1

TRNG I HC CNG NGH TP. H CH MINH

KHOA CN SINH HC MI TRNG THC PHM
Lp 12DSH02

BO CO
THC HNH HA SINH

GVHD: Nguyn Th Hai

Sinh vin: Trng Th Tho

MSSV:1211100186

Ng L Hng Duyn

MSSV: 1211100062

Cao Th Nhm

MSSV:1211100142

on Ngc King

MSSV:1211100293

V Nguyn Anh Th

MSSV:1211100192

Phan Th Thy Vy

MSSV: 1211100283

BI 1: GII THIU PHNG TH HA SINH, CCH PHA

CH CC DUNG DCH DNG TRONG PHNG TH
NGHIM HA SINH

L thuyt
Quy tc lm vic trong phng th nghim ha sinh

I.

a. An ton khi lm vic vi axit v kim

An ton khi lm vic vi axit
-

Phi lm vic trong t ht bt c khi no un nng axit hoc thc hin phn ng

vi cc axit t do
-

Khi pha long lun phi cho axit vo nc .

An ton khi lm vic vi kim

-

Kim c th lm chy da

Mang gng tay , khu trang khi lm vic vi dung dch kim

Thao tc trong t ht, mang mc n chng c phng nga bi v hi kim

b. Quy tc lm vic vi ha cht th nghim

Ha cht th nghim:
-

Cc ha cht dng phn tch, lm tiu bn, tin hnh phn ng, trong phng th

nghim c gi l ha cht th nghim.

Nhn hiu ha cht :
-

Ha cht c bo qun trong chai l, thy tinh hoc nha ng kn c nhn ghi

tn ha cht, cng thc ha hoc, mc sch, tp cht, khi lng tnh, khi lng phn
t, ni sn xut , iu kin bo qun.
Cch s dng v bo qun ha cht:
-

Khi lm vic vi ha cht , nhn vin phng th nghim cng nh sinh vin cn ht

sc cn thn trng gy nhng tai nn ng tic cho mnh cng nh cho ngi khc.

Bao gi cng axit hay baz vo nc khi pha long .

Khng ht axit hay baz bng ming m phi dng cc dng c ring nh: ng bp

cao su.

II.

Cch pha ch cc dung dch trong th nghim ha sinh:

Dung dch :
-

Dung dch l hn hp ca hai hay nhiu cht tc ng tng h vi nhau v mt

vt l v ha hc. Trong dung dch gm c cht ha tan v dung mi .

Cc n v nng dung dch:
Nng phn trm, (% )

Nng phn trm khi lng, % (w/w): l s gam cht tan c trong 100g dung

dch.

Nng trm khi lng th tch (w/v): l s g cht tan c trong 100ml dung dch.

Nng phn trm th tch th tch , % (v/v): l s ml dung cht c trong 100ml

dung dch.

Nng gam lt, (g/L): l s g cht tan c trong 1 lt dung dch.

Nng phn t g hay nng mol, (mol/L): l s phn t g ( hay s mol) cht tan

trong 1 lt dung dch.

Nng ng lng (N); l s ng lng gam (lg) cht tan c trong 1 lt

dung dch.
S ng lng cht tan = s mol (n) x h s ng lng (z)
-

H s ng lng (z) : ph thuc vo bn cht ca cht v phn ng m cht

tham gia.
Nng dung dch bo ha: l nng dung dch khi ti a cht ha tan c
mt trong dung dch.
n v nng dng trong cc php phn tch vi lng :
-

Nng mg/mL : s mg cht tan trong 1mL dung dch

Miligam phn trm , mg% : mg cht tan trong 100g dung dch

Phn nghn ,0/00 : s gam cht ha tan trong 1000g dung dch

Phn triu , ppn : s mg cht ha tan trong 1kg hay 1 lt dung dch

Phn t , ppb : s g cht ha tan c trong 1kg hay 1 lt dung dch

Cch pha dung dch c nng xc nh

a. Pha dung dch c nng theo khi lng ,% (w/w)
- Cht tan l cht rn khan
- Cht tan l cht rn ngm nc ( CuS04.5H2O..)
Khi pha dung dch cn phi tnh thm lng nc kt tinh c sn.
b. Pha dung dch long t mt dung dch m c hn
c. Pha dung dch bo ha :
Ly cht tan cn pha vo becher , thm mt t nc ct v khuy cho tan . Nu sau
khi khuy , cht tan khng tan ht lng xung th phn dung dch pha trn l dung dch
bo ha. Nu cht tan tan ht , thm cht tan v tip tc khuy , c nh th cho n khi
cht tan khng cn tan c no.
d. Pha dung dch c nng % theo th tch :
Cht tan l cht rn khan

Cn lng cht tan cn thit , chuyn sang bnh nh mc , dng nc ct ha tan v

nh
mc n th tch ng.
-

Cht tan l cht rn ngm nc

Khi pha dung dch ta cn phi tnh lng kt tinh c sn ging nh phn a
-

Cht tan dng lng : mt s cht tan dng lng nh HCl, H2S04
Ngy nay a s cc dung dch th nghim c pha ch theo nng khi lng- th

tch (w/v).
i.
-

Pha dung dch nng phn t gam:

Cht tan l cht rn khan :

Mun pha dung dch nng 1M ca mt cht no , ta tnh khi lng phn t cht
theo n v gam. Cn chnh xc lng cht tan , qua phu cho vo bnh nh mc c
dung tch 1 lt . Cho vo tng lng nc ct nh , lc ha tan hon ton v a nc
ct ti mc . Chuyn dunh dch sang bnh cha, lc trn u ng nht.
Khi phi un dung dch ha tan, hoc qu trnh ha tan c ta nhit th phi ch
nhit tr li bnh thng (nhit khng kh) ri mi thm nc ti vch nh mc.
-

Cht tan l cht rn ngm nc : khi tnh lng cht tan cn cn phi tnh lun c

khi lng cc phn t nc.

