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INVESTIGATION INTO the CELLULAR ACTIONS of CARNOSINE and C-PEPTIDE Emma H

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INVESTIGATION INTO the CELLULAR ACTIONS of CARNOSINE and C-PEPTIDE Emma H Marshall Digital Scholar Theses, Dissertations and Capstones 2014 INVESTIGATION INTO THE CELLULAR ACTIONS OF CARNOSINE AND C-PEPTIDE Emma H. Gardner [email protected] Follow this and additional works at: http://mds.marshall.edu/etd Part of the Analytical Chemistry Commons, and the Biochemistry Commons Recommended Citation Gardner, Emma H., "INVESTIGATION INTO THE CELLULAR ACTIONS OF CARNOSINE AND C-PEPTIDE" (2014). Theses, Dissertations and Capstones. Paper 868. This Thesis is brought to you for free and open access by Marshall Digital Scholar. It has been accepted for inclusion in Theses, Dissertations and Capstones by an authorized administrator of Marshall Digital Scholar. For more information, please contact [email protected]. INVESTIGATION INTO THE CELLULAR ACTIONS OF CARNOSINE AND C-PEPTIDE A thesis submitted to the Graduate College of Marshall University In partial fulfillment of the requirements for the degree of Master of Science in Chemistry by Emma H. Gardner Approval by Dr. Leslie Frost, Committee Chairperson Dr. Derrick Kolling Dr. Menashi Cohenford Marshall University May 2014 Table of Contents List of Tables iv List of Figures v IRB Letter vii Abstracts viii 1. Investigation into the Cellular Actions of Carnosine 1 Introduction 1 Experimental Methods 11 Preparation of Carnosine Affinity Beads 11 Tissue Lysis 12 Protein Isolation 13 SDS PAGE Analysis of Isolated Proteins 13 Preparation of Tryptic Digests of Protein Samples from SDS-PAGE Bands 13 Analysis of Tryptic Digests of Proteins by MALDI-TOF Mass Spectrometry 14 Analysis of Tryptic Peptide Amino Acid Sequences by LC/ESI/MS 15 Determining the Effect of Carnosine on AGE Structure Formation 15 Sodium Borohydride Reduction 16 UV-VIS Absorbance Profile of Hemin 16 Results and Discussion 16 Identification of Carnosine Binding Proteins Isolated from Bovine Kidney Tissue 16 Identification of Carnosine Binding Proteins Isolated from Mouse Kidney Tissue 24 Effect of Carnosine on the Formation of AGEs 28 Analysis of Carnosine-Hemin Interactions 33 Future Work 34 References 36 2. Proinsulin C-Peptide as an Active Biomolecule 38 Introduction 38 Experimental Methods 46 Preparation of C-Peptide Affinity Beads 46 Tissue Lysis 47 Protein Isolation 47 SDS PAGE Analysis of Isolated Proteins 48 ii Preparaion of Tryptic Digests of Protein Samples from SDS-PAGE Bands 48 Analysis of Tryptic Digests of Proteins by MALDI-TOF Mass Spectrometry 49 Analysis of Tryptic Digests of Proteins by LC/ESI/MS 49 Results and Discussion 50 Results from Trial 1 Using Tris Pre-clear Beads 52 Proteins Isolated from Kidney Tissue by C-peptide Beads 53 Proteins Isolated from Liver Tissue by C-peptide Beads 58 Results from Trial 2 using Bradykinin Pre-clear Beads 61 Proteins Isolated from Liver Tissue by C-peptide Beads 63 Proteins Isolated from Kidney Tissue by C-peptide Beads 65 References 71 Appendix A 73 Results from Earlier Trials for Heart Tissue Lysates 73 iii List of Tables 1.1 Glycated peptides in Cytochrome C identified by MALDI-TOF and LC/ESI/MS/MS 9 1.2. Carnosine-binding proteins identified by mass spectrometry 20 iv List of Figures 1.1. Structure of the dipeptide carnosine 1 1.2. Structure of the heme b 2 1.3. Overview of various oxidative stress-related effects 4 1.4. Glycation 6 1.5. MALDI-TOF mass spectra of the +2 ion of bovine Cyt 10 1.6. Basic experimental protocol for protein isolation 18 1.7. SDS-PAGE gel separation of proteins 18 1.8. MALDI-TOF peptide fingerprint mass spectrum for band 1 20 1.9. Binding of haptoglobin to hemoglobin 21 1.10. MALDI-TOF peptide fingerprint mass spectrum for band 2 22 1.11. TCP-1 chaperonin with its eight different subunits 23 1.12.MALDI-TOF peptide fingerprint mass spectrum for band 3 24 1.13. The Peptide Mass Fingerprint spectra for 20 kDa 26 1.14. The structure of HBB 27 1.15. The structure of the hemoglobin 27 1.16. MALDI-TOF mass spectra of the +2 ion of glycated cytochrome c 29 1.17. MALDI-TOF tryptic peptide mass fingerprint specta of cytochrome C 30 1.18. MALDI-TOF tryptic peptide mass fingerprint spectra of cytochrome C 31 1.19. Space-filling plot of the horse cytochrome C structure 32 1.20. UV-VIS absorption profile of hemin in saturated carnosine solution 34 2.1. Structure of insulin C-peptide 38 2.2. Proinsulin structure 39 2.3. Insulin biosynthesis pathway 40 2.4. Proposed mechanism for C-peptide signaling cascade 42 2.5. Depiction of the role of C-peptide in atherosclerosis 43 2.6. Basic experimental protocol 51 2.7. SDS-PAGE Gel separation of proteins 53 2.8. MALDI-TOF peptide fingerprint mass spectrum for kidney 25 kDa 55 2.9. The amino acid sequence for E2 56 2.10. Schematic of oxidation decarboxylation of pyruvate 56 2.11. MALDI-TOF peptide fingerprint mass