modul modul modul.pdf

Laboratorium Biokimia

PENUNTUN PRAKTIKUM

BIOKIMIA

Mutakin Nyi Mekar Saptarini

Melisa Intan Barliana Wiwiek Indriyati Danni Ramdhani

LABORATORIUM BIOKIMIA FAKULTAS FARMASI

UNIVERSITAS PADJADJARAN 2015

Nama : NPM : Hari dan waktu praktikum : Kelompok : Tanda tangan :

i Laboratorium Biokimia

i Laboratorium Biokimia

ATURAN UMUM LABORATORIUM BIOKIMIA

Selama mengikuti Praktikum Biokimia, peserta diwajibkan untuk :

1. Mempersiapkan diri dengan a. Menggunakan jas lab dan sepatu tertutup selama praktikum b. Membawa tool kit

2. Bertanggung jawab terhadap peralatan yang digunakan : a. Meminjam peralatan gelas yang disimpan dalam lemari praktikum. b. Mengecek ulang peralatan tambahan dan mengembalikan alat yang

dipinjam harian setelah selesai melakukan praktikum. Jika tidak mengembalikan alat pada hari yang sama, maka alat dianggap hilang/pecah dan dimasukkan ke dalam daftar alat yang harus diganti pada akhir semester.

c. Mengganti alat yang hilang/pecah di akhir semester dengan disertai bon pembelian alat.

3. Menjaga kebersihan laboratorium : a. Menjaga kebersihan alat dan meja kerja. b. Tidak membuang batu didih atau padatan ke dalam washbak. c. Membuang sampah pada tempat yang telah disediakan. d. Mematikan keran air dan api/gas setelah selesai digunakan. e. Jika ada keraguan dalam hal keselamatan dan keamanan kerja di

laboratorium. Jangan segan untuk bertanya kepada ASISTEN, PETUGAS LAB atau PEMIMPIN PRAKTIKUM.

Jatinangor, Februari 2015

Penanggung jawab Praktikum

ii Laboratorium Biokimia

DAFTAR ISI

Halaman

Aturan umum laboratorium biokimia

Daftar isi

Keselamatan di Laboratorium

1. Ekstraksi enzim

2. Fraksinasi enzim

3. Analisis kadar protein total

4. Uji aktivitas amilase

5. Uji aktivitas protease

6. Uji aktivitas lipase

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iii Laboratorium Biokimia

KESELAMATAN DI LABORATORIUM

Aturan Keselamatan di Lingkungan Laboratorium

Keselamatan di laboratorium kimia melibatkan sikap hati-hati dan kesadaran pada potensi bahaya. Kecelakaan biasanya dapat diantisipasi dan dicegah. Jika tindakan pencegahan diikuti, maka kecelakaan akan lebih sedikit terjadi. Jumlah kecelakaan laboratorium dapat dikurangi jika setiap praktikan mengikuti semua petunjuk yang diberikan. Catatan khusus harus diambil dari petunjuk khusus untuk menghilangkan potensi bahaya.

A. Aturan Umum 1. Instruktur tidak bertanggung jawab pada kerusakan terhadap efek personal. 2. Ketika praktikan akan melakukan percobaan, instruktur harus hadir atau praktikan

harus mendapat izin dari instruktur dan praktikan ditemani oleh praktikan lain. 3. Alat gelas yang rusak atau pecah harus dilaporkan kepada instruktur. JANGAN

BERSIHKAN PECAHAN GELAS TANPA DIDAMPINGI INSTRUKTUR. 4. Peralatan elektronik, seperti hand phone atau tablet, harus dimatikan selama

praktikum.

B. Tanggung Jawab Praktikan 1. KETAHUI LETAK PERALATAN KESELAMATAN, seperti safety shower, pemadam

kebakaran, dan peralatan pertolongan pada kecelakaan. 2. LINDUNGI MATA. Pelindung mata (goggle) digunakan selama praktikum. 3. RAMBUT PANJANG HARUS DIIKAT. 4. SEPATU HARUS MELINDUNGI KAKI DENGAN SEMPURNA. Jangan

menggunakan sepatu yang terbuka. 5. GUNAKAN PAKAIAN YANG MEMBERIKAN PERLINDUNGAN MAKSIMUM.

