Unformatted text preview: Biochemistry 440, Fall 2004 Mid-term Exam #1 Page 1 Name: ID #: 1. Glycine, a diprotic acid with pKa1 = 2.2; pKa2 = 9.6, can be used as a buffer. The different species of glycine are called: GlyH2+, GlyH, and Gly-. Answer the following questions for a buffer made by mixing 750 mL of 0.1 M GlyH and 250 mL of 0.1 M Gly-. (a) Calculate the pH of the buffer. Show your work. (27pts total) (6pts) (b) What fraction of the buffer components are in the conjugate base form? Show your work.
(4pts) (c) How many moles of strong acid or strong base corresponds to 0.5 equivalent of this buffer? Show your work.
(4pts) (d) Calculate the pH after 5 mL of 10 M NaOH are added. Show your work.
(6pts) (e) Use the titration curve on the right to indicate where the buffer would be if 5 mL of 10 M HCl are added INSTEAD of the NaOH in Part (d). MARK the place on the curve CLEARLY with an “X”.
(3pts) 12 10 8 (f) The pI of glycine is 6.0. Is glycine a good buffer at pH 6? WHY or WHY NOT?
(4pts) 6 4 2 2. Page 2 The amino acid below has pK1 = 2.0 pKR = 8.0, pK2 = 10.3 and a pI = 5.0. Answer the questions about the amino acid below: (a) NAME the amino acid by its 3-letter and 1-letter code. (18 pts) H H N C COOH HC SH
+H (b) CIRCLE the protons that will be dissociated at pH 9. (c) What is the NET charge of the amino acid at each pH below? NET charge pH 1 pH 5 pH 12 (d) Give an example of a function this amino acid can perform. (e) What other amino acid(s) have sulfur in them? 3. Use the picture below to answer the following questions. (15 pts) (a) Write the sequence of the peptide using 1-letter code.
(10 pts) (b) DRAW a box around all the atoms in the second residue.
(3 pts) (c) CIRCLE all the atoms that are co-planar in the C-terminal peptide bond.
(2 pts) 4. Page 3 You discover a new protein that is secreted by bacteria living in a hot spring at 60 C. You purify the protein and make the observations listed below.
o (26 pts) 1. The protein binds to a cation exchange column at pH 8.5. 2. You have a size exclusion column that retains & separates proteins in the MW range 10 kD – 75 kD. Under native conditions, the protein is not retained by this resin. 3. You run the protein collected from the column on SDS-PAGE, but you forget to add bmercaptoethanol. You observe two bands, corresponding to 15 kDa and 70 kDa. 4. You re-run the SDS-PAGE with b-mercaptoethanol. You observe two bands of equal intensity at 35 kDa and 15 kDa. 5. When the protein is loaded onto the size exclusion column in the presence of 10 mM bmercaptoethanol, the protein elutes from the column at the position of a 50 kDa species. (a) Check ONE: c pI < 7
(3 pts) (6 pts) c pI = 7 c pI > 7 (b) List the number of subunits and their size. (c) From the data provided, deduce the quaternary structure of the native protein and its molecular size.
(9 pts) (d) Sequence analysis reveals that your protein is homologous to a protein found in E. coli. The E. coli protein is reported to have [GdnHCl]50% = 4.8 M at 25o C. Chemical denaturation experiments were performed on the two proteins at two different temperatures, 37o C and 50 o C, to give the four curves below. MATCH the curves to the correct sample.
(8 pts) SAMPLE E. coli protein at 37o C CURVE 1.0 0.9 0.8 0.7
Fraction Unfolded YOUR protein at 37o C E. coli protein at 50o C YOUR protein at 50o C A B C D 0.6 0.5 0.4 0.3 0.2 0.1 0.0 0.0 1.0 2.0 3.0 [GuHCl] (M) 4.0 5.0 6.0 Page 4 5. BPG binds to hemoglobin (Hb) with a KD = 2 x 10-2 M. (24 pts total) (a) WRITE the expression for KD in terms of concentration of Hb, BPG, and HblBPG.
(3 pts) (b) At sea level, [BPG] = 5 mM. What fraction of Hb molecules have a BPG bound? SHOW your work.
(5 pts) (c) The total concentration of Hb (bound plus unbound) in erythrocytes is 1 mM. What is the concentration of HblBPG at sea level? SHOW your work.
(5 pts) (d) What percent of BPG is actually bound to Hb? SHOW your work.
(5 pts) (e) With proper training at high altitude, BPG levels can double ([BPG] = 10 mM). Will this double the fraction of HblBPG? EXPLAIN your answer.
(6 pts) Page 5 6. If Curve C corresponds to adult Hb under normal physiological conditions at pH 7.4, which curve would correspond to the following conditions? A curve may be used more than once, or may not be used at all.
1 Condition A B C D decreased pH slightly decreased [BPG] binding of O2 to Mb increased CO2 levels
0 2 4 6 8 10 12 14 Curve Letter Fractional Saturation O2 0.8 0.6 0.4 0.2 0 fetal pO2 (kPa) fetal Hb ...
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