B .

CC PHNG PHP NH LNG PROTEIN

I. Xc nh nng protein ho tan theo Lowry

1. Nguyn tc Phng php ny da trn c s phc cht ng protein kh hn hp photphomolipden photphovonphramat (thuc th Folin ciocalteu) to phc cht mu xanh da tri c hp th cc i bc song 660nm. Cng mu ca hn hp phn ng t l thun vi nng protein trong mt phm vi nht nh. Da vo mc hp th quang hc ca protein chun, ta c th xc nh c hm lng protein trong mu nghin cu.

2. Ho cht cn dng - Dung dch A: 4g NaOH (0,1M) v 20g Na2CO3 (2%) pha trong 1000ml nc ct. - Dung dch B: 0,5g CuSO4.5H2O (0,5%) pha trong dung dch Natri Xitrat (1%) hoc trong dung dch Natri, Kali Tactrat 1%. - Dung dch C: Hn hp ca hai dung dch A v B theo t l 49:1 (pha trc khi dng). - Thuc th folin, trc khi dng pha long hai ln sao cho axit bng 1N. - Albumin huyt thanh b 1mg/ml.

3. Cch tin hnh

- Mu th nghim: ly chnh xc 0,5ml dch cha protein vi hm lng thch hp cho vo ng nghim, thm vo 2ml dung dch C, lc u yn trong 10 pht. Sau thm vo hn hp trong ng nghim 0,25ml folin pha long 2 ln, lc u v yn trong 30 pht, mu vng ca hn hp chuyn sang mu xanh da tri v t n cng mu cc i. em so mu ca hn hp trn my so mu quang in bc sng 660nm. Xc nh c tr s mt quang hc ca dung dch nghin cu. o trn my 3 ln lp li v ly tr s trung bnh. - Mu i chng: 0,5ml m cho vo ng nghim, thm vo 2ml dung dch C v bc tip theo c tin hnh nh mu th nghim.

4. Xy dng ng th chun Hm lng protein tan trong dung dch c xc nh theo phng php Lowry, dng albumin huyt thanh b (BSA) lm cht chun. Cn 0,01g (10mg albumin huyt thanh b) ho tan trong 10ml nc, ta c dung dch gc c nng l 1mg/ml. Sau pha long dung dch gc bng nc ct vi cc nng 20, 40, 60, 80, 100, 120 g/ml tin hnh xy dng th chun. - Mu th nghim: ly chnh xc 0,5ml dung dch BSA nng pha long nh trn cho vo ng nghim, thm vo mi ng 2ml dung dch C nhit phng 10 pht, sau cho 0,25ml thuc th folin (1N) em so mu vi bc sng 660nm. - Mu i chng: ly 0,5 ml nc ct cho vo ng nghim, thm vo 2ml dung dch C v cc bc tip theo c tin hnh nh mu th nghim. Qua 3 ln lp li th nghim c th xy dng ng hi quy. Kt qu c x l theo phng php thng k thng thng.

Ta c phng php hi quy :

y = 0,55066844x + 0,01791961

OD 660 nm

0,6 0,5 0,4 0,3 0,2 0,1

0 0,2 0,4 0,6 0,8 1 1,2

mg

Hm lng protein Da vo th chun, c th xc nh c hm lng protein trong mu nghin cu.

II. nh lng protein theo phng php Bradford

Hm lng protein c th c nh lng theo phng php Bradford (1976).( Cn gi l phng php so mu v ch p dng c vi cc protein tan trong dung dch nn mun xc nh hm lng protein mt mu ta phi trch ly, hm lng protein xc nh ph thuc vo hiu qu trch ly tc ph thuc vo kh nng ha tan ca protein trong dung mi trch ly ). 1. Nguyn tc:

Phng php ny da vo s thay i mu sc xy ra khi Coomassie Brilliant Blue G-250 lin kt vi protein trong dung dch axit, dng proton ha ca thuc nhum Coomassie Blue c mu da cam . Thuc nhum lin kt cht ch vi cc protein, tng tc vi c nhm k nc v cc nhm mang in tch dng trn phn t protein.Trong mi trng ca cc gc mang in tch dng, s proton ha khng xy ra v c mu xanh xut hin.

