Tetraphenylphosphonium Bromide TTP Large MoleculeSmall Molecule Interaction Program
Tetraphenylphosphonium Bromide TTP Large Molecule-Small Molecule Interaction Program. SHEA HIGH SCHOOL July 13 -17, 2009 Ms. La Roche Cesar Luzon, Janice Miranda, Maldine Santos, Estefania Tabares.
Tetraphenylphosphonium Bromide TPP. Formula: CH 3 P(C 6 H 5)4 Mol WT: 419. 30 Synonyms: Bromure de tétraphénylphosphonium; Bromuro de tetrafenilfosfonio; Classification: ORGANOPHOSPHINES. Physical state: white to off-white crystalline powder. Melting Point: 295 - 299 C Solubility in water: Soluble. Stability: Stable under ordinary conditions.
ELECTROPHORESIS Well 1: 10. 0 u. L Linearized Plasmid DNA. Well 2: 10. 0 u. L uncut Plasmid DNA Well 3: 10. 0 u. L linearized plasmid DNA w B-A. Well 4: 10. 0 u. L uncut plasmid DNA w B-A. Well 5: 5. 0 u. L Kb ladder 22 17 21 15 19 23 23 24 27 30 34 41 45
Standard Migration Curve - Salmon DNA and TPP 100, 000 10, 000 Base pairs Base parirs 1, 000 15 20 25 30 35 Distance (mm) 40 45 50
Electrophoresis 7 -16 -09 Distance (mm) Base parirs 19 23, 130 23 9, 416 27 6, 557 30 4, 361 34 2, 322 41 2, 027 45 564 125 Distance Base pairs E-linear EB-linear plasmid & TPP 22 Observations: both the linear and the uncut plasmid with TPP travelled 1 mm further showing the small molecule bonded with the DNA molecule. Distance 15000 X-uncut plasmid Base pairs 21 17000 XB-uncut & TPP 17 24 38000 9000 15 23 10000 11000
Cary Spectrophotometer Temp. Cº Absorbance ∆T DNA 25 0. 7259 35 0. 7259 45 0. 7276 55 0. 7254 60 0. 908 65 0. 9441 70 0. 9464 75 0. 9531 80 0. 9843 85 1. 0185 90 1. 0255 95 1. 0346 Temp. Cº Absorbance ∆T DNA+orbizine 25 0. 91 35 0. 91 45 1. 04 55 1. 2268 60 1. 2377 65 1. 256 70 1. 2452 75 1. 2833 80 1. 2498 85 1. 3066 90 1. 334 95 1. 3642 ∆A 10 10 10 5 5 5 5 A∆ 10 10 10 5 5 5 5 Tave 0 0. 0017 -0. 0022 0. 1826 0. 0361 0. 0023 0. 0067 0. 0312 0. 0342 0. 007 0. 0091 ∆A/∆T 30 40 50 57. 5 62. 5 67. 5 72. 5 77. 5 82. 5 87. 5 92. 5 Tave 0 0. 13 0. 1868 0. 0109 0. 0183 -0. 0108 0. 0381 -0. 0335 0. 0568 0. 0274 0. 0302 30 40 50 57. 5 62. 5 67. 5 72. 5 77. 5 82. 5 87. 5 92. 5 0 0. 00017 -0. 00022 0. 03652 0. 00722 0. 00046 0. 00134 0. 00624 0. 00684 0. 00182 ∆A/∆T 0 0. 013 0. 01868 0. 00218 0. 00366 -0. 00216 0. 00762 -0. 0067 0. 01136 0. 00548 0. 00604
Temperature vs. Absorbance - Salmon DNA 1. 6 1. 4 1. 2 1 Absorbance 0. 8 0. 6 0. 4 0. 2 0 0 20 40 60 80 100 Temperature ⁰C Temperature vs. Absorbance - Salmon DNA + TPP 1. 2 1 0. 8 Absorbance 0. 6 (nm) 0. 4 0. 2 0 0 20 40 60 Temperature (⁰C) 80 100
