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Kabagambe, Benjamin (2008) SPECTROSCOPIC INVESTIGATION OF PROTEINS. Master's Thesis, University of Pittsburgh. (Unpublished)

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Apomyoglobin is obtained from pure myoglobin by extracting out the heme group. UsingUV-visible spectrometry we are able to monitor complete removal of heme group. Thisextraction initiates the disruption of myoglobin's tertiary conformation. Apomyoglobin consistsof 8 α-helices labeled A through H. These helices unfold when the protein is subjected to pHdecrease. It is more compact at about pH 7 and unfolds as we change to more acidicenvironment. At about pH 2 we have A, G, H core with the rest of the helices unfolded. At pHvalues slightly lower than 2, A, G and H helices are also unfolded. When the protein is notdenatured, the refolding process is done by changing the pH towards neutral. At pH 2, G and Hhelices refold and at pH 4 A-helix refolds as well. We have been able to label A-helix withprotons and the rest of the protein with deuterium using H2O and D2O respectively. We havethen used temperature change to initiate the unfolding of protonated A-helix. When dissolved inD2O, the protonated A-helix at elevated temperatures, exchanges its N-H protons to N-Ddeuterons. Temperatures were elevated from 0 °C to 80 °C and changes in Am III peak intensitieswere observed using UV resonance Raman spectroscopy. These peak changes were quantifiedand used to calculate the number of N-H bonds present between 0 °C and 80 °C. The number ofN-H bonds decreased with increase in temperature indicating the unfolding of A-helix.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairAsher, Sanford Aasher@pitt.eduASHER
Committee MemberMichael, Adrianamichael@pitt.eduAMICHAEL
Committee MemberWeber, Stephensweber@imap.pitt.eduSWEBER
Date: 17 January 2008
Date Type: Completion
Defense Date: 12 October 2007
Approval Date: 17 January 2008
Submission Date: 23 October 2007
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: Apomyoglobin; Spectroscopy; UV Resonance Raman
Other ID:, etd-10232007-094038
Date Deposited: 10 Nov 2011 20:03
Last Modified: 15 Nov 2016 13:50


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