JANGAN GUNAKAN ROK PENDEK. 6. TIDAK BOLEH MAKAN DAN MINUM DI RUANG LABORATORIUM. 7. JANGAN MENCICIPI ZAT KIMIA. Setelah praktikum selesai, cuci tangan dengan

sabun. 8. JANGAN MENCIUM ZAT KIMIA SECARA LANGSUNG. Gunakan tangan untuk

menghembuskan bau zat kimia ke hidung.

iv Laboratorium Biokimia

9. Hindari kontak dengan kulit, gunakan peralatan yang memadai, dan perlakukan seluruh sampel dengan perhatian ekstrim.

10. Gunakan sarung tangan ketika bekerja dengan sampel dan zat kimia.

C. Aturan Perawatan 1. LAPORKAN ZAT KIMIA YANG TUMPAH KEPADA INSTRUKTUR. SEGERA

BERSIHKAN TUMPAHAN PADATAN DAN CAIRAN. 2. JANGAN MEMBUANG ZAT KIMIA KE BAK CUCIAN ATAU TEMPAT SAMPAH. 3. GELAS PIALA DIGUNAKAN UNTUK MENYIMPAN LARUTAN STOK. JANGAN

MENGEMBALIKAN SISA LARUTAN STOK KE DALAM WADAH ASAL, karena mencemari seluruh larutan stok.

4. BACA LABEL PADA WADAH DENGAN PRESISI. 5. JANGAN MEMASUKKAN PIPET KE DALAM WADAH LARUTAN STOK. Tuang

larutan stok ke dalam gelas piala dan masukkan pipet ke dalamnya. 6. AMBIL PEREAKSI SECUKUPNYA, TIDAK MELEBIHI YANG DIPERLUKAN. Baca

prosedur dengan presisi dan tentukan jumlah setiap pereaksi yang diperlukan. JANGAN MENGEMBALIKAN SISA PEREAKSI KE DALAM WADAH ASAL.

7. JANGAN MEMBUANG KERTAS ATAU SAMPAH PADAT KE DALAM BAK CUCIAN.

D. Prosedur Kecelakaan dan Darurat 1. Setiap praktikan melaporkan setiap kecelakaan, walaupun ringan, ke instruktur. Jika

perlu, instruktur mepaorkan-nya ke dosen. 2. Ketika terjadi kecelakaan dan tidak ada dosen, hubungi lembagaa kesehatan.

E. Kontrak Jika praktikan tidak menandatangani kontrak, maka praktikan dilarang untuk mengikuti praktikum.

Saya, yang bertanda tangan, telah membaca aturan keamanan di laboratorium dan praktikum yang diberikan dalam panduan laboratorium. Saya bertanggung jawab untuk

v Laboratorium Biokimia

mengamati praktikum dan perhatian selama ada di laboratorium. Jika saya tidak memenuhi aturan ini, maka saya bersedia tidak mengikuti praktikum dan diberi nilai NOL untuk praktikum tersebut.

Jatinangor, ................................

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EKSTRAKSI ENZIM

TUJUAN

Mengekstraksi protein, termasuk enzim yang ada dalam sampel

PENDAHULUAN

Enzim adalah biokatalisator yang berfungsi sebagai katalis dalam proses biologis. Enzim adalah molekul biopolimer yang tersusun dari serangkaian asam amino dalam komposisi dan susunan rantai yang teratur dan tetap. Enzim memegang peranan penting dalam berbagai reaksi di dalam sel. Sebagai protein, enzim diproduksi dan digunakan oleh sel hidup untuk mengkatalisis reaksi, antara lain konversi energi dan metabolisme pertahanan sel. Enzim meningkatkan laju reaksi sehingga terbentuk kesetimbangan kimia antara produk dan pereaksi. Dalam keadaan fisiologi yang normal, enzim tidak mempengaruhi jumlah produk dan pereaksi yang sebenarnya dicapai tanpa kehadiran enzim.

Enzim dapat mempercepat reaksi 108 hingga 1011 kali lebih cepat daripada reaksi tanpa katalis. Enzim merupakan katalis yang sangat efisiendan memiliki derajat kekhasan yang tinggi. Enzim menurunkan energi aktivasi suatu reaksi kimia. Enzim amilase, lipase dan protease yang merupakan enzim hidrolitik pemecah senyawa makromolekul karbohidrat, lemak dan protein. Aplikasi amilase, lipase dan protease dalam bidang industri pangan maupun nonpangan berkembang luas di Indonesia.