-

Cht tan dng lng: nu cht tan l dung dch, ta phi tnh ton da vo nng

dung dch .
Hiu chnh nng dung dch.
a. Dung dch chun.
Dung dch chun l cc dung dch c chun b sn, m bo chnh xc v c
dng nh chun cc dung dch t pha ch khi lm th nghim.
Cch pha dung dch t ng chun:
-

Dng inh thy tinh chc thng ampun, hng ln phu vo bnh nh mc, dng

bnh tia ra sch cht tan c trong ampun vo bnh nh mc 1 lit, va thm nc ct va
lc v a nc ct ti vch mc.
-

i vi cc hp cht bn vng, c thnh phn khng thay i nh NaCl, AgNO3,

acid oxalic,.. c th pha dung dch chun trc tip bng cch cn chnh xc cht cn pha,
pha long v nh mc ti th tch ng.
-

i vi cc cht nh NaOH, HCl, Na2S2O3,..khng th pha ngay c dung dch

chun, do cc cht ny thng khng bn vng v d thay i thnh phn, v vy sau khi
pha phi hiu chnh li nng .
b. Phng php hiu chnh nng dung dch

i vi cc cht d thay i thnh phn khi dng rn, nu mun pha dung dch c
nng chnh xc, ta pha dung dch c nng gn ng, sau hiu chnh nng ca
dung dch da vo phn ng vi mt dung dch chun thch hp.
i vi cc dung dch d thay i trong qu trnh bo qun, mi ln s dng li
phi xc nh li h s hiu chnh nng dung dch
Xt mt phn ng trung ha acid base, 1 ion gam H+ s phn ng vi 1 ion gam
OH-. Do :
C1V1 =C2V2
Nu gi Cp l nng dung dch nh pha v Ct l nng thc ca dung dch ta c
h s hiu chnh K:
K = Cp/Ct = Vp/Vt

III.

Dung dch m

a. nh ngha dung dch m

Dung dch m l dung dch c pH khng thay i nhiu lm khi 1 lng axit (H+ )
hoc baz (OH-) . Vy, dung dch m gm 1 cp axit baz lin hp ( axit yu v mui
ca axit yu ny hoc baz yu v mui ca baz ny) v t l ca chng s quyt nh
pH dung dch.
Trong c th sng, dung dch m ng vai tr quan trng v d nh n nh pH
ca mu bng cc h m cacbonat v phosphat.
b. Cch pha mt s dung dch m thng dng
-

Dung dch m borat

Dung dch m citrate (pH = 3,0 3,6)
Dung dch m phosphate( pH = 5,7 8,0)
Dung dch m Na2HPO4 KH2PO4 (Ph = 5,0 8,0)
Dung dch m glycine HCl (pH = 2,2 3,6)
Dung dch m Glycine NaOH (pH = 8,6 10,6)
Dung dch m axetat (pH = 3,6 5,6)
Dung dch m vn nng 0.1 M

CU HI N TP
Cu 1:
Pha 1L dung dch NaOH 40% (w/v): cn X=(40x1000)/100=400(g) NaOH kh.
Cu 2:
Nng mol ca H2SO4 l:
CM = (10.d.C%)/M = (10x97x1.84)/98 = 18.2 (mol)
Th tch H2SO4 cn s dng l:
VH2SO4 = (VH2SO4 2M. CMH2SO4)/CMH2SO4 = (2x500)/18.2 = 54.91(ml)
Lng nc cn b sung l: 500 54.91 = 445.09 (ml)
Cu 3:
Pha 250ml dung dch CuSO4 1M, cn m = n.M = 0.25 x 250 = 62.5 (g)
CuSO4.5H2O
Cu 4:H2SO4 2M = H2SO4 4N
C1.V1 = C2.V2 V2 = (1000 x 0.1)/4 = 25 (ml)
Cu 5:
-

Lng NaOH cn pha dung dch 10% l: X= ( 10x500)/100 = 50 (g)

Lng dung dch NaOH 40% cn dng l: Y = (100x50)/40 = 125 (g)

Lng nc ct thm vo: 500 125 = 375 (g)

Cu 6:
-

Lng HCl cn pha dung dch 2% l: X = (2x500)/100 =10 (g)

Lng dung dch HCl 37% cn dng l: Y = (100 * 10)/37 = 27.03 (g)
=> 27.03 /1.19 = 22.71 (ml)

Lng nc ct thm vo : 500 22.71 = 477.29 (ml)

BI 2 : NH LNG NG KH BNG PHNG

PHP ACID DINITRO-SALICYLIC (DNS)
L THUYT

Nguyn tc
Phng php ny da trn c s phn ng to mu gia ng kh vi thuc th
acid dinitrosalicylic (DNS) .Cng mu ca hn hp phn t l thun vi nng
ng kh trong mt phm vi nht nh.

