Brugia Malayi - Clark Science Center - Smith College

The Stabilizing Effect of Metalloporphyrins on Myoglobin
Metasebia Aberra
30
Deconvolution
10
Deconvolution MbMnPPIX
Cp (kcal/K mol)
25
20
15
10
5
Cp (kcal/K mol)
5
0
-5
-10
-15
-20
-25
0
10 20 30 40 50 60 70
Temperature ( o 80 90 100 110
C)
-30
10 20 30 40 50 60
Temperature ( o 70 80 90
C)
DSC of native myoglobin T m
= 72.8˚C,
DSC of Mn-substituted myoglobin T m
= 78.5˚C,
∆H = 139.0 kcal/mol, ∆C p
= 0.194 ∆H = 123.5 kcal/mol, ∆C p
= 0.124
Myoglobin is an oxygen binding protein found in the heart and skeletal muscles. It contains a metalloporphyrin prosthetic group
called a heme that consists of a porphyrin ring with an embedded Fe II. Previous studies show that the presence of the heme
group prevents the misfolding of myoglobin and the formation of other structures called amyloid fibrils. Theses structures are
similar to the amyloid fibrils formed by other proteins during the course of Alzheimer’s and Parkinson’s disease. 1 My research
focuses on identifying the specific features of the heme group that stabilize myoglobin, by substituting the heme group with
different metalloporphyrins.
For my research I used a bovine heart as a source of protein. The myoglobin was purified from the heart muscle by several
sequential steps, including centrifugation, salt precipitation, ultrafiltration, dialysis and ion-exchange chromatography. The purity
of the myoglobin preparations were assessed by their UV-visible spectra and by gel electrophoresis. Finally, the myoglobin
stability, thermal denaturation and amyloid formation were measured using differential scanning calorimetry (DSC). Based
on the information obtained by DSC, the heat capacity (ÄC), free energy (ÄG), enthalpy (ÄH) and entropy (ÄS) of myoglobin
denaturation were determined.
Using DSC, I measured the stability and thermal denaturation of native myoglobin (with heme as the prosthetic group) and
made preliminary measurements of several metal-substituted porphyrins. Native myoglobin showed maximum unfolding at 72.8 o C
and an enthalpy of 139.0 kcal/mol. My future experiments include substitution of different transition metals (Co, Cu, Fe, Mn, Sn,
Zn) as well as modified porphyrins such as deutroporphyrin and mesoporphyrin.
I have presented this research to fellow SURF participants and faculty, and plan to continue this work. (Supported by the
Howard Hughes Medical Institute)
Advisor: David Bickar
References:
1
Friedrich, R.P, 2003, Mechanism of amyloid plaque formation suggests an intracellular basis of Aâ pathogenicity, PNAS early edition, 1-6.
2012
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