Bai 7

PROTEIN

7.1 AXIT AMIN PHAN UNG VcJI NINHVDRIN

Phiin ung mau d~c trung cua ninhydrin vcri cac a-aminoaxit va ct1ang mau ph1;1
thui)c n6ng d<) axit amin la ca si'J cua vi~c sii d1,mg ninhydrin trong cac phucmg phap
djnh tinh va djnh luqng axit amin bltng ky thu~t so mau.
a) D,.,,ng c,.,,: Ong nghi~m. k~p. pipette, nbi each thiiy.

bJ Roa chat
- Thu6c thii ninhydrin: dung djch ninhydrin 1q;. trong acetone 95'¼:
- Cac dung djch axit amin: glutamic. leucin, prolin c6 nong 0.05'k
- Mi)t dung djch thuy phan {proteolysatl chua hbn hgp axit amin
cJ Tien hanli: Lay vao 5 ong nghi~m dii ghi s6 thll' ti!

- Ong thll' nhat: lml.. dung djch glutamic

- Ong tM hai: lmL clung djch leucin
- Ong thu ba: 1ml dung djch prolin
- Ong thi'.r ttc: lmL dung djch hbn hqp axit amin
- Ong thu nam: 0,5mL hon hgp axit amin va 0,5mL ntfc/ C cat
Them vao moi 6ng 0,2mL thuoc thl.i' ninhydrin va d~t cac ong nay vao noi each
thuy hay tu say d 90°C trong 10 phut. Lay ra. Chi mau va viet phan ang.

7.2 £>!NH LlJ<;jNG NITd AXIT AMIN BANG PHlldNG PHAP CHUAN E>Q FORMOL

(PHUONG PHAP SORENSEN)

a- Nguyen tiic: Cac aldehyt d~ ket hgp voi nh6m amin. Khi cho formaldehyd tac dung
voi axit amin, nh6m amin bi methylen h6a t~o thanh dlm xufit methylen imino axit.
R -CH- COOH + CH 20 - A- -CH- COOH
I I
NH N .:: CH 2
2

R - CH - COOH + NaOH - R - CH - COONa + H2 0

1 I
N = CH N = CH -,
2

Hqp chat t!}.o thanh c6 tinh axit m!}.nh hon axit amin tl,f do, cac nh6m cacboxyl
cua cht'.mg de dang djnh phan b~ng ki~m. qua d6 gian tiep tinh duqc hcqng nita amin
cua cac axit amin c6 trong dung djch.
Protea 5&