THC HNH
I.

Dng c -Ha cht:

1. Dng c
-

My o mu

Cuvette d = 1 cm, V = 4ml

ng nghim c np v cc dng c thy tinh khc

Bp in

2. Ha cht
-

Mu rau qu cha ng (da)

-Thuc th DNS

-Dung dch glucose chun 1mg/ml (bo qun lnh)

II.

Tin hnh

1. Chit xut ng trong thc vt

-

Chun b mu thm 20g

Gi nh

Vt ly nc cho vo bnh nh mc

Thm nc ct 100ml

Pha long mu h s n (20,50,100,200 ln)

2. Dng ng chun v xc nh hm lng ng

Ha cht

ng1

ng2

ng3

ng ng5

ng6

ng7

4
Glucose 1mg/ml

0.2

0.4

0.6

0.8

Nc ct(ml)

0.8

0.6

0.4

0.2

Mu pha long(ml)

0.2

0.4

0.6

0.8

DNS (mol)

OD540nm

0.333

0.712

1.233

1.469

1.623 0.179

o bc sng 540nm

CH : tin hnh cc mu dng ng chun v th nghim ng thi

ng chun nng ng kh (mg/ml)

vi mt quang OD540nm
2
1,8

1,6

OD540nm

1,4
1,2
1
y = 1,7206x + 0,0347
R = 0,9756

0,8
0,6
0,4
0,2
0
0

0,2

0,4

0,6
C

0,8

1,2

10

Tnh kt qu:
T phng trnh th ng cong chun tnh c mg/ml ng kh trong dung dch
ng pha long.
Nhn vi h s pha long n c lng ng trong 1 ml dung dch gc
Chn h s pha long n sao cho OD nm trong gii hn ng chun
Da vo phng trnh ng tuyn tnh gia nng ng kh (mg/ml) vi mt
quang OD540 trn ta tnh c hm lng ng kh P trong mu phn tch X.

y = 1,7206x + 0,0347 x =

0,0347
1,7206

0,1790,0347
1,7206

= 0.084

Tnh hm lng ng trong nguyn liu (mg/g) = X * n * V/m = 0.084 * 25 * 100/20 = 10.5

11

CU HI N TP
Cu 1: ng kh l cc ng cha nhm aldehyde (-CHO) hoc ketone (-CO) nh
glucose , fructose ,arabinose , maltose , lactose (saccharose , trehalose khng phi ng
kh.
Cu 2: p dng phng php o ng kh trong trng hp phn tch nng
ng kh trong dung dch.
Cu 3 : Sai s h thng do nhng l do:
- Ha cht chun b cho th nghim khng c chun.
- Do vn chuyn mu khng ng cch
- Thao tc o khng chnh xc
- Ghi khng chnh xc ch s
- dng c cuvete khi o OD
- Ht ha cht khng chnh xc
Cu 4 : Khi o OD my bo over l do mu qu m c cn phi pha long li.
Ga tr OD nm trong khong t 1 n 5 .V nu cao qu hoc thp qu ng chun s
b lch.
Cu 5: Trong bi thc hnh chn ng glucose dng ng chun .iu ny
khng lun ng .v ng kh c nhiu loi nh fructose, maltose..

12

BI 3: NH LNG NIT TNG S BNG PHNG

PHP KJELDAHL
1. nh ngha:
- Tt c cc dng nit c trong c th hay trong cc m c gi l nit tng s.
Nit c trong cc thnh phn amino acid ca protein l nit protein. Nit khng c trong
thnh phn protein nh ca cc mui NH4+, cc amino acid t do, cc peptide, urea v
cc dn xut ca urea, cc alkaloid, cc base purin v pyrimidine,.. l nit phi protein
Nit tng s = Nit protein + Nit phi protein
- m tng s hay protein tng s l nit tng s nhn vi h s chuyn i. H s
ny ph thuc vo hm lng nito trong protein. Thng thng nito chim 16% protein
nn h s chuyn i thng s dng l 100/16 = 6.25
- m tng s = Nito tng s X h s chuyn i
- Bng di y biu din h s chuyn i c th cho nhiu i tng mu khc
nhau.
MU
Ngun gc ng vt
Ht bng
u phng
u nnh
Ht hng dng
Cm da
Ht m
Bp
Go
La m

H S CHUYN I
6.25
5.30
5.46
5.71
5.3
5.3
5.3
6.25
5.95
5.83

2. Nguyn tc:
a) V c ha mu.
- Trc tin mu c v c ha bng H2SO4 c nhit cao v c cht xc tc.
Cc phn ng ca qu trnh v c ha xy ra nh sau:
2H2SO4 2H20 + 2SO2 + O2