ALAT DAN BAHAN

- Buah: pepaya, nenas, apel, jeruk, tomat - Aquades - Larutan dapar fosfat (PBS) 5 mM pH 6,8 suhu 4 C - Gelas ukur - Gelas piala 100 mL dan 500 mL - Labu takar 250 mL


- Batang pengaduk - Timbangan - Botol kaca kapasitas 500 mL

PROSEDUR PENYIAPAN PEREAKSI

Larutan dapar fosfat (phosphate buffer solution, PBS) 5 mM pH 6,8: Pada 51,0 mL larutan kalium dihidrogen fosfat (27,2 g/L) ditambahkan 49,0 mL larutan dinatrium hidrogen fosfat (71,6 g/L). Sesuaikan pH hingga 6,8 jika diperlukan. Simpan pada suhu 2-8 C.

PROSEDUR

1. Sampel dikupas (pepaya dan nenas) dibersihkan di bawah air mengalir, kemudian dengan aquades.

2. Keringkan sampel dengan tissue. 3. Timbang 50 g sampel, kecuali jeruk dan tomat 25 g. 4. Tambahkan 50 mL PBS 5 mM pH 6,8, kecuali jeruk dan tomat tanpa penambahan PBS

5 mM pH 6,8. 5. Haluskan sampel dengan mortir. 6. Saring ekstrak ke dalam labu ukur 25 mL. 7. Genapkan volume hingga 25 mL dengan PBS 5 mM pH 6,8 melalui ekstrak buah. 8. Pindahkan ekstrak ke dalam botol kaca dan beri keterangan (kelompok, hari, tanggal). 9. Ekstrak kasar yang dihasilkan disimpan dalam lemari pendingin.

HASIL PENGAMATAN

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KESIMPULAN

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DAFTAR PUSTAKA

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PERTANYAAN

1. Jelaskan prinsip ekstraksi protein? 2. Jelaskan fungsi PBS 5 mM pH 6,8 pada ekstraksi protein? 3. Apakah PBS 5 mM pH 6,8 dapat diganti dengan bufer lain? Jelaskan!

JAWABAN PERTANYAAN

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FRAKSINASI ENZIM

TUJUAN

Pemisahan fraksi enzim berdasarkan ukuran molekul protein

PENDAHULUAN

Kromatografi filtrasi gel memisahkan protein berdasarkan ukuran molekul. Campuran protein dimasukkan ke dalam kolom filtrasi gel yang berisi matriks kromatografi dengan ukuran pori-pori tertentu. Protein dielusi dengan dapar, dikumpulkan sebagai fraksi individual dan dianalisis secara terpisah. Filtrasi gel dapat digunakan untuk memisahkan protein berdasarkan perbedaan ukuran molekulnya atau menjadi protein tanpa garam (yaitu menghilangkan cemaran dengan berat molekul rendah seperti garam, asam amino, peptida, dll).

Fraksinasi protein menggunakan kromatografi filtrasi gel


Pemisahan beragam protein dalam sampel disebabkan oleh perbedaan kemampuannya untuk memasuki pori-pori. Secara umum, protein besar tidak dapat memasuki pori-pori gel dan akan terelusi dengan cepat dari kolom. Protein yang lebih kecil akan memasuki dan meninggalkan pori-pori gel secara berulang-ulang dan akan tertinggal dalam kolom lebih lama. Karenanya, protein akan terelusi berdasarkan penurunan ukuran molekul.

Matriks Gel Filtrasi Gel (FG) Komersial

Tipe Gel Rentang Fraksinasi (Berat Molekul) Sephadex G-25 1,000-5,000

Bio-Gel P-60 3,000-60,000 Sephadex G-75 3,000-70,000

Sephadex G-100 4,000-100,000 Bio-Gel P-100 5,000-100,000

Sephadex G-200 5,000-250,000 Bio-Gel P-200 30,000-200,000

Sephacryl S-300 10,000-1,500,000 Sepharose 2B 70,000-40,000,000

Standar Berat Molekul Protein

Protein Berat Molekul Cytochrome c 11,700 Myoglobin 16,800 Trypsinogen 24,000 Carbonic anhydrase 29,000 Ovalbumin 45,000 Hemoglobin 64,500 Bovine serum albumin 66,000 Transferrin 74,000 Immunoglobulin G 158,000 Fibrinogen 341,000 Ferritin 470,000 Thyroglobulin 670,000


PROSEDUR PEMBUATAN PEREAKSI

Larutan dapar fosfat (phosphate buffer solution, PBS) 0,05 M pH 6: Larutkan 6,8 g natrium dihidrogen fosfat (BM 137,99) dalam 900 mL aquades, atur pH hingga pH 6 dengan larutan natrium hidroksida 5 N. Genapkan volume hingga 1000 mL.