2. Vt liu v dng c: - Cc mu protein. - Dung dch mu( thuc nhum) + Coomassie Brilliant Blue G-250 (CBB G-250)100mg + Ethanol (EtOH) (absolute) 95%50ml + H3PO4 85% + H2O - Pipet man (Hng Gilson Php). - My o quang ph Shimadzu UV 200s (Japan). - ng nghim

3. Cch tin hnh 3.1. Xy dng ng cong chun biu din nng protein chun (BSA): * Quy trnh phn tch:

Dung dch protein chun BSA (Bovine Serum Albumin) 10-100g protein/ml dung dchNaCl 0,15M

ng th nghim: 0,1ml dung dch protein chun ln lt cc nng t 10-100g, thm 5ml dung dch thuc nhum, trn u, phn ng xy ra nhit phng.

ng i chng: 0,1ml dung dch NaCl 0,15M, thm 5ml dung dch thuc nhum, trn u, phn ng xy ra nhit phng.

c quang ph bc sng 595nm sau 2 pht n 1 gi (cuvet 3cm).

*Quy trnh vi phn tch:

Dung dch protein chun BSA (Bovine Serum Albumin) 1,0-10,0 g protein/ml dung dchNaCl 0,15M ng th nghim: 0,1ml dung dch protein chun ln lt cc nng t 1,0-10,0g, thm 1ml dung dch thuc nhum, trn u, phn ng xy ra nhit phng.

ng i chng: 0,1ml dung dch NaCl 0,15M, thm 1ml dung dch thuc nhum, trn u, phn ng xy ra nhit phng.

c quang ph bc sng 595nm sau 2 pht n 1 gi (cuvet 3cm). => V th biu din s bin thin mt quang (OD) , theo nng protein chun BSA tng ng vi gi tr OD 595nm. (2 th tng ng vi 2 dung dch protein chun)

ng cong tiu chun ny c s dng xc nh hm lng protein trong cc mu cha bit, cng c xy dng tng t theo phng php Lowry.( hnh 7.2 v 7.3 trang 88 sch thc hnh ha sinh hc-Nguyn Vn Mi) 3.2. nh lng protein camu:

Ht 0,1ml mu protein cn phn tch vo ng nghim Cho thm t 1-5ml thuc nhum, lc u, ch khong 10 pht o quang ph bc sng 595nm

=> Da vo th chun xc nh hm lng protein trong mu theo cng thc sau:

X=[C].a
Trong : + X: s g protein c trong mu + [C]: nng protein theo th (g/ml). + a: pha long dung dch. Mun xc nh hm lng protein kh ta tnh:

P=X.V/m
Trong : + X: s g protein c trong mu + V: thtch dung dch trch ly; ml + m: khi lng mu; g

Lu : - Thuc nhum phn ng vi protein to dung dch c mu xanh. - Cng mu t l thun vi nng protein c trong dung dch. - y l th nghim vi hp cht mu, nn ng nghim v cuvet phi c loi b cht mu bng axeton, sau ra hoc ngm trong HCl 0,1M. nh gi: Phng php ny c nhy cao, cho php pht hin 5-200g protein/ml, d thc hin v tit kim thi gian.

III. nh lng protein bng phng php quang ph:

1. Nguyn tc :

Phng php ny l cc axit amin thm nh tryptophan , tyrosine , phenylalanine trong chui polypepetid c ph hp th bc sng cc i bc sng 282nm. hp th tia cc tm thay i ty loi protein , nhng h s tt o c (ngha l hp th ca dd protein 1% vi ng truyn qua 1cm),cho mi protein cho php tnh nng ca protein tinh sch , s hp th tia cc tm : protein hp th tia cc tm bc sng 240-280nm , axit

amin nhn thm :tryptophan , tyrosine , phenylalanine c ph hp th 280nm . ng thi chng cng hp th bc sng thp hn 215-230nm hp th ny ph hp cho peptid khng c tryptophan , tyrosine. Nu protein tinh sch th nng tuyt i ca n tnh theo gi tr o c . Nu protein khng tinh sch th nng ca protein tng s c tnh tng i t hp th . Phng php ny khng dung c cho cc dd c nng protein thp <0.1mg/ml hoc khi c mt nhiu cht khc m hp th cng 1 vng cc tm hoc khi protein trong dch huyn ph ch khng phi trong dd . 2. Nguyn liu v thit b : DD protein o m ha tan protein My quang ph UV c cuvet thch anh ng sang truyn qua 1cm 3. Tin hnh :
Li tm mu loi b cc phn t hoc phc hp khc c trong th

huyn ph t bc sng 280nm my quang ph v iu chnh hp th v khng vi cuvet cha m c hp th ca mu hoc trong cng mt cuvet hoc cuvet khc cng cp . Nu gi tr thu c > 2.0 th pha long mu (1/5 hoc 1/10) hoc ng sang truyn ngn hn (2nm) cho n khi s c nm trong khong 0.1-0.5 Lp li bc 2 v 3 260nm

 Tnh t s hp th 260 / 280nm. T s ny nn nh hn 0.6 .

