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ANALISIS ASAM AMNIO DAN PROTEIN

ANALISIS ASAM AMNIO DAN PROTEIN. Abdul Rohman Fakultas Farmasi UGM. Bahan Pembelajaran Analisis asam amino dan Protein. Pendahuluan Analisis Asam amino Titrimetri Gasometri Spektrofotometri Kromatografi. Asam amino.

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ANALISIS ASAM AMNIO DAN PROTEIN

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  1. ANALISIS ASAM AMNIO DAN PROTEIN Abdul Rohman FakultasFarmasi UGM

  2. BahanPembelajaranAnalisisasam amino dan Protein • Pendahuluan • AnalisisAsam amino • Titrimetri • Gasometri • Spektrofotometri • Kromatografi

  3. Asam amino • AA merupakan monomer penyusun protein. Kebanyakan protein disusunoleh 20 asam amino • Sumberutama diet AA padamanusiaberasaldari protein, yang secaraenzimatisakandidigestiuntukmembebaskanAA-nya • AA dapatberadadalambentukbebasatauterikatdalampeptidaatauprotein menentukanmetodeanalisisdanpenyiapansampelnya

  4. What is amino acid? What is amino acid? Amino Acid: aminated carboxylic acid (R-COOH) R group

  5. Examples -amino acetic acid -amino propanoic acid ,-2 amino caproic acid

  6. Classification of Amino Acid 1. By the location of Amino-group ： / / -AA 2. By its acidity： neutral/acidic/ basic AA ratio of Amino-group to carboxylic group 3. By whether containing phenyl group aromatic / non aromatic AA 4. By its occurrence in protein Protein / non protein AA 5. By polarity of R group : polar / apolar side chain AA 6. By its nutrient value to human: EssentialAA and non-essential AA

  7. 20 -AA commonly found in proteins

  8. Continued

  9. continued

  10. continued

  11. TitrasiFormol

  12. Gasometri Gugusalfa amino primer dariasam amino bereaksidenganasamnitritdanmenghasilkan gas nitrogen (N2). Asamnitritinidibuatdenganmereaksikannatriumnitritdenganasamasetat. Gas nitrogen yang terjadidimurnikandenganmengalirkannyapadakaliumpermanganat, untukselanjutnyadikumpulkandandiukurvolumenya.

  13. Spektrofotomeri UV Aromatic AAs absorb light in the near ultraviolet (230-300nm). phenylalanine tyrosine Note: This UV absorption property of protein is solely determined by the content of these 3 aromatic AAs. However, far ultraviolet (190nm)absorption of protein stems from the peptide bond. tryptophan

  14. UV Spectra

  15. Derivative spectrophotometry

  16. Spektroskopivisibel Ninhidrin Metode yang dikembangkan

  17. Ninhidryn FRIEDMAN, M. J. Agric. Food Chem. 2004, 52, 385−406

  18. ContohMetode yang dikembangkan

  19. High performnce liquid Chromatography

  20. Detektor • Detektor UV-Vis merupakandetektor yang paling umumdigunakanuntukanalisisbahanmakananmenggunakan KCKT. • AA dapatdideteksisecaralangsungdengan UV karena AA mampumenyerapdipanjanggelombangantara 190 – 210. Meskipundemikian, karenakebanyakanpelarutdanjugakomponenmatrikssampel yang lain jugamenyerapdidaerahini, maka AA harusdiderivatisasiterlebihdahulusebelumdianalisisdengan KCKT menggunakandetektorspektrofotometer UV-Vis. • Derivatisasi AA dapatdilakukansebelumpemisahandikolomkromatografi (pre-column derivatization) ataudilakukansetelah AA keluardarikolom (post-column derivatization).

  21. Beberapaagenpenderivat yang digunakan Dideteksidipanjanggelombang 570 nm

  22. Dansilklorida Dansil-AA dideteksidenganfluoresensi (λeks 360 nm; λemisi = 470 nm) dandengan UV (λmaks 250 nm) Source: Snyder et al., 1997

  23. Dabsilklorida Turunandabsilmenunjukkanserapanmaksimaldi 420 nm (daerahtampak), sertasangatstabil, danbatasdeteksinyaberadadi level pmol. Turunaninitetapstabilsetelahpenyimpananselama 4 minggudisuhuruang.

  24. 1-fluoro-2,4-dinitrobenzena (FDNB)

  25. Fenilisotiosianat Karenahasilderivatisasibersifattidakberfluoresensi, makateknikderivatisasidengan PITC dideteksidenganspektrofotometri UV-Vis, yang umumnyadilakukandi 254 nm (λmaks = 269 nm). Meskipunderivatisasiinitidaksepekadibandingkandenganteknikderivatisasi yang menghasilkanderivat yang dideteksidenganfluoresen, akantetapiproduk PTC-AA bersifatsangatmenyerap UV, sehingga AA dengan level pmoldapatdideteksi. Batas sensitifitasagenpenderivatiniadalah 50 pmolpadarasiosinyal/derau (S/N) 2,5

  26. orto-ftalaldehid Derivat yang dihasilkanantara AA dengan OPA dapatdideteksidengan UV padapanjanggelombang 330 nm, akantetapiuntukmenghasilkansensitifitas yang tinggi, makadigunakandetektorfluoresendenganλeks = 330 nm dan λ em = 430 nm.

  27. Dietil 2(etoksimetiliden)propandioat Deteksi UV: 280 nm

  28. Kromatogramstandar AA (atas) dan AA dalamsampelmadu (bawah). Deteksi UV = 280 nm). Konsentrasistandar AA (mg/kg) diberikandalamtanda kurung.1 = histidin (6,4); 2 = arginin (15,4); 3= asparagin (20,8); 4 = glutamin (41,8); 5 = serin (9,9); 6 = asamaspartat (25,3); 7 = asamglutamat (12,7); 8 = treonin (8,0); 9 = glisin (5,1); 10 = β-alanin (3,5); 11 = asam γ-aminobutirat (2,4); 12 = α-alanin (7,0); 13 = prolin (948,3); 14 = tirosin (15,7); 15 = metionin (3,5); 16 = valin (4,7); 17 = cistein (15,1); 18 = triptofan (7,2); 19 = ornitin (2,11); 20 = fenilalanin (93,2); 21 = isoleusin (14,5); 22 = leusin (15,5); 23 = lisin (7,8) • Source: Rebane, R. and Herodes, K. 2010. AnalyticaChimicaActa 672: 79–84

  29. Gas Chromatography Principle: Non-volatile AA Volatile AA-derivate

  30. Automatic AA analyzer From pH2.2 to pH 6.4

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