PROSEDUR

a. Persiapan bahan pengepak 1. Timbang 0,5 g sephadex G-100 dilarutkan dalam 60 mL air bebas ion sambil diaduk

dengan magnetic stirer perlahan selama 30 menit, kemudian didiamkan selama 3 hari pada suhu dingin atau selama 3 jam pada suhu 90C.

2. Kemudian matriks dicuci dengan 50 mL PBS 0,05 M pH 6 dan diagitasi sampai gelembung udara hilang.

b. Pembuatan kolom

1. Tuang matriks gel sephadex G-100 secara perlahan tapi kontinyu. Jika terbentuk rongga udara, bagian luar kolom diketuk sehingga rongga udara tersebut hilang.

2. Lakukan ekuilibrasi kolom dengan mengalirkan sejumlah PBS 0,05 M pH 6 untuk mencuci kolom.

c. Separasi sampel

1. Tuang 5 ml sampel protein pada bagian atas kolom, diamkan 5 menit hingga sampel memiliki kesempatan untuk memasuki kolom.

2. Tuang bufer fosfat 0,05 M pH 6 hingga memenuhi atas kolom 3. Elusi perlahan dengan laju 30 tetes/menit 4. Fraksi ditampung dengan volume 3 ml 5. Ukur absorbansi pada 280 nm

dari fraksi individual untuk mendeteksi protein


HASIL PENGAMATAN

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DAFTAR PUSTAKA

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PERTANYAAN

1. Jelaskan prinsip fraksinasi protein menggunakan kromatografi filtrasi gel! 2. Jelaskan fungsi pengembangan matriks gel! 3. Jelaskan mengapa fraksi yang mengandung protein dapat dideteksi menggunakan

spektrofotometer UV-vis pada panjang gelombang 280 nm!

JAWABAN PERTANYAAN

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ANALISIS KADAR PROTEIN TOTAL

TUJUAN

Menentukan kadar protein total pada fraksi protein

PENDAHULUAN

Metode yang umum digunakan untuk menentukan kadar protein antara lain metode Biuret, Bradford, dan Lowry. Masing-masing metode memiliki kekurangan dan kelebihan. Pemilihan metode yang terbaik dan tepat untuk suatu pengukuran tergantung pada beberapa faktor, yaitu jumlah material atau sampel yang tersedia, waktu yang tersedia untuk melakukan pengukuran, dan spektrofotometer yang tersedia (sinar tampak atau UV).

Reaksi Biuret dilakukan dengan membasakan larutan sampel dengan NaOH, kemudian ditambahkan larutan tembaga sulfat (CuSO4) encer. Reaksi ini menunjukkan adanya senyawa-senyawa yang mengandung gugus amida asam (-CONH2) yang berada bersama gugus amida asam yang lain atau gugus yang lain seperti -CSNH2, -C(NH)NH2, -CH2NH2, -CRHNH2, -CHOHCH2NH2, -CHOHCH2NH2, -CHNH2CH2OH, -CHNH2CHOH. Sehingga, reaksi Biuret tidak spesifik untuk protein. Zat selain protein yang mengandung gugus amida asam atau malonamida juga dapat memberikan reaksi positif yang ditandai dengan timbulnya warna merah-violet atau biru-violet.

Intensitas warna larutan tergantung pada konsentrasi protein dalam sampel. Penentuan protein dengan metode Biuret dilakukan dengan mengukur optical density (OD) pada panjang gelombang 560 580 nm. Konsentrasi protein dalam sampel ditentukan melalui persamaan garis kurva baku/standar dari protein standar yang diketahui konsentrasinya. Persamaan garis ini melukiskan hubungan antara konsentrasi protein dengan OD pada panjang gelombang terpilih.


