Malaysian Journal of Analytical Sciences Vol 18 No 2 (2014): 306 -320

 

 

 

KAJIAN AWALAN TERHADAP PENULENAN DAN PENGENALPASTIAN ASID AMINO AROMATIK L-DOPA DARIPADA KAKI PEREKAT KUPANG HIJAU AIR TAWAR MALAYSIA  

 

(Preliminary Study on Purification and Identification of Aromatic Acid Amino L-DOPA from Malaysia Freshwater Green Mussel Byssus)

 

Saiful Irwan Zubairi¹*, Wan Rosmaryana Wan Musa¹, Syed Anuar Faua’ad Syed Mohammad²

 

¹Pusat Pengajian Sains Kimia & Teknologi Makanan,

Fakulti Sains & Teknologi,

Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan, Malaysia

²Jabatan Kejuruteraan Bioproses,

Fakulti Kejuruteraan Kimia & Kejuruteraan Sumber Asli,

Universiti Teknologi Malaysia, 81310 UTM Skudai, Johor Darul Takzim, Malaysia

 

*Corresponding author: saiful-z@ukm.edu.my

 

 

Abstrak

L-DOPA (L-3,4-dihidroksifenilalanin) adalah sejenis asid amino aromatik yang dapat dikesan melalui kaedah pengekstrakan larutan berasid dan penulenan daripada perekat kaki kupang hijau. Oleh yang demikian, objektif utama kajian ini adalah untuk membuat pencirian dan penulenan asid amino L-DOPA dengan menggunakan kaedah kromatografi penurasan gel Sefadak G-200 melalui penggunaan dua jenis larutan fasa bergerak gel berasid dan beralkali. Penghancuran atau penghomogenan sampel perekat kaki kupang dilakukan dengan menggunakan mortar dan alu beserta penambahan cecair nitrogen bagi memecahkan struktur protein perekat kaki kupang hijau. Sampel yang dihancurkan dicampurkan dengan asid perklorik 0.7%, 1.0%, dan 1.5% (v/v) (pra-rawatan sampel) sebelum proses proses pengekstrakan dijalankan. Seterusnya, proses pengekstrakan dilakukan dengan mengempar sampel pada 11,000 rpm selama 10 min pada suhu 10 ^oC. Supernatan S1 diambil dan ditambah dengan aseton dan asid sulfurik pekat, kemudiannya diempar untuk kali kedua untuk pengambilan pellet dan dilarutkan menggunakan larutan fasa bergerak sebelum proses penulenan dijalankan. Proses penulenan dijalankan menggunakan dua jenis fasa bergerak iaitu asid asetik 5% (v/v) dan NaOH 1M. Bacaan nilai penyerapan (abs) untuk fraksi sampel ekstrak berprotein ditulen dianalisa pada jarak gelombang di antara 214 nm ke 400 nm menggunakan UV-spektrofotometer. Bacaan penyerapan (abs) tertinggi akan dipilih bagi proses pengesanan dan pembuktian kewujudan asid amino L-DOPA di dalam fraksi tertulen. Proses verifikasi dilakukan dengan menggunakan kromatografi cecair berprestasi tinggi (HPLC) dan kromatografi lapisan nipis (TLC). Keputusan ujikaji menunjukkan penggunaan asid perklorik 0.7% dan asid asetik 5% bagi proses pra-rawatan dan cecair fasa bergerak proses penulenan masing-masing menghasilkan profil abs efluan yang tertinggi (menghampiri nilai 1.0) pada jarak gelombang 260 nm. Analisis TLC membuktikan kewujudan beberapa asid amino penting selain L-DOPA iaitu tirosin dan fenilalanin pada masa (jam) penggumpulan efluan ke-78. Manakala, analisis HPLC menunjukkan kepekatan L-DOPA adalah yang tertinggi (2.94 ± 0.12 mg/l) secara signifikan (p<0.05) berbanding pada masa penggumpulan efluan yang lain.

 

Kata kunci: L-3,4-dihidroksifenilalanin (L-DOPA), perekat kaki, kupang hijau, gel Sefadak G-200, TLC, HPLC

 

Abstract

L-DOPA (L-3, 4-dihydroxyphenylalanine) is a type of aromatic amino acid which can be detected by using acidic extraction and purification method involving adhesive byssus green mussel protein. The main objective of this study is to identify and purify the aromatic amino acid L-DOPA via the utilization of gel Sephadex G-200 filtration chromatrography based on two types of acidic and basic mobile phase solution. The crushing and homogenizing for adhesive byssus green mussel were conducted using a mortar and a pestle with the aid of liquid nitrogen. The samples that had been crushed were then mixed and dissolved in perchloric acid 0.7%, 1.0% and 1.5% (v/v) (pre-treatment) prior to the extraction process. The extraction was carried out by centrifuging the extracts at 11,000 rpm for about 10 mins and at a temperature of 10 ⁰C to obtain supernatant S1. The supernatant was mixed with acetone and sulphuric acid and centrifuged for the second time to produce a pellet and then it was dissolved in the respective mobile phase solutions prior to purification process. Purification was later performed using two mobile phase solutions which were acetic acid 5% (v/v) and NaOH 1M. The absorbance (abs) value of each purified protein extract fractions was collected and analysed at 214 nm to 400 nm with the help of UV-spectrophotometer. The highest abs value was selected for identification and verification of amino acid L-DOPA in the purified solution. Verification was carried out by utilizing high performance liquid chromatography (HPLC) and thin layer chromatrography (TLC). The results showed that the use of 0.7% (v/v) perchloric acid and 5% (v/v) acetic acid for pre-treatment process and mobile phase solution of purification process respectively, yielded the highest effluent abs profile at a wavelength of 260 nm. TLC analysis proved the existence of several important amino acids besides L-DOPA which were tyrosine and phenylalanine after 78 hrs of collection of effluents. Meanwhile, the analysis of HPLC revealed the highest concentration of amino acid L-DOPA (p<0.05) as compared to the other collected effluents.

 

Keywords: L-3,4-dihydroxyphenylalanine (L-DOPA), adhesive byssus, green mussel, gel Sephadex G-200, TLC, HPLC

 

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