BCH 4053 PRE-TEST 1 GROUP NAME _____________________
May 29, 1996

This test is take-home and open book, and it is
intended that all members of the group contribute to
completing it. Only one copy is to be submitted by
the group, and all members who participated should
sign their names below. Test is due by 1:30 pm on
Monday, June 3.

__________________________ __________________________

__________________________ __________________________ Page Points

1 _____
2 _____
3 _____
4 _____
5 _____
6 _____

Total _____

Points

(12) 1. Draw a titration curve for the dipeptide Val.His on the graph below.
Locate and identify the points on the curve corresponding to pK1,
pK2, and pK3. Calculate the approximate pI value and locate its
position on the curve. Indicate the pH region on the graph in which
at least 25% of the histidine side chain is charged.

þ
13 Ãþ
³ pI =
12 Ãþ
³
11 Ãþ
³
10 Ãþ
³
9 Ãþ
³
8 Ãþ
³
7 Ãþ
pH ³
6 Ãþ
³
5 Ãþ
³
4 Ãþ
³
3 Ãþ
³
2 Ãþ
³
1 ÃÄÄÄÄÄÄÄÄÄÄÄÄÄÄÁÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÁÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÁþ
1 2 3

equivalents of base ----->

(5) 2. Acetoacetic acid, a ketone body produced by the liver and used as a
fuel by other tissues, has a pK of 3.58. What fraction of the
acetoacetic acid molecules in urine having a pH of 4.48 would be in
the protonated, uncharged form? (Note: I'm looking for fraction,
not ratio).

(10) 3. You have solutions of (a) 1.0 M NaOH and (b) 1.0 M H3PO4, and you
would like to prepare 2 L of 0.05 M phosphate buffer at pH 7.0.
Calculate the volume of solutions (a) and (b) and water that must be
combined to produce the buffer. (Assume volumes are additive.)
For H3PO4: pK1 = 2.12; pK2 = 7.21; pK3 = 12.67

(10) 4. Given the following data on three different proteins:

Protein: hemoglobin chymotrypsinogen urease

Molecular Weight (M) 64,500 23,250 482,000
Diffusion Coefficient (D) 6.9 9.5 3.5
Isoelectric pH (pI) 6.8 9.5 5.0

Indicate in the blanks which of the three proteins will:

____________________ (a) Elute first from a gel filtration column.

____________________ (b) Elute first from a diethylaminoethyl
cellulose ion exchange column.

____________________ (c) Have the smallest frictional coefficient
(f).

____________________ (d) Migrate fastest upon electrophoresis in
sodium dodecyl sulfate (SDS).

____________________ (e) Migrate fastest to the anode in a native
electrophoresis experiment at pH 6.0.

(18) 5. Underline the following peptides which are negatively charged at
pH 7.0. Circle each amino acid which is non-polar.

gln.val.tyr.ala lys.arg.glu.trp

met.his.leu.asp cys.pro.gly.asn

F-A-D-S M-I-C-K

(6) 6. Describe two methods of cleaving disulfide bonds in proteins prior to
sequencing.

7. Consider the following peptide: AEMKHS

(5) (a) Calculate the pI. (You may either use actual pK values from page
113, or the approximate values you were asked to learn. On the
regular test you would use approximate values.) Sketch enough of the
structure to show how you arrive at your calculation.

(6) (b) Treatment with cyanogen bromide produces two peptides. You want to
separate these peptides from starting material and from each other.
Predict the order of elution of the two product and one starting
peptide from a cation exchange column eluted with a buffer of
increasing pH. Explain your reasoning.

(4) (c) Draw the structure of the first two PTH-amino acids produced in the
first two rounds of the Edman degradation of the above peptide. PTH-
amino acids are analyzed by reversed phase HPLC, similar to the
analysis of PTC amino acids (Fig. 4-20, p. 4-23), in which the
derivatives are eluted in order of decreasing polarity (most polar
first). Predict the order of elution of these two PTH-amino acids
from the HPLC column.

(10) 8. The conformation of a peptide bond can be described by plotting a
Ramachandran map, in which the þ and è angles are plotted against
each other.

(a) The þ angle represents rotation about which bond?

(b) The è angle represents rotation about which bond?

alpha helix (right handed), á-sheet (parallel), collagen helix

(6) 9. The fraction of ligand binding sites (þ) on myoglobin and hemoglobin
can be expressed by the relationship

þ = (pO2)n/[(pO2)n + (P50)n]

where pO2 is the pressure of oxygen, P50 is the oxygen pressure
required to occupy 50% of the sites, and n is the Hill coefficient.
For myoglobin, P50 = 1 torr and n = 1.
For hemoglobin, P50 = 26 torr and n = 2.8.

Calculate þ for the two proteins in tissues where pO2 = 30 torr.

(8) 10. The following graph shows three idealized curves that describe
oxygen binding to hemoglobin. Identify by letter the curve or curves
which represent the situations described in the questions below.
þ
³
³ A
³
³ B
Y ³
³ C
³
³
ÀÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄÄþ
pO2

(a) A comparison of fetal hemoglobin to normal hemoglobin A could be
represented by comparing
______ to _______.

(b) Increasing the concentration of BPG in the red cell will cause
a shift in oxygen binding similar to the shift from

______ to _______.

_______ to _______.

(d) Increasing the pH of the hemoglobin solution from 7.2 to 7.4
will cause a shift in oxygen binding similar to the shift from

_______to _______.