Reaksi antara pereaksi Biuret dengan ikatan peptida

ALAT DAN BAHAN

- Labu ukur 100 mL - Tabung reaksi - NaOH 0,1 N - Na2CO3 - K-Na-tartrat - CuSO4 - Kasein


Pereaksi Biuret : 100 mL Na2CO3 2% dalam larutan NaOH 0,1 N ditambah 1 mL CuSO4 1% dan 1 mL K Na tartrat 2%


PROSEDUR

1. Penentuan panjang gelombang maksimum (perwakilan kelompok)

a. Timbang 50 mg kasein, larutkan dengan aquades dalam labu ukur 100 mL dan genapkan hingga tanda batas (konsentrasi = 50 mg/100 mL = 500 g/mL).

b. Pipet 5 mL larutan kasein dan encerkan dengan aquades dalam labu ukur 10 mL (konsentrasi 250 g/mL).

c. Pipet 1 mL larutan kasein 250 g/mL dan masukkan ke dalam tabung reaksi. d. Tambahkan 9 mL pereaksi Biuret, diamkan selama 10 menit (konsentrasi 25 g/mL). e. Ukur absorbansi larutan kasein pada panjang gelombang 400-800 nm dengan

spektrofotometer. Blangko berisi 1 mL aquades dan 9 mL pereaksi Biuret. f. Gambarkan spektrum absorbansi terhadap panjang gelombang. g. Tentukan panjang gelombang maksimum kasein.

2. Pembuatan kurva baku larutan kasein (perwakilan kelompok)

a. Siapkan larutan kasein dalam 22 tabung reaksi sehingga diperoleh konsentrasi bertingkat dari 50-500 g /mL (duplo).

Pengenceran larutan kasein untuk pembuatan kurva baku

No mL kasein (500 g/mL) mL H20 konsentrasi

kasein (g/mL)

1 2 3 4 5 6 7 8 9

10 11

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0

1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,1 0

0 50 100 150 200 250 300 350 400 450 500

b. Pada setiap tabung, tambahkan 9 mL pereaksi Biuret,diamkan selama 10 menit. c. Absorbansi setiap tabung diukur dengan spektrofotometer pada panjang gelombang

maksimum (550 nm). d. Buat kurva baku yang menunjukkan hubungan antara absorbansi dan konsentrasi. e. Tentukan persamaan garis kurva baku.


3. Pengukuran fraksi protein

a. Pipet 1 mL fraksi protein protein, masukkan ke dalam tabung reaksi. b. Tambahkan 9 mL pereaksi Biuret, diamkan selama 10 menit. c. Ukur absorbansi fraksi, sehingga konsentrasi protein dalam fraksi protein perlu

diperhitungkan dengan pengenceran (1 : 10).

HASIL PENGAMATAN

Spektrum absorbansi terhadap panjang gelombang (print)


Absorbansi larutan kasein untuk pembuatan kurva baku

No mL kasein

(500 g/mL) mL H20

mL pereaksi

Biuret

konsentrasi

kasein

(g/mL) Absorbansi

1

2 0 1,0 9 0

3

4 0,1 0,9 9 5

5

6 0,2 0,8 9 10

7

8 0,3 0,7 9 15

9

10 0,4 0,6 9 20

11

12 0,5 0,5 9 25

13

14 0,6 0,4 9 30

15

16 0,7 0,3 9 35

17

18 0,8 0,2 9 40

19

20 0,9 0,1 9 45

21

22 1,0 0 9 50


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DAFTAR PUSTAKA

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PERTANYAAN

1. Jelaskan mengapa kasein dapat digunakan sebagai baku protein pada pembuatan kurva baku!

2. Jelaskan apakah kasein dapat digantikan oleh protein lain sebagai baku protein? 3. Berapa jumlah asam amino dalam kasein? 4. Berdasarkan jumlah asam amino kasein, berapa jumlah ikatan peptida dalam kasein!

JAWABAN PERTANYAAN

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UJI AKTIVITAS AMILASE

TUJUAN

Menentukan aktivitas amilase dari fraksi protein

PENDAHULUAN

Enzim amilase dapat memutuskan ikatan glikosidik ( 14) di antara satuan glukosa yang membentuk polimer, seperti pati. Pati terdiri atas amilosa dan amilopektin, yang tersusun atas satuan glukosa rantai lurus dan rantai bercabang. Hasil hidrolisis pati oleh amilase adalah glukosa.

Gula dengan sifat pereduksi (karena adanya gugus aldehid atau keto yang potensial) disebut gula pereduksi, seperti glukosa, galaktosa, laktosa, dan maltosa. Aktivitas amilase dapat ditentukan dengan metode CarawaySomogyi menggunakan iodin/kalium iodida (1959). Metode ini menggunakan spektrofotometer yang mengukur penurunan absorbansi pati yang terjadi karena aktivitas amilase yang mendegradasi pati pada ikatan glikosidiknya.

