Optimizing Digestion of Hemoglobin Using Chymotrypsin and Assessing Efficiency by Capillary Electrophoresis Amneek Randhawa and Dr. Golfam Ghafourifar Department of Chemistry, University of the Fraser Valley, 33844 King Rd, Abbotsford, BC, V2S 7M8 Goals • • Determine an optimal enzyme-to-substrate ratio to digest hemoglobin in its denatured and native form using free and immobilized chymotrypsin enzyme Using findings from the separation procedure of the capillary electrophoresis instrument to discover differences of crosslinking reagents—formaldehyde and glutaraldehyde, denatured vs. native hemoglobin, and free vs. immobilized chymotrypsin (CT) Enzyme to Substrate Ratio Digestion of Hemoglobin Introduction • The use of immobilized enzymes to digest protein substrates holds increased experimental advantage in proteomic studies1. Digestion Procedure: § Molecular weight: 64 kDa and an oxygen transport metalloprotein4 § Iron containing protein possesses four polypeptide chains, each specifically wrapped around a heme group4 “The Crystal Structure of Human Deoxyhaemoglobin at 1.74 A Resolution.” Research conducted investigated the efficiency of hemoglobin digestion in its native and denatured form using free and immobilized chymotrypsin enzyme. 1:1 vs. 100:1 DAD1 A, Sig=200,16 Ref=off (AMNEEK\FOR30009.D) mAU 14 37°C, 4 hours 12 Collect Supernatant Store at -20°C Immobilized CT Enzyme Chymotrypsin • Immobilized, or • Free + Hemoglobin • Denatured, or • Native 6 4 Peptide Mapping using CE room temp 3 hrs, room temp Centrifuge 3.7 hrs, room temp FA Particle min Figure 6 Denatured Hb (0.1733 mM) digested with GA/CT (0.001733 mM) 37°C, 4 hours *Required volumes added depending on ratio Store at -20°C 5 4 Free CT Enzyme 3 2 Denatured vs. Native Hemoglobin 1 0 Figure 7 Denatured Hb (0.325 mM) digested with Free CT (0.0325 mM) 5 10 15 20 25 min Figure 8 Denatured Hb (1.3 mg) digested with Immobilized CT/GA (1.3 mg) • Use of denatured hemoglobin allows for a more accessible substrate, resulting in a greater digestion efficiency • GA/CT 1:1 (E:S) ratio showed enhanced digestion efficiency compared to 100:1 • Crosslinking reagent glutaraldehyde portrayed better results for a digestion procedure compared to formaldehyde • Versatile and possessing greater effectiveness immobilized enzyme proved ideal digestion for proteomic studies over the use of free enzyme 9. Decant 10.Wash with PBS X3 11. Wash with H2X3 12. Add 200 μL H2O 13. Freeze enzyme particle GA Particle 6. Wash with 500mM NaCl X3 7. Wash with PBS X3 8. Freeze the particle Future Work Figure 2 Native Hemoglobin proteolysis Native hemoglobin (0.65 mM) digested with Free CT (0.0065 mM) at 37°C for 4 hours Immobilized agents and enzymes will be used to fabricate IMERs (immobilized enzyme microreactors). The IMER will later be coupled to a capillary electrophoresis instrument, allowing for the analysis of large biomolecules through on-line digestion of proteins. • Optimization of the IMER: • Increase/Decrease pressure • Increasing time of the digestion—increasing interaction between immobilized enzyme and denatured hemoglobin • Increase time of fabrication—immobilized enzyme interacts with the capillary for a longer period of time Glutaraldehyde vs. Formaldehyde 4. Discard supernatant 5. Wash particle with PBS X3 Denaturation of Hemoglobin DAD1 A, Sig=200,16 Ref=off (AMNEEK\FOR30009.D) DAD1 A, Sig=200,16 Ref=off (AMNEEK\GLU00001.D) mAU mAU Chymotrypsin immobilized with crosslinking reagents: Glutaraldehyde & Formaldehyde used to digest denatured hemoglobin 5 3 2 14 12 10 8 6 4 References 1 2 0 120 μL iodoacetamide (100 mM in water) 25 DAD1 A, Sig=200,16 Ref=off (AMNEEK\GLU00001.D) 4 1. 8 mg BSA in 800 μL ammonium bicarbonate 50°C containing 8 M 15 urea minutes 2. 120 μL dithiothreitol (45 mM in water) 20 Free vs. Immobilized Enzyme Figure 1 Denatured Hemoglobin proteolysis Denatured hemoglobin (0.65 mM) digested with Free CT (0.0065 mM) at 37°C for 4 hours 2. 78 μL FA 15 mAU 40 μL 1. 0.0325 grams CT in 1mL PBS 10 Conclusion 8. 200 μL of glycine 5. Wash particle with phosphate buffer X3 6. Wash with NaCl 7. Wash with phosphate buffer 0 The free CT electropherogram displays increased noise, with uncertainties as peaks are not definitive. Immobilized CT electropherogram has greater # of peaks which are more defined w/o noise GA/FA—crosslinking reagents 2 hrs 2 Figure 5 Denatured Hb (1.3 mg) digested with GA/CT (1.3 mg) Glutaraldehyde (GA) and Formaldehyde (FA) 4. Decant the supernatant 37°C for 4 hours 8 5 Advantages of Immobilized Enzymes: 1. Increased enzyme-to-substrate ratios1 2. Improved protein digestion procedures with little to no autoproteolysis1 3. Reusability of the immobilized enzyme particle1 1. 40 μL 1.3 mM CT 2. 78 μL GA 3. 297 μL Phosphate buffer • 1:1: Better digestion—smaller peaks and no noise • 100:1: no defined peak + noise 10 5 10 15 20 25 Figure 3 Denatured Hb. (1.3 mg) digested using Immobilized CT/ Glutaraldehyde (1.3mg) at 37°C for 4 hours Denatured Hb. Extensively used for the immobilization of enzymes, glutaraldehyde serves as a reliable crosslinking reagent. Figure 1.3 displays a 1:1 enzyme to substrate digestion. min Glutaraldehyde displayed greater digestion efficiency. This is supported by the presence of smaller peaks on the electropherogram for denatured hemoglobin digested using immobilized CT/Glutaraldehyde 0 5 10 15 20 25 Figure 4 Denatured Hb. (1.3 mg) digested using Immobilized CT/ Formaldehyde (1.3 mg) at 37°C for 4 hours Formaldehyde is used to trap the CT enzyme to support proteomic analysis and detection of interactions b/w substrate and enzyme3. Figure 1.4 displays a 1:1 enzyme to substrate digestion. min 1. 2. 3. 4. Ghafourifar, Golfam. “Research Interests.” Golfam Ghafourifar Lab, www.golfamlab.com/publications/. Perutz, et al. “The Crystal Structure of Human Deoxyhaemoglobin at 1.74 A Resolution.” J.Mol.Biol., www.rcsb.org/pdb/explore/jmol.do?structureId=4hhb&bionumber=1&jmolMode=HTML5. Hoffman, Elizabeth A., et al. “Formaldehyde Crosslinking: A Tool for the Study of Chromatin Complexes.” Journal of Biological Chemistry, 30 Oct. 2015, www.jbc.org/content/290/44/26404.full. “Metalloprotein.” Metalloprotein - an Overview | ScienceDirect Topics, www.sciencedirect.com/topics/ biochemistry-genetics-and-molecular-biology/metalloprotein.