13

- Oxi to thnh trong phn ng li oxi ha cc nguyn t khc. Cc phn t cha

nito di tc dng ca H2SO4 to thnh NH3. V d cc protein b thy phn thnh amini
acid, cacbon v hidro ca acid amine to thnh CO2 v H2O, cn nito c gii phng
di dng NH3 kt hp vi H2SO4 d to thnh (NH4)2SO4 tan trong dung dch.
2NH3 + H2SO4 (NH4)2SO4
- Cc nguyn t P, K, Ca, Mg,chuyn thnh dng oxide: P2O5, K2O, CaO,
MgO,
b) Chng ct m:
- ui NH3 ra khi dung dch bng NaOH
(NH4)2SO4 + 2NaOH Na2SO4 + H2O + 2NH3
- NH3 bay ra c lm lnh bin i thnh NH4OH ri vo bnh hng, bnh hng
cha H2SO4 0.1N
2NH4OH + H2SO4 (NH4)2SO4 + 2H2O + H2SO4 (d)
c) Chun H2SO4 d
Chun H2SO4 d bng NaOH 0.1N
H2SO4 (d) + NaOH Na2SO4 + H2O
d) Tnh kt qu
Hm lng % nito tng s c tnh theo cng thc:
N(%) = {1.42/1000*(V1 V2)*100/m}*n
Trong :
-

V1: s ml H2SO4 cho vo trong bnh

V2: s ml NaOH 0.1N chun
m: s gam mu hay ml mu sau khi chun li
n: h s pha long mu khi v c ha mu a vo chng ct

1.42: h s nito, c 1ml H2SO4 dng trung ha NH4OH th tng ng vi

1.42mg nito
Bi tp: pha long 50 ln sa ti nguyn cht Vinamilk bng bnh nh mc
100ml, sau chng ct m vi lng H2SO4 cho vo bnh l 20ml v chun bng
NaOH 0.1N. Tnh hm lng N tng s trong mu, tnh ra protein tng s.
V c ha mu

14

2NH3 + H2SO4 + (2g xc tc) + to (NH4)2SO4

2ml mu

5ml

dd trong sut

Chng ct m

(NH4)2SO4 + 2NaOH Na2SO4 + H2O + 2NH3

100ml

35ml

NH3 + H2O NH4OH

2NH4OH + H2SO4 (NH4)2SO4 + 2H2O + H2SO4 (d)
20ml

Chun H2SO4 d

H2SO4 (d) + NaOH + 3 git Tashiro Na2SO4(xanh l m) + H2O

14,25ml
Chun cho thy lng NaOH 0.1N phn ng l V2= 14.25ml
Ta c: V1 = 20ml, m = 100ml, n = 50 ln
p dng cng thc ta tnh c:
N(%) = {1.42/1000*(V1 V2)*100/m}*n = {1.42/1000*(20 14.25}*100/100}*50
= 0.40825%
Nito (tng s) = Nito tng s X h s chuyn i = 0.40825 x 6.25 = 2.5515625

Dd trc chun

Dd sau chun

15

CU HI N TP
Cu 1) Gii thch ngha cc bc trong th nghim
a) V c ha mu.
- Oxi to thnh trong phn ng li oxi ha cc nguyn t khc. Cc phn t cha
nito di tc dng ca H2SO4 to thnh NH3.NH3 kt hp vi H2SO4 d to thnh
(NH4)2SO4 tan trong dung dch.
2NH3 + H2SO4 (NH4)2SO4
b) Chng ct m:
- ui NH3 ra khi dung dch bng NaOH
- (NH4)2SO4 + 2NaOH Na2SO4 + H2O + 2NH3
- NH3 bay ra c lm lnh bin i thnh NH4OH ri vo bnh hng, bnh hng
cha H2SO4 0.1N
- 2NH4OH + H2SO4 (NH4)2SO4 + 2H2O + H2SO4 (d)
c) Chun H2SO4 d
- Chun H2SO4 d bng NaOH 0.1N
- H2SO4 (d) + NaOH Na2SO4 + H2O
Cu 2. Tho lun cc yu t dn n sai s.
Ht khng chnh xc mu
Chun khng chnh xc
Sai s ca my chng ct
Cu 3. V sao phi bo m NaOH trong bnh chng ct m d?
V NaOH d th s chuyn tt c (NH4)2SO4 thnh NH3
Cu 4. Nu thay H2SO4 trong bnh hng bng acid boric (H3BO3) th kt qu c thay
i g khng?
Ta c: 0.1N H2SO4 = 0.05M H2SO4
Ta chuyn qua phng trnh v 1.42 l h s nito, c 1ml H2SO4 dng trung ha
NH4OH th tng ng vi 1.42mg nito. V ly 1ml nn nhn thm cho 10-3

2NH4OH
0.1*10-3

H2SO4
0.05*10-3

mN=0.1*10-3 * 14.2

(NH4)2SO4 + 2H2O + H2SO4 (d)

16

3NH4OH

+ H3BO3 (NH4)3BO3 + 3H2O + H3BO3 D

0.15*10-3*14.2

0.05*10-3

mN=0.15*10-3 * 14.2= 2.13*10-3

V vy nu thay thnh acid boric th h s s l 2.13
Cu 5. Nu chun bng NaOH 0.05N th kt qu N tng s thay i th no?
p dng cng thc: C1V1 = C2V2
V1, C1 c nh v V2 phi chun
M C2 gim 1 na th V2 tng th tch gp i Cng thc thay i