Struktur 3D -amilase


Mekanisme kerja -amilase

ALAT DAN BAHAN

- Tabung reaksi - Beaker glass - Batang pengaduk - Gelas ukur - Pipet tetes - Pati larut - Kalium dihidrogen fosfat - Dikalium hidrogen fosfat - HCl - Iodin - Kalium iodida



- Larutan pati larut 1%: larutkan 1 g pati larut dengan aquadest dalam labu ukur 100 mL (konsentrasi 10 mg/mL).

- Larutan HCl 10%: masukkan 10 mL HCl pekat ke dalam labu ukur 100 mL yang telah berisi 50 mL aquades, genapkan volume hingga 100 mL dengan aquades.

- Larutan iodin-kalium iodida (iodin Lugol): Larutan 5% terdiri atas iodin 5 % dan KI 10% dicampur dalam aquades dan memiliki kandungan iodin total 126,5 mg/mL.

- Larutan bufer fosfat 0,1 M pH 3,5: Larutkan 13,6 g kalium dihidrogen fosfat dalam 900 mL aquades. Cek pH dan atur pH hingga dicapai pH 3,5 dengan asam fosfat, encerkan hingga 1000 mL dengan aquades.

- Larutan bufer fosfat 0,1 M pH 4,5: Larutkan 13,6 g kalium dihidrogen fosfat dalam 900 mL aquades. Cek pH dan atur pH hingga dicapai pH 4,5 dengan asam fosfat, encerkan hingga 1000 mL dengan aquades.

- Dapar fosfat 0,1 M pH 5,6: larutkan 0,908 kalium dihidrogen fosfat dengan aduades dalam labu ukur 100 mL dan genapkan hingga tanda batas (larutan I). Larutkan 1,161 g dikalium hidrogen fosfat dengan aduades dalam labu ukur 100 mL dan genapkan hingga tanda batas (larutan II). Campurkan 94,4 mL larutan I dan 5,6 mL larutan II. Cek pH dan atur pH hingga dicapai pH 5,6.

- Larutan dapar fosfat 0,1 M pH 7,5: Larutkan 13,6 g kalium dihidrogen fosfat dalam 900 mL aquades. Cek pH dan atur pH hingga dicapai pH 7,5 dengan larutan kalium hidroksida 300 g/L, encerkan hingga 1000 mL dengan aquades.

- Larutan dapar amonium klorida pH 9,5: Larutkan 33,5 g amonium klorida dalam 150 mL aquades, tambahkan amonia pekat dan encerkan hingga 250 mL dengan aquades. Simpan dalam wadah polietilen.

PROSEDUR

a. Gelatinisasi pati larut 1. Penambahan 40 mL pati larut 1% pada 50 mL aquades mendidih dalam beaker

glass, sambil diaduk. 2. Genapkan hingga 100 mL dengan air. 3. Larutan pati tergelatinisasi dibiarkan dingin pada suhu kamar (konsentrasi pati 4

mg/mL).


4. Ambil 1 mL larutan pati tergelatinisasi, encerkan hingga 100 mL dengan aquades. Larutan ini digunakan sebagai larutan stok (substrat) untuk pengujian (konsentrasi 0,04 mg/mL = 40 g/mL).

b. Pembuatan kurva standar pati (duplo) 1. Tabung reaksi diisi 2,5 mL larutan stok, 1,75 mL dapar fosfat 0,1 M pH 5,6 dan 0,75

mL aquades. 2. Ambil campuran reaksi (variasi volume, lihat tabel) dan pindahkan ke tabung reaksi

lain berisi 1,5 mL HCl 10% untuk menghentikan reaksi. 3. Tambahkan 1,5 mL indikator (larutan iodin-kalium iodida). 4. Absorbansi dibaca pada 620 nm. 5. Buat blanko. Blanko dibuat dengan komposisi yang sama dengan campuran reaksi,

kecuali tanpa penambahan indikator.

c. Uji aktivitas amilase pada variasi suhu (duplo) 1. Tabung reaksi diisi 2,5 mL larutan stok, 1,75 mL dapar fosfat 0,1 M pH 5,6 dan 0,75

mL ekstrak amilase. 2. Campuran reaksi diinkubasi pada 27, 37, 47, 57, dan 67 C selama 30 menit. 3. Ambil 1 mL campuran reaksi dan pindahkan ke tabung reaksi lain berisi 3 mL HCl

