Bioanalytical Chemistry
Final Exam
1. Total protein in an unknown sample was estimated using the Lowry and Bradford assays.
Results were 33 ±2 μg/mL from the Lowry assay and 21 ±1 μg/mL from the Bradford assay,
using stock solutions of bovine serum albumin (BSA) as standards in each assay. The
unknown sample was then thoroughly oxygenated, and the assays were repeated. The Lowry
method yielded 22± 1 μg/mL, while the Bradford results did not change. Why did the results
of the Lowry assay decrease by 33%?
2. An enzyme has a molecular weight of 47 kDa and a preparation that is 100% pure has a
specific activity of 700 I.U./mg, at 25 °C in a pH 7.5 phosphate buffer. The enzyme is known
to be monomeric (one active site per enzyme molecule).
(a) Calculate the mass of the pure enzyme preparation needed in 100mL of buffer (saturated
with substrates) to produce a solution that will initially consume substrate at the rate of 5×107
M/min.
(b) Calculate kcat for the enzyme.
(c) A second preparation of this enzyme was tested for activity. In 100 mL of buffer, 1.6 mg
of the solid enzyme was dissolved. This enzyme solution was mixed with a substrate solution
in the ratio 500:1500 μL, and in the final solution, all substrates were present in great excess
of their Km values. Absorbance spectroscopy showed that 0.32mM substrate was consumed
per minute. Calculate the specific activity of the enzyme preparation.
3. Immunogold labeling is to be used prior to examination of a specimen by TEM. The
primary antibody is directed against a known cell surface antigen. You must decide whether
to use a gold nanoparticle-labeled second antibody to the primary antibody, or a gold
nanoparticle-labeled protein A preparation for staining prior to imaging. Which of these
would you choose, considering selectivity and signal-to-noise issues?
Good Luck!
PhD. Chem. R. Esra DEMİRDÖĞEN