BI 4. NH LNG PROTEIN BNG PHNG PHP

BRADFORD

L thuyt
1. Nguyn tc
Phng php ny da trn s thay i bc sng hp thu cc i ca thuc nhum
Coomassie Brilliant Blue khi to phc hp vi protein. Trong dung dch mang tnh acid,
khi cha kt ni vi protein th thuc nhum c bc sng hp thu cc i 465 nm; khi
kt hp vi protein th thuc nhum hp thu cc i bc sng 595 nm. hp th
bc sng 595 nm c lin h mt cch trc tip ti nng protein.
xc nh protein trong mu, u tin ta xy dng mt ng chun vi dung
dch protein chun bit trc nng . Dung dch protein chun thng l bovine
serum albumin (BSA) . Sau khi cho dung dch protein vo dung dch thuc nhum, mu
s xut hin sau 2 pht v bn ti 1 gi. Tin hnh o dung dch bng my quang ph k
ta c ODx, hp th s t l vi lng trong mu. Thc hin mt i chng vi nc
ct (OD0). Ly gi tr OD = ODx - OD0. Lng protein mu trong dung dch o c xc
nh bng cch chm trn ng chun theo OD, ly gi tr ca im trn trc honh.
chnh l lng protein mu trong dung dch o.

17

Cng thc phn t ca Coomasie Brilliant Blue G-250

THC HNH
1. Dng c - ha cht
1. Dng c
- My o quang ph
- ng nghim (14)
- Pipette 5ml, 1ml (c th thay bng pipetteman)
- ng h bm giy
- Giy thm
- Gi ng nghim
- Bnh tia ng cn
2. Ha cht
- Cn 900
- Dung dch protein chun: cn 10 mg albumin bng cn phn tch, pha trong 1 ml
nc ct, lc u cho tan. Gi -200C. Khi dng pha long ra 100 ln, c dung dch c
nng 0,1 mg/ml.
- Dung dch thuc th Bradford: dd thuc th c thnh phn trong 100ml nh sau:
Coomassie Brilliant Blue: 0,005g

18

Methanol: 4,7g
Phosphoric acid 85%: 8,5g
Phm mu Coomasie Brilliant Blue c lm tan trong ethanol trong mt chai
ng mu ti c np. B sung phosphoric acid v chnh ti 100ml bng nc ct. Lc
u, gi 40C
- Dung dch cn xc nh hm lng protein (phng th nghim cung cp)
2. Tin hnh
- Thu dch trch ly protein nh trong bi enzyme (5 g malt trch ly bng nc thu
c V = 100 ml). Pha long mu vi h s pha long n ln c mu phn tch (mu
X).
- Lp mt lot 6 ng theo s th t 0, 1, 2, 3, 4, 5 v 1 ng nghim cha mu cn
phn tch X

- Dng ng h bm giy, canh thi gian 0 pht cho 5ml thuc th vo ng nghim
0, lc u yn. thi im 1 pht cho 5ml thuc th vo ng nghim 1, lc u
yn,... c tip tc cho n ht.

19

ng nghim

Nc ct (ml)

0.9

0.9

0.7

0.6

0.5

Albumin chun (0,1mg/ml)

0.1

0.2

0.3

0.4

0.5

TT Bradford

Nng Albumin (g /ml)

10

20

30

40

50

4.14

OD595nm

0.076 0.210 0.376 0.439 0.485 0.045

- Tnh thi gian ng 0 c 20 pht, tin hnh o hp thu ca dung dch bc

sng 595 nm. Ta c gi tr OD0, thi im 21 pht o ng 1 (OD1),... tng t c
cch mt pht o cho ht cc ng. Ghi li gi tr OD.
3. Tnh kt qu:
- V ng tuyn tnh gia nng protein (g/ml) vi mt quang OD595nm
NG CHUN BSC PHNG PHP BRADFORD
0,6

0,5

OD 595nm

0,4

0,3

y = 10,514x + 0,0015
R = 0,9675

0,2

0,1

0
0

10

20

30

40

50

C (g /ml)
60

- Da vo phng trnh ng tuyn tnh gia nng protein (g/ml) vi mt

quang OD595 trn ta tnh c hm lng protein P trong mu phn tch X.

20

= 10,514 + 0.0015 =

0,0015 0,045 0,0015

=
= 4,14 . 103
10,514
10,514

= . = 4,14 . 103 . 25 = 0,1035

tnh tan hm lng protein trong 1g mu cn:
Protein (g/g) =

= 0,1035

100
10

= 1.035

CU HI N TP:
1. Gii thch ngha cc bc trong th nghim.
- Pha long mu vi h s pha long n ln c mu phn tch
Phi gim bt nng protein trong mu, khi o nng quang khng b vt
qu gii hn cho php.
- Lp mt lot 6 ng theo s th t 0, 1, 2, 3, 4, 5 v 1 ng nghim cha mu
cn phn tch X.
Phi pha cc nng khc nhau lp ng chun. xc nh protein trong
mu.
- Dng ng h bm giy, canh thi gian 0 pht cho 5ml thuc th vo ng
nghim 0, lc u yn. thi im 1 pht cho 5ml thuc th vo ng
nghim 1, lc u yn,... c tip tc cho n ht.
Phng php ny da trn s thay i bc sng hp thu cc i ca thuc nhum
Coomassie Brilliant Blue khi to phc hp vi protein.
- Tnh thi gian ng 0 c 20 pht, tin hnh o hp thu ca dung dch
bc sng 595 nm. Ta c gi tr OD0, thi im 21 pht o ng 1 (OD1),...
tng t c cch mt pht o cho ht cc ng. Ghi li gi tr OD
Trong 20 pht thuc nhum kt hp vi protein khi thuc nhum hp thu cc
i bc sng 595 nm.

