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- (25 pts) Insulin is an important hormone produced by the pancreas that allows cells to process glucose. Diabetes mellitus, affecting 16 million people in the U.S., is a condition in which insulin production or utilization is impaired. Current treatment for diabetes often involves subcutaneous injection of insulin.
a) Access the RCSB Protein Data Bank at http://www.pdb.org/pdb. Perform a search on insulin and access the human insulin two disulfide model to answer the following:
i) (2 pts) What is the primary chemical structure for insulin?
A chain (21 residues): GIVEQSCTSISSLYQLENYCN B chain (30 residues): FVNQHLCGSDLVEALYLVCGERGFFYTKPT
ii) (3 pts) What is the secondary structure of insulin?
The A chain exhibits an alpha helix formed by residues 13-16. The B chain forms an alpha helix with residues 9-18. The fractional alpha helical content for insulin is thus 14/51 = 0.27.
Disulfide bonds between cysteine residues A7-B7 and A20-B19 bond the A and B chains.
iii) (2 pts) From the Ramachandran plot analysis, what is the range of psi, phi values for most of the amino acid residues in insulin?
From the Ramanchandran plot, most of the residues are found in the range: φ = -45 to -120°, ψ = 0 to -80°.
b) (2 pts) Could insulin be readily radiolabeled with 125 I for detecting its adsorption?
(^125) I will react with tyrosine residues in proteins. Insulin has 4 tyrosine residues and
thus in principle can be radiolabeled with 125 I. In practice, the tyrosine at A appears to be the only one accessible for labeling (see P. Iozzo et al., Nucl. Med. Biol. 29, 73 (2002), and refs. therein).
Numerous research efforts underway are investigating controlled release approaches that would eliminate the need for daily insulin injections. Such strategies typically involve exposure of insulin to synthetic material surfaces. To investigate the effects of adsorption on the secondary structure of insulin, Mollmann et al. ( Eur. J. Pharm. Sci. 2006 , 27 , 194) performed circular dichroism (CD) measurements on insulin in phosphate buffered saline (PBS) solution and adsorbed to the surface of colloidal Teflon particles 215 nm in diameter and stabilized in water with a small fraction of negatively charged sulfate groups on their surface. The CD data are shown in the figure below.
c) (3 pts) What is the effect of adsorption on the secondary structure of insulin?
Image removed for copyright reasons.
See Fig. 2 in Mollmann, Susanne H., Lene Jorgensen, Jens T. Bukrinsky, Ulla Elofsson,
Willem Norde, and Sven Frokjaer. "Interfacial Adsorption of Insulin: Conformational Changes
and Reversibility of Adsorption." Eur J Pharm Sci 27 (2006): 194-
The alpha helical content of insulin decreases upon adsorption while the random coil content increases. This is observed through the decreased negative ellipticity at 222 nm and decreased positive ellipticity at 192 nm (Note that a random coil exhibits a large negative ellipticity at 197 nm.)
d) (4 pts) From the CD data provided, calculate the fractional α helical content for insulin in solution and adsorbed to the Teflon particles.
−[ ]θ 222
The helical content can be determined using the equation: f α − helix^ =
The molar ellipticity at 222 nm is ~ -12,000 for insulin in solution, giving f α = 0.30, in good agreement with 0.27 determined above from the structure database. Adsorbed to a surface, the [θ] 222 ~ -9000, giving f α = 0.23.
e) (2 pts) Why might changes in the secondary structure of insulin be important to consider in developing controlled delivery strategies?
If insulin denatures upon contacting a synthetic surface before being administered, its activity in vivo may be reduced.
One must be careful in performing the experiment, however. At high [P], if KU is
large compared to K F so that C is large compared to ( K F − KU )[ PF ] (small values of
[PF], then: K (^) F [ PF ] ν (^) F ≈ C
so that a linear increase in fluorescence intensity with [PF] might still be observed. The authors provided insufficient details on their experiments to determine whether their conclusion is valid or not.
- (20 pts) Prosthetic vascular grafts are commonly employed in the treatment of atherosclerosis, as a means to bypass occluded blood vessels. Read through the text on pp. 479-483 (1st^ ed.: pp. 420-422) to assist in answering the questions below.
a) (2 pts) What materials are currently used in commercial vascular grafts?
Dacron (polyethylene terephthalate, PET) is commonly used for large diameter vessels while expanded polytetrafluoroethylene (ePTFE) is used for synthetic grafts of diameter below < 8 mm.
b) (2 pts) What is the most common reason for failure of synthetic vascular grafts of small diameter?
Thrombosis is the chief source of failure in small diameter grafts, as even moderate deposits on the inner surface of the graft can restrict blood flow.
c) (4 pts) Describe the expected synthetic/biological material interface at the interior and exterior surfaces of a vascular graft many months post-implantation.
The interior of the graft will typically have an inner lining of aggregated platelets and fibrin, except near the sutured ends of the graft where endothelial cells migrate into the device to create a neointima. The exterior of the graft will exhibit a histology that is typical of the foreign body response, namely a fibrous capsule encompassing the device, incorporating foreign body giant cells adjacent to the device surrounded by connective tissue containing fibroblasts and capillaries.
Fibrinogen is known to play an important role in clot formation. Makogonenko et al. ( Biochemistry 2002 , 41 , 7907) investigated the interaction of adsorbed fibrinogen with fibronectin, employing surface plasmon resonance (SPR). The figure below shows fibronectin adsorption data at 22 °C on a gold surface coated with fibrinogen (dashed line, 100 nM fibronectin), and the same surface after subsequently being treated with thrombin (solid lines, 0.25 – 3.0 nM fibronectin).
Image removed for copyright reasons. See Fig. 2(a) in Makogonenko, E., G. Tsurupa, K. Ingham, and L. Medved. "Interaction of Fibrin(ogen) with Fibronectin: Further Characterization and Localization of the Fibronectin-Binding Site." Biochemistry 41 (2002): 7907-7913.
d) (2 pts) What do the units of SPR response indicate?
The response is measured in arc seconds, the shift in the angle where maximum energy transfer occurs to the Au film. This value increases with fibronectin adsorption because the index of refraction at the surface is increased.
e) (2 pts) Explain why fibrinogen binds fibronectin only after exposure to thrombin.
Thrombin catalyses the cleavage of fibrinogen to form fibrin. The results suggest that the fibronectin binding sites on fibrinogen are only accessible when fibrinogen is cleaved.
f) (2 pts) From the inset of kobs data obtained from the fits to the SPR data for thrombin- exposed fibrinogen, estimate the value of the dissociation constant KD.
Noting kobs =^ kd +^ k^ a [ P ] , ka should be given by the slope and k d by the y-intercept
of the straight line fit.
KD = k (^) d / k (^) a ≈ 0.3 nM
