General Biochemistry 4115
General Biochemistry 4115 4115
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This 3 page Class Notes was uploaded by Thomas Salazar on Sunday September 18, 2016. The Class Notes belongs to 4115 at Virginia Polytechnic Institute and State University taught by Dr. Richard Helm in Fall 2016. Since its upload, it has received 12 views. For similar materials see General Biochemistry in Biochemistry at Virginia Polytechnic Institute and State University.
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Date Created: 09/18/16
Note set #2 – week 4 General Biochemistry PROTEIN ISOLATION and CHARACTERIZATION Proteins can be salted out of a sample with ammonium sulfate CHROMATOGRAPHY: “Running a mixture of protein(s) through a column with the intent of differentiating by various means o Different types are defined by if they isolate proteins using particle size, fractionation speed, pressure o Size exclusion chromatography is based on molecule size o Ion exchange, ect… SDS-PAGE: inactivates proteins, lose quaternary structure! “coats” all proteins with a negative charge heat proteins to inactivate and loosen bonds alkylate disulfide bonds block off disulfide cysteines with bulkier compounds run analysis based solely on molecular mass (proteins are now all negative so charge is irrelevant). o Band Identification of SDS-PAGE 1. Cut bands out, put in test tube 2. Remove the stain from the gel 3. Block thiols and dehydrate 4. Rehydrate with trypsin Endoproteinases to hydrolyze peptide bonds 5. Peptides release from gel, run mass spec against a database of peptides ISOELECTRIC FOCUSING o Proteins are loaded on a gel that has an immobilized pH gradient built in o Proteins migrate based on pI, then stop o Hydrophobic membrane proteins will precipitate out when reaching their pI o 2-D analysis: Isoelectric trial + SDS-PAGE = charge + mass analysis CHROMATOGRAPHY TYPES o Size-exclusion: apparatus includes pump column, UV detector, fraction collector Maintains quaternary structure! Column has specifically sized pores Larger molecules don’t fit in pores go through column faster Smaller molecules interact with pores go through column slower o Ion-exchange Column material is charged +/- Cation exchange columns interact with positively-charged proteins Anion exchange columns interact with negative-charged proteins o ions “stick” to the column, so based on the pKa of the protein, you can customize the chromatography to bind only certain proteins at certain pH’s o After the target protein(s) are isolated in the column, adding a salt solution “washes out” all ions in the column. o Affinity Chromatography Ligand is attached to column beads binds selectively to specific proteins Note set #2 – week 4 General Biochemistry Process: add proteinwashelute with imidazole Most common type is the HIS tag: Adding several Histidine to the terminus of a protein sequence WESTERN BLOTTING: o SDS-PAGE o Transfer proteins to nitrocellulose membrane o Block the rest of membrane with non-antibody proteins wash o Add antibody #1 to bind target proteinswash o Add antibody #2; binds to #1, and allows for chemical rxns. on #2 that can act as chemical “handles.” Quantitative western blotting uses pixelated digital analysis of western blots, based on intensity of bands normalized against “unchanging” proteins such a beta-actin or beta-tubulin and standards for comparison. HEMOGLOBIN Proteins have dynamic shapes They have microenvironments that can affect pKa and ligand binding activity Non-covalent interactions can influence proteins function and form The effects of allosteric binding can be modeled using technology Hemoglobin is used in mammalian O deliv2ry, CO cyclin2, and other molecular transport o Erythrocytes = red blood cells = ~25% of the cells in a human body o Hemoglobin is not an enzyme, it is a transporter protein can bind a total of 4 2 molecules also carries CO2, NO, and some other molecules has a “sigmoidal” affinity curve, due to allosteric effects and varies based on the amount of cooperativity occurring in the molecule o Myoglobin stores O in 2uscular environments has a mono-peptide structure, and holds only 1 O m2lecule however, it has a stronger affinity for oxygen than hemoglobin Cooperativity of Heme groups: O b2nding to a heme group moves the Iron atom closer into the ring, which pulls on HIS 87. The resultant flexing of the molecule puts the protein from a “tense” T-state, into a “relaxed” R-state. The flexing of the heme group gives subsequent heme groups higher affinity for O 2 Oxy-hemoglobin (Hb-O2), is a much stronger acid than deoxy-hemoglobin (deoxy- Hb) o The following rxn. occurs in the presence of excess acid Hb-O 2 H deoxy-Hb + O 2 SO the things that favor deoxy form: Note set #2 – week 4 General Biochemistry o Acidic environments reduce Hb’s affinity for binding oxygen. This is known as the Bohr effect o CO H 2,Cl , 2,3-BPG 2,3-BPG binds in the cavity inside the tetramer of Hb This binding makes it harder for oxygen to bind to the heme group CO r2acts to form carbonic acid, creating more acidic environment o When it encounters an energy-consuming environment (working muscles), Hb- O 2avors the release of O a2 the site HEMOGLOBIN VARIATIONS o Sickle cell anemia Abnormal shaped crescent blood cells Comes from a single amino acid mutation GV Impinges circulation, but creates a higher resistance to malaria o Nitric Oxide (NO) Neurotransmitter, derived from arginine Extremely high affinity for Hb; however, it reacts with CYS92 thiol group in Hb before it reacts with heme Mechanism for transport not well understood Binds to form other S-nitrothiol groups o A 1C Formed by glycating a heme group Acts as a primary indicator/marker for diabetes
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