21

2. Tho lun cc yu t dn n sai s.

Do sai thao tc trong tin hnh th nghim.
Mu protein chun b khng c chun.
Ha cht chun b cho th nghim khng c chun.
Do vn chuyn mu khng ng cch
Ly mu khng ng theo th tch yu cu
Ghi khng chnh xc ch s.
Thao tc o khng chnh xc
My o OD khng chun.
3. p dng phng php bradford cho trng hp no
Phng php ny da trn s thay i bc sng hp thu cc i ca thuc
nhum Coomassie Brilliant Blue khi to phc hp vi protein.Hnh thnh hp
cht mu c kh nng hp thu nh sng bc sng 595nm, cng mu t l
vi nng protein trong dung dch.
Tit kim thi gian v d thc hin.
Cng mu t l nng protein trong dung dch nng cng cao th mu
xanh cng m khng ph thuc nng acid amin ca dung dch.
Phng php c nhy cao cho php pht pht hin ti vi protein 5-200 g/
ml Nhy hn phng php Lowry gp 4 ln.
t b nh hng bi cc cht thng gp trong t bo gp chng hn nh mui.
4. Trng hp mu th rn cn phi x l mu nh th no?
Cn m = 5-10g mu rn nghin nh, cho vo bnh nn dung tch 250ml, b sung 50ml
nc ct v 10ml dung dich m phosphat pH = 4,9. Gi hn hp nhit 30oC trong
thi gian 1 gi c khuy o nh k. Lc, ra thu hi dung dch sao cho V = 100ml. Bo
qun dung dch gc 2 4oC trong thi gian mt ngy.

22

BI 5: PHNG PHP XC NH HOT TNH ENZYME

L THUYT:
I.

Enzyme v n v o hot tnh ca enzyme.

a. nh ngha
Enzyme l cht xc tc sinh hc c bn cht l protein v c tnh c hiu cao. Mi
enzyme c kh nng xc tc cho mt hoc mt s phn ng nht nh. Hot ng hay
hot tnh ca enzyme cng mnh th lng c cht c chuyn ha hoc lng sn
phm to thnh trn mt n v thi gian cng ln. V vy c th nh gi hot tnh xc
tc ca enzyme bng cch xc nh tc chuyn ha c cht hoc tc tch ly sn
phm phn ng.

V nguyn tc c th hai nhm phng php chnh sau:

o lng c cht b mt i hay lng sn phm c to thnh trong mt thi

gian nht nh ng vi mt nng enzyme xc nh.

o thi gian cn thit thu c mt lng bin thin nht nh ca c cht hay
sn phm tng ng vi mt nng enzyme xc nh.
thc hin c mc ch trn, nhiu phng php phn tch khc nhau c s
dng: phng php o quang ph, o phn cc, p sut, nht, phng php sc k
v phng php ha hc.
Mc ch xc nh hot tnh enzyme l xc nh s n v hot tnh. Mt n v hat
tnh enzyme c nh ngha theo nhiu phng php.
b. n v hot tnh ca enzyme:
n v n v quc t (Enzyme Unit, vit tt U)
Do Hip hi Ha sinh Quc t (International Union of Biochemistry IUB) nh
ngha:
Mt n v chun ca enzyme (1 U) l lng enzyme xc tc chuyn ha c
1 mol c cht sau 1 pht iu kin tiu chun.

23

1 U = 1 mol sn phm = 1 mol c cht (10-6 mol)/pht

Katal:
Nm 1979, Hi ng Danh php ca IUB khuyn co nn s dng Katal lm n v
c bn ca hot tnh enzyme.
Mt Katal l lng enzyme xc tc chuyn ha c 1 mol c cht sau 1 giy iu
kin tiu chun.
1 Kat = 1 mol c cht/ giy = 60 mol/ pht = 60 x 106 mol/ pht = 6 x 107 U
1 U = 1/60 x 10-6 Kat = 16,67 nKat (nanokatal)
n v Katal c khuyn co v nm trong h n v o lng Quc t (SI).
n v t t: n v hot tnh da vo s thay i c tnh hn hp phn ng, v
d s thay i c, nht ...trong mt n v thi gian. Trng hp c cht v sn
phm l mt hn hp phc tp th p dng n v hot tnh ny.

II.

Hot tnh ring ca enzyme.

Hot tnh ring ca mt ch phm enzyme c trng cho tinh sch ca ch phm
enzyme. Hot tnh ring c biu th bng s n v enzyme/mg protein ( U/mg
protein) hoc (Kat/kg protein), trong hm lng protein c xc dnh bi phng
php Lowry hoc Bradford.

