioc

yana

tes.

2.Co

nfor

mat

iona

l an

alys

isfo

r de

term

inin

g st

ereo

chem

istr

y.

Pr

imar

y,

seco

ndar

y,

tert

iary

an

d qu

ater

nary

str

uctu

res

(α-h

elix

sha

pes,

ple

ated

she

ets,

s-s

and

s-h

linka

ges

etc.

) ar

e be

ing

dete

rmin

ed m

ainl

yby

X-

RAY

crys

tallo

grap

hy.

Prot

ein

Stru

ctur

es

Prim

ary

Stru

ctur

e of

Pro

tein

Prim

ary

Stru

ctur

e:du

e to

cov

alen

t pep

tide

bond

s of

indi

vidu

alam

ino

acid

Seco

ndar

y St

ruct

ure

of P

rote

in

Seco

ndar

y St

ruct

ure:

due

to

hydr

ogen

bon

ding

bet

wee

n pe

ptid

e bo

nds.

Kin

ds o

f Sec

onda

ry S

truct

ure:

1.a

-Hel

ix2.

Plea

ted

shee

ts st

ruct

ure

A.

Para

llel

B.

Ant

i-par

alle

l

Terti

ary

Stru

ctur

e of

Pro

tein

Terti

ary

Stru

ctur

e:

aggr

egat

ion

of in

divi

dual

pr

otei

n th

roug

h hy

drog

en,

ioni

c, h

ydro

phob

ic a

nd

disu

lfide

bon

ds

Qua

tern

ary

Stru

ctur

e of

Pro

tein

Qua

tern

ary

stru

ctur

e A

ggre

gatio

n of

se

vera

l pep

tide

chai

ns to

form

a

defin

ite

mol

ecul

e by

io

nic

bond

, hy

drog

en b

ond,

an

d/or

hy

drop

hobi

c bo

nd

Sepa

ratio

nte

chni

ques

for

prot

eins

1. S

epar

atio

nby

diff

eren

tials

olub

ility

char

acte

rist

ics:

isoe

lect

ric

prec

ipita

tion

2. S

epar

atio

nby

adso

rptio

n:io

nex

chan

gech

rom

atog

raph

yan

dH

PLC

3. S

epar

atio

nby

size

: Ultr

afilt

ratio

nan

ddi

alys

is

4. S

epar

atio

nby

elec

trop

hore

sis

Ele

ctro

phor

esis

:D

efin

ition

: “M

igra

tion

of c

harg

edm

olec

ules

in a

so

lutio

nth

roug

han

ele

ctri

calf

ield

”A

. In

poly

acry

lam

ide

gel e

lect

roph

ores

is,

prot

eins

are

forc

edto

mig

rate

in a

queo

usbu

ffer

sth

roug

ha

solid

mat

rix(

poly

acry

lam

ide

gels

) und

erth

ein

fluen

ceof

an

elec

tric

alfie

ld. M

igra

tion

orm

obili

tyof

spec

ific

prot

eins

is a

func

tion

of th

ene

t cha

rge

on m

olec

ule,

as w

ella

s m

olec

ular

size

, sha

pe.

B.In

isoe

lect

ric

focu

sing

, ph

grad

ient

sare

form

edus

ing

amph

olyt

es(b

uffe

rso

lutio

ns),

and

prot

eins

mig

rate

toth

elo

catio

nin

the

grad

ient

at w

hich

ph=t

heir

isoe

lect

ric

poin

t.C

.Cap

illar

yel

etro

phor

esis

:cap

illar

ytu

bing

is u

sed

in

plac

eof

gel

s

Vis

ualis

atio

nof

Pro

tein

sFl

uore

scen

cem

icro

scop

yaf

terr

eact

ing

with

a dy

e(A

NS-

anili

neon

apht

alen

esu

lfoni

cac

id)

whi

chre

nder

sonl

ypr

otei

nsflu

ores

cent

is u

sed.

Stai

ning

inte

nsity

is in

fluen

ced

byco

mpo

sitio

nal

diff

eren

cesi

n pr

otei

n an

dal

soby

stru

ctur

alch

ange

sdue

topr

oces

sing

.