spectrum for kidney 50 kDa 57 2.12. Basic reaction catalyzed by long-chain specific acyl-CoA dehydrogenase 58 v 2.13. MALDI-TOF peptide fingerprint mass spectrum at liver 50 kDa 59 2.14. MALDI-TOF peptide fingerprint mass spectrum for liver 100 kDa 61 2.15. The amino acid structure of the peptide Bradykinin 62 2.16. SDS-PAGE Gel separation of proteins 63 2.17. MALDI-TOF peptide fingerprint mass spectra for liver 50 kD 64 2.18. MALDI-TOF peptide fingerprint mass spectrum for kidney 40 kD 65 2.19. MALDI-TOF peptide fingerprint mass spectrum for kidney 25 kD 67 2.20. The Matrix Mascot search results for the matched peptide sequences 67 2.21. CID mass spectrum recorded on the (M+2H) +2 ion at m/z 919 68 2.22. Structure of lipoic acid 70 A.1. SDS-PAGE gel of proteins isolated from mouse heart 74 A.2. MALDI-TOF peptide fingerprint mass spectrum for 55 kDa 75 A.3. MALDI-TOF peptide fingerprint mass spectrum for 30 kDa 76 A.4. The reaction catalyzed by GAPDH as part of the glycolysis cycle 77 vi Office of Research Integrity May 9, 2014 Emma H. Gardner 1133 Emerald Rd Charleston, WV 25314 Dear Ms. Gardner: This letter is in response to the submitted thesis abstract entitled “ Investigations into the Cellular Actions of Carnosine and Proinsulin C-Peptide.”After assessing the abstract it has been deemed not to be human subject research and therefore exempt from the oversight og the Marshall University Institutional Review Board (IRB). The Code of Federal Regulations (45CFR46) has set forth the criteria utilized in making this determination. If there are any changes to the abstract you provided then you would need to resubmit that information to the Office of Research Integrity for a review and determination. I appreciate your willingness to submit the abstract for determination. Please feel free to contact the Office of Research Integrity if you have any questions regarding the future protocols that may require IRB review. Sincerely, Bruce F. Day, ThD, CIP Director vii Abstracts 1. Investigation into the Cellular Actions of Carnosine Carnosine is a dipeptide composed of beta-alanine and histidine found exclusively in long- lived animal tissues. The cellular action of carnosine is still under extensive investigation; however, it has been proposed to have a role as an anti-oxidant and oxygen free radical scavenger, a physiological buffer, a heavy metal chelator, and has been implicated as an anti- aging agent. 2,4 Our lab has been studying the interaction between carnosine and heme by analyzing both the effect carnosine has on the glycation of the heme containing protein cytochrome c and the interaction of carnosine with free hemin. We have observed that the addition of carnosine to glycated cytochrome c inhibits the formation of advanced glycation end product (AGE) structures. We have also found that the addition of carnosine to a solution of free hemin drastically increases the solubility of hemin and causes a shift of the λmax in its UV-Vis absorbance profile. We propose that carnosine is a natural intracellular heme chelator and that this interaction may act as a signal for cellular antioxidant processes. We have designed an experiment to identify proteins capable of binding to carnosine and heme-chelated carnosine. Using mass spectral techniques, we were able to identify a protein isolated from kidney tissue lysates using carnosine affinity beads. By identifying a carnosine protein binding partner, we gained a better understanding of the cellular action of carnosine that may eventually lead to the development of novel drugs for the treatment of a variety of diseases in which oxidative injury and cellular stress occurs. viii 2. Proinsulin C-Peptide as an Active Biomolecule Proinsulin C-peptide was thought to be an inert species whose only purpose was for proper binding of insulin; however, now C-peptide is believed to be an important, biologically active molecule that can improve some of the complications associated with diabetes mellitus. In addition, type-1 patients who are able to maintain C-peptide levels have fewer complications and better glycemic control. The goal of this project was to gain a better understanding of the biochemical mechanism of action of C-peptide by identifying cellular proteins in varying types of tissue cell lysates capable of binding to the proinsulin C-peptide. This experiment was performed via creating C-peptide affinity beads by attaching C-peptide to NHS magnetic beads via the peptide’s N-terminus. Once attached, cellular lysates of varying tissue types were incubated with the affinity beads. Any bound proteins are eluted, separated by reducing SDS- PAGE, digested with trypsin, and identified via Protein Mass Fingerprint spectra obtained from the MALDI-TOF mass spectrometer. We were able to successfully identify several proteins from mouse kidney, liver, and heart tissue lysates that were isolated with the C-peptide affinity beads.
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