10% untuk menghentikan reaksi. 4. Tambahkan 3 mL indikator (larutan iodin-kalium iodida). 5. Absorbansi dibaca pada 620 nm, jangan lupa konsentrasi pati terhidrolisis dikalikan

dengan faktor pengenceran. 6. Buat blanko. 7. Jumlah pati terhidrolisis per satuan waktu ditentukan dari kurva standar konsentrasi

pati (substrat) terhadap absorbansi.

d. Uji aktivitas amilase pada variasi pH (duplo) 1. Tabung reaksi diisi 2,5 mL larutan stok, 1,75 mL dapar pH 3,5; 4,5; 5,6; 7,5; 9,5 dan

0,75 mL ekstrak amilase. 2. Campuran reaksi diinkubasi pada 37 C selama 30 menit. 3. Ambil 1 mL campuran reaksi dan pindahkan ke tabung reaksi lain berisi 3 mL HCl

10% untuk menghentikan reaksi. 4. Tambahkan 3 mL indikator (larutan iodin-kalium iodida). 5. Absorbansi dibaca pada 620 nm, jangan lupa konsentrasi pati terhidrolisis dikalikan

dengan faktor pengenceran.


6. Buat blanko. 7. Jumlah pati terhidrolisis per satuan waktu ditentukan dari kurva standar konsentrasi

pati (substrat) terhadap absorbansi.

HASIL PENGAMATAN

Absorbansi dan konsentrasi larutan pati tergelatinisasi (kurva baku)

Tabung ke-

Volume campuran reaksi (mL)

Volume aquades (mL) Absorbansi konsentrasi

1

2 0 1,0

1

2 0,2 0,8

1

2 0,4 0,6

1

2 0,6 0,4

1

2 0,8 0,2

1

2 1,0 0

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Absorbansi dan konsentrasi pati terhidrolisis pada variasi suhu

Suhu (C) Tabung Absorbansi Konsentrasi pati Konsentrasi pati dikalikan faktor pengenceran

27 1

2

37 1

2

47 1

2

57 1

2

67 1

2

JANGAN LUPA DIKALIKAN DENGAN FAKTOR PENGENCERAN

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Absorbansi dan konsentrasi pati terhidrolisis pada variasi pH

pH Tabung Absorbansi Konsentrasi pati Konsentrasi pati dikalikan faktor pengenceran

4 1

2

6 1

2

7 1

2

8 1

2

10 1

2

JANGAN LUPA DIKALIKAN DENGAN FAKTOR PENGENCERAN

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KESIMPULAN

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DAFTAR PUSTAKA

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PERTANYAAN

1. Jelaskan mekanisme kerja amilase! 2. Jelaskan tujuan dilakukan variasi suhu dan pH inkubasi pada uji aktivitas amilase! 3. Jelaskan mekanisme kerja larutan HCl 10% sebagai penghenti aktivitas enzim!

JAWABAN PERTANYAAN

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UJI AKTIVITAS PROTEASE

TUJUAN

Menentukan aktivitas protease dari setiap fraksi protein

PENDAHULUAN

Protease (peptidase atau proteinase) merupakan enzim golongan hidrolase. Protease mendegradasi protein menjadi molekul yang lebih sederhana, yaitu oligopeptida pendek atau asam amino melalui reaksi hidrolisis pada ikatan peptida. Protease bersifat esensial dalam metabolisme protein. Kebanyakan protease disintesis dalam bentuk zimogen yang tidak aktif.

Fungsi protease dalam tubuh adalah membantu pencernaan protein dalam makanan, menggunakan kembali protein-protein intraseluler, koagulasi sel darah, dan akivasi berbagai jenis protein, enzim, hormon, serta neurotransmiter. Ada enam jenis aktivitas katalitik spesifik dari enzim peptidase, yaitu serin, treonin, sistein, aspartat, glutamat, dan metalo-peptidase.

Endopeptidase bekerja dengan menghidrolisis ikatan peptida pada bagian tengah rantai polipeptida. Contoh endopeptidase adalah kimotripsin, tripsin, dan pepsin. Ketiga jenis enzim ini banyak digunakan dalam industri berbasis teknologi pangan.

Struktur 3D enzim peptidase yang berasal dari fungi


Mekanisme kerja protease pada ikatan peptida

Pengukuran aktivitas protease menggunakan metode Bergmeyer & Grassl 1983. Prinsip metode ini adalah kasein sebagai substrat akan dihidrolisis oleh protease dengan bantuan air menjadi peptida dan asam amino. Laju pembentukan peptida dan asam amino merupakan ukuran aktivitas katalisis protease.