Derivative Plot - ∆A/∆T vs. Average Temperature Using the Cary Spectrophotometer Samon DNA 0. 04 0. 035 0. 03 0. 025 0. 02 0. 015 0. 01 0. 005 0 0 10 20 30 40 50 60 70 80 90 100 -0. 005 Salmon DNA +TPP 0. 02 0. 015 0. 01 0. 005 0 0 -0. 005 -0. 01 20 40 60 80 100
MAJOR GROOVE MINOR GROOVE INTERCALATING
TEST ORIENTATION MAJOR GROVE MINOR GROVE INTERCALATING DNA and TPP X -582. 33 -582. 68 -574. 78 DNA and TPP Y -1203. 054 -611. 55 -572. 25 DNA and TPP Z -582. 55 -574. 72 -572. 89
Smart spec. Temp. C° Absorbance (nm) Column 1 Column 2 Column 3 Column 4 DNA ΔT ΔA Tave ΔA/ΔT 50 0. 706 5 0. 01 52. 5 0. 002 55 0. 716 5 0. 001 57. 5 0. 0002 60 0. 717 5 0. 074 62. 5 0. 0148 65 0. 791 5 0. 128 67. 5 0. 0256 70 0. 919 5 0. 121 75 0. 0242 80 1. 04 10 0. 008 82. 5 0. 0008 85 1. 048 5 0. 542 87. 5 90 1. 59 5 -1. 59 45 Temp. C° Absorbance (nm) Column 1 Column 2 Column 3 Column 4 DNA+TPP ΔT ΔA Tave ΔA/ΔT 50 1. 249 5 0. 178 52. 5 0. 0356 55 1. 427 5 0. 185 57. 5 0. 037 60 1. 612 5 0. 028 62. 5 0. 0056 65 1. 64 5 0. 064 67. 5 0. 0128 70 1. 704 5 0. 042 72. 5 0. 0084 80 2. 017 10 0. 277 82. 5 0. 0554 85 2. 294 5 0. 095 87. 5 0. 019 90 2. 389 5 0. 009 91 0. 0018 Absorbance at 260 nm
Temperature vs. Absorbance - at wavelength of 260 nm Salmon DNA 1. 8 1. 6 1. 4 1. 2 1 Absorbance 0. 8 0. 6 0. 4 0. 2 0 40 50 60 70 80 90 100 Temperature ⁰C Temperature vs. Absorbance at wavelength of 260 nm Salmon DNA + TPP 3 2. 5 2 1. 5 Absorbance (nm) 1 0. 5 0 40 50 60 70 Temperature ⁰C 80 90 100
Derivative Plot - ΔA/ΔT vs. Average Temperature Salmon DNA Using the Smart. Spec Plus 0. 03 0. 025 0. 02 0. 015 0. 01 0. 005 0 0 20 40 60 80 100 Derivative Plot - ΔA/ΔT vs. Average Temperature Salmon DNA using the Smartspec Plus 0. 06 0. 05 0. 04 ΔA/ΔT 0. 03 0. 02 0. 01 0 0 20 40 60 Average Temperature 80 100
DISCUSSION: • Hyperchem analyses led to the prediction that tetraphenylphosphonium Bromide would bond to DNA. The experimental data shows a shift in the melting curve when TPP is added, agreeing with this prediction • No significant changes were made in the experimental procedures. • Light banding in electrophoresis gels suggest using a larger amount of the • Samples. Practice pipetting into wells is suggested. • Repeated trials of both experimental procedures are recommended for further research. • The melting point of Salmon DNA was 70⁰C on the Smart Spec and 59⁰C on the Cary spectrophotometer. • The difference in the melting point could be due to the fluctuating temperatures with the Smart Spec Plus. • The melting point of the Salmon DNA with the small molecule TPP: Cary appears to be 49⁰C. There are three other indications of melting in different phases. Smart Spec – points to two melting points, 68⁰C and 83⁰C.
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