2-Practice Problems Key - 1. Determining protein...

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1. Determining protein concentration You want to measure the concentration of a peptide solution using absorbance at 280 nm. You prepare a 10-fold dilution of your protein sample, pipet it into a cuvette with a 1 cm pathlength and measure the A 280 with a spectrophotometer. You know that your peptide has the following sequence: WHYWITCHESLIKEWINDYDAYS Your measured absorbance is 0.2314. What is the concentration of your original peptide solution? (Be careful that the units cancel properly). Hint: Go to http://www.expasy.ch/tools/protparam.html , enter your protein sequence and hit the “Compute parameters” button. Scroll through the information on the next page and find the Extinction Coefficient ( ε ). Check out some of the other useful information on this page! ε = 20970 M -1 cm -1 A = ε lc c = (0.2314) / [(20970 M -1 cm -1 )(1 cm)] c = 11.03 μ M * 10 (don’t forget the 10-fold dilution!) = 110.3 μ M 2. Identifying the sequence of unknown peptides You have isolated a new pentapeptide (5 residues) from a plant. You get the following information through a series of experiments: a. The pentapeptide strongly absorbs light at 280 nm b. The pentapeptide has a net charge of “-0.5” at pH 10.50. c. Bubbling oxygen through the solution results in the formation of decapeptides (10 residues) d. Treatment of the peptide with 1-fluoro-2,4-dinitrobenzene, followed by complete hydrolysis and chromatography, yielded only free amino acids and the following derivative: NH 3 + CH C H 2 O - N Give the one letter code for a pentapeptide that fits all these criteria. Draw the structure of the peptide in the form that would predominate at pH 7.0. One possible peptide would be W-C-R-K-G. The sequence of the residues doesn’t matter. See structure on next page.
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H 3 N CH C CH 2 O HN H N CH C CH 2 O SH H N CH C CH 2 O CH 2 CH 2 NH C NH 2 NH 2 H N CH C CH 2 O CH 2 CH 2 CH 2 NH 3 H N CH C H O O 3. Your first protein purification You’ve just joined a research lab and your first task is to purify the enzyme lactate dehydrogenase (LDH) from bovine heart. You pick up 10 kg of beef heart from a
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This note was uploaded on 02/21/2010 for the course MCB 102 taught by Professor Staff during the Spring '08 term at University of California, Berkeley.

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2-Practice Problems Key - 1. Determining protein...

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