Post on 08-May-2018
transcript
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.
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.
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
.