A researcher performed a spectrophotometric titration experiment to study the bi
ID: 227818 • Letter: A
Question
A researcher performed a spectrophotometric titration experiment to study the binding capacity that human serum albumin (HSA, 67,000 Da), a transfer protein found in human blood, has for a novel molecular probe, EO550 (MW=691.85 g/mol). When bound to HSA, EO550 absorbs light strongly at 550 nm. When unbound, both HSA and EO550 have negligible absorbance at 550 nm. The researcher prepared eight individual samples, each containing 0.74 mg of HSA dissolved in 150 L of a pH 7.4 phosphate buffered saline solution. To each of the eight samples, a different volume of 2000.0 M EO550 was added and the absorbance was measured at 550 nm. The observed absorbance measured at 550 nm for each added volume of the EO550 solution is shown in the table on the right. As more EO550 is added to the HSA solution, the absorbance increases as more EO550 binds to available sites on the HSA protein. When the binding sites are fully occupied, the absorbance will no longer increase (level off) as more EO550 is added.
A) What is the corrected absorbance when 13.30 L of the EO550 solution is added to the 150.0 L HSA solution?
B) Construct a graph of the corrected absorbance (y-axis) versus the volume of titrant added (x-axis). What is the estimated end point of the titration? Calculate the mass of EO550 added to reach the end point.
C) Approximately how many molecules of EO550 can bind to a single HSA protein?
Mapoob Sapling Learning spectrophotometric titration ex A researcher performed a study the binding capacity that human serum albumin (HSA, 67,000 Da), a transfer protein found in human blood, has for a novel molecular probe, EO550 (MW-691.85 g/mol). When bound to HSA, EO550 absorbs light strongly at 550 nm. When unbound, both HSA and EO550 have negligible absorbance at 550 nm ent to Volume )Observed EO550 Added Absorbance 3.70 8.60 9.50 10.40 0.210 0.232 0.251 The researcher prepared eight individual samples, each containing 0.74 g of HSA dissolved in 150 of a pH 7.4 phosphate buffered saline lution. To each of the eight samples, a different volume of 2000.0 EO550 was added and the absorbance was measured at 550 nm observed absorbance measured at 550 nm for each added volume of the EO550 solution is shown in the table on the right. As more EO550 is added to the HSA solution, the absorbance increases as more EO550 binds to available sites on the HSA protein. When the binding sites are fully occupied, the absorbance will no longer increase (level off) as more EC 12.40 13.30 15.40 0.257 0.256 0.255 is added A) What is the corrected absorbance when 13.30 of the E0550 solution is added to the 150.0 d HSA solution? Number Corrected absorbance(13.30 A.) = Scroll down to view the rest of the question. B) Construct a graph of the corrected absorbance (y-axis) versus the volume of titrant added (x-axis). What is the estimated end point of the titration? Calculate the mass of EO550 added to reach the end point. - Previous Give Up & View SolutionO Check Answer Next Exit HintExplanation / Answer
When unbound both HAS and EO550 at 550nm have negligible absorbance.
HAS = 67,000 Da
EO550 = 691.85 gm
Blank absorbance = 0.0334
Corrected absorbance = Observed absorbance – Blank absorbance
Corrected absorbance at 11.20L = 0.254 – 0.0334 = 0.2206
B)
End point will be given by intersection of two lines.
0.0208 x + 3 * 10-5 = 8 * 10-5 x + 0.2211
End point, x = 10.67 µL6
Moles of EO550 = 10.67 µL * 1950 µM * 10-6 M/µM = 0.0208 µmoles
Mass of EO550 = 0.0208 * 691.85 = 14.39 µg
C)
Molecules of EO550 = 0.0208 µmoles * (10-6 mol/µmol) * NA (6.022 x 1023)
= 1.252 x 1016
Moles of HSA = 0.70 mg * (10-3 g/mg) / (67000 g/mol) = 1.044 x 10-8 moles
Molecules of HSA = 1.044 x 10-8 NA(6.022 x 1023)
= 6.286 x 1015
Molecules of EO550 that can bind to single HSA protein
= 1.252 x 1016 / 6.286 x 1015
= 1.99 2
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