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LAMPIRAN
Lampiran 1. Komposisi media International Streptomyces Project 2 (ISP2)
Bahan Media Produksi (%w/v) Yeast extract Malt extract Glucosa Akuades 0,04 0,10 0,04 1,00 L
Lampiran 2. Komposisi larutan dalam reaksi uji ATPase
Bahan Jumlah
Master Mix (50 L/sumur) H2O
0,1 M MOPS 0,1 M MgCl2 0,1 M ATP
Enzim RNA helikase HCV
38,5 L 5,0 L 0,5 L 1,0 L 5,0 L Dye solution ATPase (100 L/sumur)
H2O
0,081% malachite green
5,7% ammonium molybdate dalam 6 N HCl 2,3% polivinil alkohol
2 2 1 1
Lampiran 3. Kurva standar Bovine serum Albumin (BSA) [BSA] mg/mL Abs 562 nm 0 0 25 0.078 125 0.2495 250 0.383 500 0.649 750 0.911 1000 1.179 y = 0,0011x + 0,0623 R2 = 0,9929 0,00 0,20 0,40 0,60 0,80 1,00 1,20 1,40 0 200 400 600 800 1000 Konsentrasi BSA (mg/mL) A 562
Lampiran 4. Kurva Standar Fosfat (Uji ATPase) Konsentrasi K2HPO4(mM) Sebelum NaSitrat Setelah NaSitrat 0.0 0.000 0.000 0.1 0.149 0.102 0.2 0.261 0.239 0.4 0.507 0.417 0.6 0.738 0.622 0.8 0.990 0.834 1.0 1.200 1.022
Kurva Standar ATPase Stock 1 mM Sebelum NaSitrat y = 1.197x + 0.0193 R2 = 0.9992 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0.0 0.2 0.4 0.6 0.8 1.0 Konsentrasi K2HPO4 O D 6 2 0 /4 0 5
Kurva Standar ATPase Stock 1 mM Setelah NaSitrat y = 1.0207x + 0.0103 R2 = 0.9989 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.2 0.4 0.6 0.8 1.0 Konsentrasi K2HPO4 O D 6 2 0 /4 0 5
Lampiran 5. Perhitungan Aktivitas RNA Helikase HCV
Satu unit aktivitas RNA helikase HCV adalah jumlah mol fosfat (P) yang dilepaskan dari ATP per menit per mg protein.
Keterangan:
Volume (v) = 5 mL
Waktu inkubasi (t) = 45 menit
Contoh perhitungan: A620/405 mM PO4 mmol P/min/mL mmol P/min/mg Sebelum Na-Sitrat 80x 1,4497 1,4102 5,2780 7,0373 100x 1,2237 1,1888 5,5616 7,4155 Setelah Na-Sitrat 80x 1,081 1,0490 3,9260 5,2347 100x 0,902 0,8736 4,0871 5,4494 otein mg zim dilutionEn mL v t MWPO mMPO mgprotein mmolP Pr 1 ) ( 1 1 min/ / 3 4 3 4
Lampiran 6. Kurva Standar Bobot Molekul Relatif BM log BM Rf 250 2,40 0,50 150 2,18 0,45 100 2,00 1,25 75 1,88 1,60 50 1,70 2,30 37 1,57 3,00 25 1,40 4,00 20 1,30 4,50 15 1,18 5,30 10 1,00 6,20 y = -0,201x + 2,2157 R2 = 0,9911 0,0 0,5 1,0 1,5 2,0 2,5 0 1 2 3 4 5 6 7 Rf L o g B M
Lampiran 7. Larutan Bufer Bufer HCl – KCl
Larutan A : 0,2 M KCl (14,91 g dalam 1000 mL akuades) Larutan B : 0,2 M HCl
50 mL larutan A + x mL larutan B ditambahkan akuades sampai 200 mL
x pH x pH 97,0 1,0 20,6 1,7 78,0 1,1 16,6 1,8 64,5 1,2 13,2 1,9 51,0 1,3 10,6 2,0 41,5 1,4 8,4 2,1 33,3 1,5 6,7 2,2 Bufer asetat
Larutan A : 0,2 M larutan asam asetat (11,55 mL dalam 1000 mL akuades)
Larutan B : 0,2 M larutan Na-asetat (16,4 g C2H3O2Na atau 27,2 g C2H3O2Na·3H2O dalam 1000 mL akuades)
x mL larutan A + y mL larutan B ditambahkan akuades sampai 100 mL
x y pH x y pH 46,3 3,7 3,6 20,0 30,0 4,8 44,0 6,0 3,8 14,8 35,2 5,0 41,0 9,0 4,0 10,5 39,5 5,2 36,8 13,2 4,2 8,8 41,2 5,4 30,5 19,5 4,4 4,8 45,2 5,6 25,5 24,5 4,6 Bufer Tris-HCl 50 mM Tris bufer
Tris HCl (g/L) Tris Base (g/L) pH
6,61 0,97 7,4
6,06 1,39 7,6
5,32 1,97 7,8
Bufer Borat-Boraks
Larutan A : 0,2 M larutan asam borat (12,4 g dalam 1000 mL akuades)
Larutan B : 0,05 M larutan borax (19,05 g dalam 1000 mL akuades; setara dengan 0,2 M Na-Borat)
50 mL larutan A + x mL larutan B ditambahkan akuades sampai 200 mL
x pH y pH 2,0 7,6 22,5 8,7 3,1 7,8 30,0 8,8 4,9 8,0 42,5 8,9 7,3 8,2 59,0 9,0 11,5 8,4 83,0 9,1 17,5 8,6 115,0 9,2
Lampiran 8. Pengendapan Protein Inhibitor dengan Amonium Sulfat
Banyaknya amonium sulfat (gram) yang ditambahkan ke dalam 1000 mL larutan ekstrak kasar protein inhibitor dengan kejenuhan amonium sulfat 65% dihitung berdasarkan tabel berikut:
From To S1% S2% 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 27 55 84 113 144 176 208 242 277 314 351 390 430 472 516 561 608 657 708 761 5 27 56 85 115 146 179 212 246 282 319 357 397 439 481 526 572 621 671 723 10 28 57 86 117 149 182 216 351 287 325 364 405 447 491 537 584 634 685 15 28 58 88 119 151 185 219 255 292 331 371 413 456 501 548 596 647 20 29 59 89 121 154 188 223 260 298 337 378 421 465 511 559 609 25 29 60 91 123 157 191 227 265 304 344 386 429 475 522 571 30 30 61 92 126 160 195 232 270 309 351 393 438 485 533 35 30 62 94 128 163 199 236 275 316 358 402 447 495 40 31 63 96 130 166 202 241 281 322 365 410 457 45 31 64 97 132 169 206 245 286 329 373 419 50 32 65 99 135 172 210 250 292 335 381 55 33 66 101 138 175 215 256 298 343 60 33 67 103 140 179 219 261 305 65 34 69 105 143 183 224 266 70 34 70 107 146 186 228 75 35 72 110 149 190 80 36 73 112 152 85 37 75 114 90 37 76 95 38 Sumber: Scope (1987)