Asam amino yang terbentuk harus dipisahkan dari substrat yang tidak terhidrolisis. Pemisahan dilakukan dengan penambahan TCA. TCA menyebabkan produk yang mengandung peptida dan asam amino akan larut dalam TCA, sedangkan protein yang tidak terhidrolisis akan mengendap. TCA juga menginaktifkan enzim protease. Asam amino tirosin dan triptofan yang larut dalam TCA dan bereaksi dengan pereaksi Folin menghasilkan warna biru. Penambahan Na2CO3 bertujuan untuk medapatkan pH sekitar 11,5 yang merupakan pH optimum untuk intensitas dan stabilitas warna. Warna yang terbentuk diukur absorbansinya pada panjang gelombang 578 nm dengan alat spektrofotometer Uv-Vis.

ALAT DAN BAHAN

- Tabung reaksi - Spektrofotometer UV-vis - Kuvet - Labu ukur 10 mL dan 25 mL - Inkubator - Pipet ukur 10 mL - Aquades - Tri kloroasetat (TCA) - Kasein


PROSEDUR

a. Pembuatan Kurva Standar Kasein (perwakilan dari setiap kelompok) 1. Larutkan 50 mg kasein dengan aquades dalam labu ukur 100 mL. 2. Lakukan pengenceran, hingga diperoleh larutan kasein standar dengan konsentrasi

4, 8, 12, 16, dan 20 mg/100 mL. 3. Ambil 0,6 mL setiap larutan, tambahkan 5 mL larutan TCA 3,5%. 4. Kontrol dibuat dari 0,6 mL aquades ditambah 5 mL larutan TCA 3,5%. 5. Ukur absorbansi setiap larutan dengan spektrofotometer pada 275 nm.


6. Buat kurva standar yang menunjukkan hubungan antara kadar kasein dan absorbansi

b. Uji aktivitas lipase pada variasi suhu 1. Siapkan tabung sampel berisi 0,5 mL substrat kasein 0,5%. 2. Siapkan tabung kontrol berisi 0,6 mL aquades. 3. Semua tabung diinkubasi selama 5 menit pada suhu 27, 37, 47, 57, dan 67 C. 4. Tambahkan 0,1 mL fraksi protein. 5. Inkubasi dilanjutkan selama 30 menit pada suhu 27, 37, 47, 57, dan 67 C. 6. Tambahkan 5 mL larutan TCA 3,5% pada semua tabung. 7. Sentrifugasi pada kecepatan 6000 G dan suhu 4 C selama 10 menit. 8. Ambil filtrat dan ukur absorbansi produk hasil reaksi pada 280 nm. 9. 1 unit aktivitas enzim kaseinolitik didefinisikan sebagai jumlah enzim yang

menghasilkan 1 g tirosin pada kondisi standar.

c. Uji aktivitas lipase pada variasi pH 1. Siapkan tabung sampel berisi 0,5 mL substrat kasein 0,5%. 2. Siapkan tabung kontrol berisi 0,6 mL aquades. 3. Semua tabung diinkubasi selama 5 menit pada suhu 37 C. 4. Tambahkan 0,1 mL fraksi protein pada masing-masing tabung sampel. 5. Tambahkan 1,4 mL larutan dapar pH 3,5; 4,5; 5,6; 7,5 dan 9,5. 6. Inkubasi dilanjutkan selama 30 menit pada suhu 37 C. 7. Tambahkan 5 mL larutan TCA 3,5% pada semua tabung. 8. Sentrifugasi pada kecepatan 6000 G dan suhu 4 C selama 10 menit. 9. Ambil filtrat dan ukur absorbansi produk hasil reaksi pada 280 nm. 10. 1 unit aktivitas enzim kaseinolitik didefinisikan sebagai jumlah enzim yang

menghasilkan 1 g tirosin pada konsidi standar.

HASIL PENGAMATAN

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KESIMPULAN

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DAFTAR PUSTAKA

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PERTANYAAN

1. Jelaskan mengapa TCA dapat mengendapkan protein! 2. Jelaskan tujuan dilakukan variasi suhu dan pH inkubasi pada uji aktivitas protease! 3. Jelaskan mengapa tirosin digunakan sebagai standar dalam perhitungan aktivitas

protease!

JAWABAN PERTANYAAN

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