III.

Phng php xc nh hot tnh enzyme.

Ch :
-

Cn trch nhng yu t c th bin tnh protein enzyme.

Cc thng s nhit , pH, nng ion v thnh phn dung dch m nh hng

ln hot tnh enzyme. Th hot tnh enzyme phi c tin hnh trong iu kin thch
hp nh iu kin sinh l, k tn tr thc phm hoc k m hot lc c th t ti u.
-

Vi nhng enzyme cn c cht hot ha hoc cht n nh th phi cho cc cht

ny vo enzyme trc khi cho c cht vo hn hp phn ng.

-

Nng c cht gii hn thch hp, tha bo ha enzyme, khng qu cao

km hm enzyme, c cht c chuyn ha 20%-30%.

24

Thi gian xc nh hot tnh thng 5-30 pht.

Khi xc nh hot tnh phi lm mu i chng( enzyme b bt hot trc khi tip

xc vi c cht) song song vi th nghim.

Enzyme amylase v phng php xc nh hot tnh.

IV.

a. Khi qu v enzyme amylase.

b. Cc phng php o hot tnh enzyme amylase.
-

Nguyn tc chung da trn c s thy phn tinh bt bi enzyme trong dd enzyme

nghin cu thnh cc dextrin c phn t lng khc nhau. o cng mu to thnh

gia tinh bt v cc sn phm thy phn ca n vi iod bng my so mu s tnh c
hot tnh ca enzyme.
-

n v amylase (theo Smith v Roe) l ln enzyme cn thit thy phn hon

ton 10 mg tinh bt sau thi gian phn ng 30 pht trong iu kin th nghim.

THC HNH:
1. Dng c - ha cht:
a. Dng c.
-

My lc.

My o quang ph, cuvette dy 1cm.

B iu khin.

Bnh nh mc.

ng nghim.

Pipette 5ml, 1ml.

ng h bm giy.

Giy thm.

Gi ng nghim.

Bnh tia ng cn.

b. Ha cht:

25

Dung dch m.
-

Dung dch m

Dung dch m acetate pH= 4,7 (xc nh hot tnh enzyme nm mc): trn 1 th

tch CH 3COOH 1N vi 1 th tch CH 3COONa 1N. Kim tra pH.

-

Dung dch m phosphate pH= 4,9 (xc nh hot tnh enzyme ca malt): trn 10

ml dung dch Na 2HPO 4 1/15 M vi 990 ml dung dch KH 2PO 4 1/15 M nhn c 1 l
dung dch m phosphate pH = 4,94. Kim tra pH.
-

Dung dch m glycine NaOH 0,1 M pH = 10 (dng xc nh hot tnh

amylase kim.
-

Dung dch HCl 0,1N .

Dung dch iod: ha tan 30 mg KI v 3 mg I2 v i mt lng nh nc ct. Lc nh

hn hp ha tan hon ton, sau chuyn dung dch sang b nh nh mc 1000 ml, b
sung nc ct n vch mc. Bo qun dung dch trong b nh mu nu ch ti.
-

Dung dch tinh bt 1%: h a tan 1 g tinh b t (theo cht kh tuyt i) vi 50 ml

nc ct trong bnh nh mc 100 ml, lc u. t v o bp cch thy ang si, lc li n

tc cho n khi tinh bt tan ho n ton. Sau lm ngu i v b sung 10 ml m acetate
pH = 4,7 (hoc 10 ml dung dch m phosphate pH = 4,9) b sung n c ct n vch
mc, lc u. Dung dch c chun b trong ngy s dng.
-

Dung dch enzyme gc.

Trch ly enzyme:
Enzyme c trch ly t ch phm hay t bo, m ng thc vt bng dd m c pH
thch hp nh
-

Trch ly enzyme t vi khun: Cn m= 0,1g ch phm dng a thy tinh ch

nh ch phm trong cc dt 50ml cho vo bnh nh mc 100ml + H2O ct lc v

trch ly enzyme bo qun 2-4 / 1 ngy.
-

Trch ly enzyme nm mc: Cn m=5g ch phm dng a thy tinh ch nh

ch phm trong cc dt 50ml cho vo bnh nh mc 100ml + H2O ct +dd m

26

Acetate pH=4,7 gi nhit 30 trong 1 gi lc, ra, trch ly ly enzyme V=

100ml bo qun 2-4 / 1 ngy.
-

Trch ly enzyme t malt: Cn m=5-10g ch phm xay nh cho vo bnh

nh mc 100ml + H2O ct +dd m phosphase pH=4,9 lc, ra, trch ly ly enzyme

V= 100ml bo qun 2-4 / 1 ngy.
Pha long enzyme:
H s pha long : n= 50,100,200
Xc nh hot tnh th nghim:
Ha cht

Th nghim

i chng

Mu trng

Dd Tinh bt 1%

2 ml

2 ml

Nc ct

2ml

1ml

1ml

1ml

Dd NaCl 3%

0,5ml

0,5ml

0,5ml

40 , 10 pht t iu kin enzyme hot ng tt.