Nu t s cao hn cho bit protein khng sch , c ln tp cht khc c bit vi axit nucleic . Nng mu = ( hp th 280nm/h s tt 280nm)*10mg/l Vi mt hn hp cc protein hoc vi bt c mt loi protein no m khng bit h s tt th tnh nh sau :

Nng protein = 1.55* hp th 280nm 0.77* hp th 260nm

IV . nh lng nit tng s bng phng php KJELDAHL 1. Nguyn tc :

Qua 2 giai on : Giai on v c ha : Nito tng s l tt c cc dng nito c trong mu . Khi t nng mu cn phn tch vi H2SO4 m c c mt ca cht xc tc , tt c cc dng m c trong mu u bin thnh dng v c (NH4)2SO4 tan trong dung dch. Giai on ct m: Sau khi v c ha . Ta ui NH3 ra khi dd bng NaOH . Lng NH3 c li cun bng my Parnas v c dn n mt erlen c cha mt lng tha H2SO4 bit chnh xc nng . (NH4)2SO4 + NaOH = Na2SO4 + H2O +NH3 H2O + NH3 + H2SO4 = H2SO4 d + (NH4)2SO4

nh lng H2SO4 cn li bng NaOH , qua tnh c lng nito c trong mu H2SO4 d + NaOH = Na2SO4 + H2O

2. Nguyn liu ha cht :

- Bt u nnh, bt xng, bt c, nc tng, nc mm v.v - Hn hp cht xc tc K2SO4 / CuSO4 - H2SO4 m c - H2SO4 0,1N - NaOH 0,1N - Nc ct 2 ln - Phenolthalein - Metyl 3. Tin hnh : 3.1. V c ha : Tin hnh trong t Hotte Cho mu vo bnh Kjendahl : ht 1ml nc mm hoc nc tng (ch sao cho nguyn liu khng dnh vo thnh c bnh). Thm 10ml H2SO4 m c v 0.5 g xc tc . Cht xc tc c th dung 1 trong nhng hn hp sau:
1 K2SO4 : CuSO4 : Se (100 : 10 : 1) 2 CuSO4 : K2SO4 (1 : 3)

3 Se kim loi (0.05g)

Sau khi thm xc tc un nh hn hp , trnh si tro v ch un mnh khi hn hp hon ton chuyn sang dch lng . Trong qu trnh un thnh thong lc nh , trng kho lo sao cho khng cn 1 vt en no ca mu nguyn liu cha b phn hy cn st li trn thnh bnh . un cho ti khi dd trong bnh hon ton trng. Chuyn ton b dd v c ha xong vo bnh nh mc 100ml , thm nc ct cho n vch nh mc . Ch phi lm ngui v lc u. 3.2. Ct m :( tm hiu th nghim ha sinh) 3.2.1. Ra sch bnh Parnas Wagner. 3.2.2. Ct m Cho 10ml dd H2SO4 0,1N + 3 git Phenolphthalein vo erlen E. Ht 10ml dd v c ha cho vo phu C. un si A. Cho 10ml NaOH d vo C. nh phn H2SO4 0,1N d = dd chun NaOH 0,1N . 4. Tnh ton 4.1 Tnh h s hiu chun F ca dd NaOH - Ly 10ml H2SO4 0.1N chun cho vo erlen , nh phn bng NaOH 0.1N - Tnh nng thc t ca dd NaOH em nh phn - F l t s gia nng thc t v nng l thuyt ca NaOH 4.2 Tnh kt qu :

Hm lng nito trong mu c tnh theo cng thc :

X = (a-b.F).0.0014.100.1000/10.V
Trong : X : hm lng nito tnh bng g/l a : s ml H2SO4 em hp th NH3 b : s ml NaOH 0.1N tiu tn khi chun H2SO4 tha V :s ml mu em v c ha 0.0014 : lng gam nito ng vi 1ml H2SO4 F : h s hiu chnh nng dd NaOH 0.1N Tnh hm lng protein th thng qua hm lng nito tng Nito trong vt liu sinh hc ch yu l nito protein ngoi ra cn c 1 lng nh nito trong cc thnh phn khc gi l nito phi protein . Nito tng = Nito protein + Nito phi protein Hm lng protein trong cc vt liu sinh hc c tnh bng cch nhn hm lng nito protein vi 1 h s chuyn i xc nh do hm lng nito c t l n nh t 15 18% protein , i a s protein l 16% Trong cc nguyn vt liu sinh hc do hm lng nito phi protein nh v vic tch ring rt phc tp nn theo quy c ngi ta tnh hm lng protein theo nito tng v gi l protein th hay protein tng . Cng thc tnh hm lng phn trm protein th c trong i a s cc nguyn liu l :

Protein (%) = Nito (%) x 6.25

Ring ng cc v u c h s chuyn i l 5.7 Sa c h s chuyn i l 6.38.

The End