PHN TCH 3-CHLOROTYROSINE BNG GCMS 1. Mu: - Ha tan protein bng m pH 7.4 hoc pH 3.8.

- Oxy ha protein bng HOCl - Cho 2 mL dd protein + 0.5 mL HOCl nng t 0 200 mM. - Thm ni chun 3-Cl-(C13) tyrosine. - Thm ni chun (C13) tyrosine. 2. Dy chun: - Chun 3-Cl-Tyr nng t 2.5 10 g/mL v 25 100 g/mL. - Chun Tyr nng t 2.5 10 g/mL v 25 100 g/mL. - Thm ni chun 3-Cl-(C13) tyrosine. - Thm ni chun (C13) tyrosine. 3. Thy phn: - Thy phn mu v chun bng HCl 6M (0.1% phenol, 0.1% Na2SO3). - Trung ha bng NaOH 10N. - Chnh pH v 1.5. 4. Kh mui bng ct trao i cation: - Ct c nhi bng Dowex 50WX8 (cng sut trao i 1.7 meq/mL). - Ra ct: 3x3mL HCl 1M 3x3mL H20 3x3mL NaOH 1M 3x3mL H20 - Conditioning: 3x3mL HCl 1M 3x3mL H20 - Load 2mL mu - Ra ct: 3x3mL H20 -Thu mu bng 2x3mL NH3 10% - Cho bay hn NH3 bng kh N2. 5. Dn xut: Mu sau khi cho bay hi n kh c to dn xut bng: - 100 L trifluoroethanol:pyridine (80:20) - 10 L ethylchloroformate - 150 L chloroform c cha 2% ethylchloroformate 6/ GC/MS: 3-Cl-Tyr: m/z (441/447), (352/358), (314/320) Tyr: m/z (318/324), (280/286) 7. Tng hp ni chun 3-[13C6] chlorotyrosine: - Ha tan L-[13C6] tyrosine trong m phosphate pH2 (50mM Na2HPO4 + 100 mM NaCl) - Thm NaOCl, 60 pht 370C. - Thm trifluoroacetic acid (TFA). - 3-[13C6] chlorotyrosine c tch bng HPLC reverve-phase abs 276nm. Dung mi A: 0.1% TFA trong nc, pH 2.5. Dung mi B: 0.1% TFA trong methanol, pH 2.5.

Cc cng thc

3-Chlorotyrosine

Ethyl chloroformate

Trifluoroethanol

Dowex 50WX8 Dung dch m PBS pH 7.4: (7.89 g NaCl + 0.204g KH2PO4 + 2.865g Na2HPO4 + 0,201 g KCl) 1L Acetate pH 3.8: (0.1M Acetic acid + 0.013M Natri acetate) 1L Cu hi: 1. Ti sao phi thm ni chun Tyrosine trong khi ch cn phn tch nng 3-Cl-Tyr? 2. Thy phn protein bng acid ct lin kt peptide, vy tc dng ca 0.1% phenol v 0.1% Na2SO3 l g? 3. Vit cc phng trnh phn ng trong qu trnh trao i cation (bc 4). 4. Tc dng ca pyridine v chloroform c cha 2% ethylchloroformate? 5. Tnh mass chromatography (441, 352, 314) v (318, 280) ca cc dn xut. Ch vit cng thc sau khi kt hp Cl-Tyr vi Ethyl chloroformate v Trifluoroethanol thnh C14H15O5F3NCl nn ch c mass 370, trong khi thy tnh ra l C17H19O7F3NCl, mass 441!!! 6. C phi tc dng ca TFA l lm ngng qu trnh phn ng gia NaOCl v Tyr? Vit phng trnh. 7. Vai tr ca dung mi A v B khi chy HPLC reverve-phase. 8. Tnh meq/mL ca dung dch m pH 7.4 v pH 3.8.