Name: ________________________

Name: _________________________

Biological Sciences 4087

Exam I

2/20/03

Total: 100 points

1.(14pts) The release of H⁺ upon oxygen binding to hemoglobin is due largely to deprotonation of His 146(b). The pK_a of this histidine is higher than normal because it forms and electrostatic bond with another amino acid in the T state of hemoglobin.

A. Write the complete name of one amino acid that can form an electrostatic bond with histidine with its side chain.

glutamate or aspartate

B. Draw the structure of the amino acid you named in part A.

see Fig. 5-5

C. The pK_a of the R group of histidine is 6.5. Using this pK_a, calculate the ratio of the base to the acid form of histidine at pH 7.4.

pH = pK_a + log [A^-]/[HA]

7.4 = 6.5 + log [A^-]/[HA]

0.9 = log [A^-]/[HA]

7.9 = [A^-]/[HA]

D. Lowering the pH of blood causes the O₂ affinity of hemoglobin to (CIRCLE ONE):

INCREASE

DECREASE

REMAIN THE SAME

2.(6pts) For this peptide: MALVICGGAMSPE

A. Write the complete name of the N-terminal amino acid.

methionine

B. Write the complete name of the C-terminal amino acid.

glutamate

C. Write the complete name of the amino acid which can form a disulfide bond with another peptide.

cysteine

3.(11pts) A. CIRCLE the peptide(s) that will form an a helix at pH 7.

POLYALANINE

POLYGLUTAMATE

POLYPROLINE

B. Name a protein which has quaternary structure.

hemoglobin, aspartate transcarbamoylase

C. Name and briefly explain a technique which can be used to determine the primary structure of a peptide.

Edman degradation-PITC is used to sequentially label and identify the amino acids starting at the N-terminal of the peptide

4.(6pts) Describe the ELISA assay.

Wells in a plastic tray are coated with sample. The protein to be detected, if present, will bind to the plastic. Unoccupied sites are blocked with casein. The first antibody is added. Unbound antibody is washed away, and a second antibody against the first antibody is added. This antibody is linked to an enzyme. The substrate for the enzyme is added, and wells containing the antigen-antibody complexes will be colored.

5.(21pts) A. Describe how to make an antibody which acts as an enzyme.

Synthesize a transition state analog for the reaction you want to catalyze and raise monoclonal antibodies to this transition state analog. Test the monoclonal antibodies for enzymatic activity.

B. A V vs. [S] plot for this antibody-enzyme would be (CIRCLE ONE):

HYPERBOLIC

SIGMOIDAL

SINUSOIDAL

C. If V_max for the reaction catalyzed by the antibody is 10mM/sec at a total antibody (enzyme) concentration of 0.1mM, what is k_cat (turnover number)?

V_max = k_cat x [E_t]

10 mM/sec = k_catx (0.1 mM)

k_cat = 100 sec^-1

D. Draw a Lineweaver-Burk plot for this reaction assuming V_max is 10mM/sec and K_mis 1mM. Label the axes of your plot. You may draw it on the back of this page.

see p. 267

6.(12pts) Draw the complete structure of the dinucleotide UG. You may draw it on the back of this sheet.

see pp. 327 and 329

7.(6pts) A. What is the function of DNA polymerase I in DNA replication in E. coli?

DNA polymerase I removes the RNA primers and replaces them with DNA.

B. What is the function of DNA polymerase I in base-excision repair in E. coli?

DNA polymerase I removes the apyrimidinic ribose and some neighboring nucleotides and replaces them with DNA.

8.(8pts) Describe the Sanger method of DNA sequencing.

The unknown DNA is used as a template for DNA synthesis, and dideoxynucleotides labeled with fluorescent dyes are used to generate random terminations. The fragments generated are separated by size on a polyacrylamide gel and detected using a laser.

9.(16pts) Define:

A. van der Waals interactions-weak noncovalent interactions between transient dipoles on neighboring atoms

B. motif-supersecondary structure; arrangements of several elements of secondary structure

C. GroEL/GroES-chaperonins in E. coli which aid in the folding of proteins

D. BPG-allosteric effector of hemoglobin which stabilizes the T state and lowers O₂ affinity