Dd enzyme

1ml

40 , 30 pht xy ra phn ng
HCl 1N

1ml

1ml

1ml

Dd enzyme

1ml

1ml

Chuyn sang bnh nh mc 100ml.

Nc ct

n vch

n vch

n vch

Iod

0,5ml

0,5ml

0,5ml

27

Tnh kt qu:

00 :
0: .
=

00 0
20
00

1,0720,106
20
1,072

= 18,022

18,022 50 100
=
= 901,1
)=

10
10 10

Hat tnh ring (n v hat tnh/mg protein) = hat tnh ton phn/mg protein
(Bradford).
Trong : n = h s pha long
V= th tch dch trch ly, ml.
m= khi lng enzyme em trch ly, g

28

CU HI N TP
Cu 1: Phn bit cc dng chnh ca enzyme amylase v ngun thu chng.
C 3 dng chnh:
--amylase (Endo -1,4-glucanase; EC 3.2.1.1) c trong nc bt, ht ha tho ny
mm, ty tng, nm mc, vi khun
--amylase (Exo -1,4-glucanase; EC 3.2.1.2) c nhiu thc vt (ht c)
-Glucoamylase
Cu 2: Bn cht ca hot tnh enzyme l g? Nu cc phng php o hot tnh ca
enzyme
Bn cht: hot tnh ca enzyme cng mnh th lng c cht c chuyn ha hoc
lng sn phm to thnh trn mt n v thi gian cng ln.
Hai nhm phng php chnh sau:
- o lng c cht b mt i hay lng sn phm c to thnh trong mt thi gian
nht nh ng vi mt nng enzyme xc nh.
- o thi gian cn thit thu c mt lng bin thin nht nh ca c cht hay sn
phm tng ng vi mt nng enzyme xc nh.
Cu 3: C cht v sn phm ca enzyme -amylase l g? Da trn pp no ngi ta
chn pp xc nh hot tnh nh trong th nghim?
C cht ca enzyme -amylase l tinh bt. Enzyme ny phn gii lin kt 1,4glycoside gia chui polysaccharide (hot tnh endoamylase) to thnh maltose, dextrin
phn t thp.
Nguyn tc chung da trn c s thy phn tinh bt bi enzyme trong dung dch
enzyme nghin cu thnh cc dextrin c phn t lng khc nhau. o cng mu to
thnh gia tinh bt v cc sn phm thy phn ca n vi iode bng my so mu s tnh
c hot tnh enzyme.
Cu 4: C th s dng pp o ng kh bng DNS acid o hot tnh enzyme amylase khng? Ti sao?

29

V DNS to mu vi ng kh cn trong bi l o hot tnh enzyme -amylase l tinh

bt to mu vi lugol.
Cu 5: Cho bit nh ngha n v hot tnh theo Smith v Roe. n v ny c ging
n v quc t khng? Ta c th chuyn n v Smith v Roe thnh n v quc t khng
nu gi nguyn pp xc nh nh trong bi.
n v amylase (theo Smith v Roe) l lng enzyme cn thit thy phn hon
ton 10 mg tinh bt sau thi gian phn ng 30 pht trong iu kin th nghim.
n v ny khng ging n v quc t.

BI 6 :TCH CC SC T QUANG HP BNG PHNG PHP

SC K GIY HOC BN MNG
I.

L THUYT
NGUYN TC

Hn hp cc phn t sc t c th phn tch thnh cc n cht bng cc phng php

sc k , trong sc k giy hoc sc k bn mng l n gin hn c . Cc phng php
da vo kh nng tng tc khc nhau ca cc phn t sc t vi phn t pha tnh v kh
nng li ko ca dung mi .
DNG C -HA CHT
-

Dng c .

Phu lc.

Giy lc .

Bn mng nhm ph silica gel G254.

Bnh tam gic 250ml.

ng nghim c np hay bnh cha dung mi sc k.

30

Mao qun chm mu hay micropipette u tp 100.

nguyn liu ha cht.

L rau ci b xi hay cc loi rau xanh khc.

Ethanol 96 %.

Acetone.

Ether du ha .

Cn 95%.

II.

TIN HNH.

Nghin l 10g trong 10ml ethanol 95% un si 5 pht.

Lc.

Ct giy lc thnh di c u nhn .k vch tip mu v vch dng.

Thm mu ln vch bng micropipette.

Nhng giy lc trong dung mi .

dung mi chy ln vch dng.

nh du cc vt sc t bng bt ch.

o khong cch t vch sc t n vch thm mu .

31

TNH KT QU
Rf=7,5/8
TR LI CU HI
Cu1 : phn cc ca cc dng sc t quang hp trong hnh 6.1 lcholorophyll A v
cholorophyll b c phn cc ln hn b-carotene , b-carotene c phn cc thp , cn
zeaxanthin c phn cc ln hn b-carotenen nhng nh hn cholorophyll A v
cholorophyll B
Cu2 :Phi dng ethan 95% trch ly sc t m khng dng nc v cc sc t ch tan
trong dung mi hu c , khng tan trongnc
Cu3 :Gi tr Rf ca sc t l 7,5/8 . Tn sc t c gi tr Rf = 7,5/8 l zeaxanthin