Khi dung p/u{(Jng plu!J:p nay c&11 chu y nhil~J8 diem sau:
- E>~ cho phan a~ t.!lo metl")._ylen iminoaxit tie'n hanh hoan toan. can c6 du m9t it
dung dich formaldehyd va phan t1ng trung hoa hoim toan cac nh6m cacboxyl dugc thtJc
hien khi pH moi truang d?t tcri 9,2+9,5.
- Arginin khong phan ung va:i formaldehyd cho nen khong tinh khi chuan bang kiem.
- Tirozin cho ket qua cao han vi nioai nh6m carboxyl, nh6m phenol cung tac
d1,mg vcri kiern. Mqt so axit amin cho ket quii it hem nhu prolin.
- Neu trong dung djch c6 chtra nhiiu NH 3 , nhat la trong cac djch thuy phan
protein thi phai lo~i trlt NH 3 truac trong chan khong a nhi~t 40°C.
- Cac muoi amon nhu NH 4 Ct, (NH 4 hS04 ciing tac d1,mg vcri formaldehyd t?o thanh
hexamethylen tetramin va axit. Axit nay sau d6 dtt<;tc trung hoa bd'i lciem:
2(NH 4 hS04 + 6CH 2 0 (CH2)sN4 + 2H2S04 + 6H20
4NH 4CI + 6CH 20 (CH 2)6 N 4 + 4HCI + 6H20
- Dung djch nghien cuu c6 mau d~m phai pha th~t loang vi neu mau d~m thi rat
kh6 nh~n biet Slf thay doi mau khi chu§°n dQ.
- Dung phvang phap nay ket qua chi th~t dung doi vcri trucrl1g h~p cua axit
monoamino monocacboxylic. De ket qua dung vcri m9i truang hgp (khi trong dung djcb
ngoai cac ax.it monoamino monocacboxylic ra con c6 chua cac ax.it monoamino dicacboxylic
va diamino monocacboxylicl c~n phai trung hoa dung dich den pH 7 truac khi phan tich.
· De xac ninh diem bit dl1u (pH 7J va diem ket thuc <pH 9.2l. ngttdi tr1 <lung.
phuang phap so mau. T~o ra hai thang mau chuan a pH 7 va pH 9,2 r6i dtta pH cua
mau ve 7 (so sanh mau vcri ma u chufo pH 7) sau c16 djnh phan den pH 9,2 (so sanh
mau vai mau chuan pH 9,2).
b-D,,mg c~
- Blnh dtnh mtrc lOOmL - Erlen lOOmL
- Pipette lmL; 20ml - Burette 25ml
- Ong dong lOmL - Becher l00mL
e- Hoa chat
- Dung dich d~m pH 7; pH 9,2 - Formol trung tinh
- NaOH 0,05N - Phenolphtalein
- H2S04 0,05N - Bromthymol blue
d- Ca.ch tie'n hanli
Clwa'n bi thang" mau co pH 7 va pH 9,2:
Lay hai binh non c6 dung tich nhu nhau:
- Cho vao binh tM nhat: 20mL dung cijch c6 pH 7 va 5 gi9t bromthymol bleu 0,04~.
- Cho vao binh thu hai: 20mL dung dich c6 pH 9,2 ; 5 gi9t brnmthymol bleu 0.04%
va 3 gi9t phenolphtalein 0,5%.
Khi d6 dung cijch trong binh 1 c6 mau xanh h,ic nh~t, dung cijch trong b1nh 2 c6 mau
tim xanh. Mau cua cac dung cijch tren giu trong binh kin c6 the hen trong ni.i"a thang.
 Bai"?
56

Tien hanh djnh phan mliec

Xac dinh ham luqng nito amin t~ong nuoc mlm.
Nt.toc mam la m9t dung dich thuy phan protein c6 the cqnh luc;mg nit11 b§.ng
pht!cmg phap chufln d9 formol. Do nuac m!m c6 mau t6i cAn phai pha th~t loang mai
co the so mau cua chat chi thi.
Lay vao blnh dinh rnuc 100 lmL nude m~m. dung nuoc cat dinh muc thanh
lO0mL, lac deu.
Lay vao binh non co cimg dung tich voi 2 binh mau chuan 20mL djch mau pha
loang W hinh cqnh muc, them 5 gi9t bromthymol blue. Neu dung dich c6 mau xanh d1.ie1ng
thi them ti.tng gi9t HCI hay H2 SO4 0,05N, neu dung clich c6 mau vang thi them Wng gi9t
NaOH 0,05N cho den khi dung dich co mau ung voi mau cua binh 1 co pH 7,0.
Them ba gi9t phenolphtalein 0,5'K; 4mL formol tiung tinh r6i chuan d9 biing NaOH
0,05N cho den khi hon hqp c6 mau ililg voi mau cua dung cqch trong b'inh 2 co pH 9,2.
Song song tien hanh lam thi nghi~m kiem chung, thay dung cijch nghien cuu
bilng nliCIC cat.
e- Tinh hit qua
86 gam nitO axit amin CO trong 1 lit nt{OC mam:
(a - b) x T x 0,0007 :, 100 x 1000
X=----------
20 x V
---
tr1mg do: x - ILr(/ng gam nito axit amin co trong; l' lit mrdr mAm.
r, - sii 111 ( , Nc1OH 0 ,0GN ch'1n g c1e chu:"{n dung- cli1:h thi nghi~m .

b - so 111.L NaOH 0,05N dung de chuan dung d\ch kiem chung.

'l' - h~ sf; hi~u chinh nong c1.'ia dung dich NaOH dem dung so v&i nong di) chuan
V - so 11,L mJ<k m {i m cho vao h1nh dinh muc.
0 ,00<J7: so gain nitff (!ng vc:'li lmL NaOH -0,05N