Biochemistry 1 Lecture Notes PDF

Bio-organic Toolbox - Biochemistry 1 2 Biochemistry 1 Topics Carbohydrates (Starch isolation and hydrolysis) Proteins (Characterization of isolate casein from milk) Enzymes (Starch isolation and hydrolysis) Sources Lectures Books: - Biology: A Gl...

Bio-organic Toolbox - Biochemistry 1 2 Biochemistry 1 Topics Carbohydrates (Starch isolation and hydrolysis) Proteins (Characterization of isolate casein from milk) Enzymes (Starch isolation and hydrolysis) Sources Lectures Books: - Biology: A Global Approach, Campbell et al., 12th ed. - Organic Chemistry, Bruice et al., 8th ed. - Principles of Biochemistry, Lehninger et al., 7th ed. (recommended) 3 Program 4 How is your grade determined? 5 Tips for studying Make notes; lectures are used as a guideline Use other sources for extra explanation: Google, Khan Academy, etc. (check your sources!) Refresh earlier theoretical concepts Don't fall behind Ask for help from peers or teacher 6 Week 1 lecture 1: key principles biochemistry macromolecules (monomers and polymers) carbohydrates casein experiment lecture 2: polysaccharides 7 Two key principles biochemistry 1) Always relate in vitro (in glass) experiments to in vivo (in the living organism) 2) Macromolecules are constructed of monomers synthesis requires energy (anabolism) break down releases energy (catabolism) 8 in vitro vs in vivo DNA restriction analysis Evaluating phenotype of a genetically modified plant Enzymatic assay Effect of medicine on mice CO2 uptake of isolated chloroplasts Cancer cells in a culturing tube Artemia in well plate exposed to metal 9 Example: problem with relation in vitro and in vivo Discovery of DNA Polymerase Polymerization rate of DNA in vitro was too slow to account for the doubling time Arthur Kornberg KhanAcademy 10 Two hypotheses 1) Researchers discovered the wrong enzyme 2) The activity of the enzyme changed when studied outside the cell 11 Hypothesis verification 1) Researchers discovered the wrong enzyme 2) The activity of the enzyme changed when studied outside the cell The enzyme that was discovered was not a DNA Polymerase, but a DNA repair enzyme Moral: always relate work back to cellular reality. Ask whether in vitro studies make sense within the context of the living cell (in vivo) 12 Synthesis and breakdown 13 Synthesis and breakdown of macromolecules Vecteezy.org 14 15 Four classes of organic compounds 16 Terminology sugars and peptides 1 monomer: amino acid, monosaccharide 2 monomers: dipeptide, disaccharide 3 monomers: tripeptide, trisaccharide 4 monomers: tetrapeptide, tetrasaccharide Etc 2-20 monomers: oligopeptide, oligosaccharide >20 monomers: polypeptide, polysaccharide 17 The synthesis and breakdown of polymers Monomers a building blocks of polymers Polymer synthesis occurs by condensation reaction, specifically a dehydration reaction Polymers are disassembled to monomers by hydrolysis. Enzymes are macromolecules that speed up the dehydration/hydrolysis processes (by lowering the activation energy) 18 19 20 Protein monomer = amino acid α What's so acidic about the amino acid? 21 Synthesis of peptide 22 The diversity of polymers Each cell has thousands of different kinds of macromolecules Macromolecules vary among cells of an organism, vary more within a species, and vary even more between species An immense variety of polymers can be built from a small set of monomers There are approximately 1300 different enzymes in a human cell 23 Four classes of organic compounds 24 Sugars = carbohydrates Monosaccharides have molecular formulas that are usually multiples of CH2O Glucose (C6H12O6) is the most common monosaccharide in nature Monosaccharides are classified by - The location of the carbonyl-group (C=O; aldehyde vs ketone) - The number of carbons in the carbon skeleton 25 KhanAcademy 26 Aldoses C4 epimers 27 Ketoses 28 Glucose enantiomers 29 Enantiomers: D and L monosaccharides 30 Hexose enantiomers, diastereomers and epimers D-talose and L-allose differ only at one assymetrical carbon 31 Epimers 32 Forms of glucose In aqueous solutions many sugars form rings​! 33 34 Glucose D-Glucose is the most common monosaccharide in nature Serves as fuel (energy) Often stored in cells as polymer 35 36 Preparation casein experiment 3 steps: Isolation of casein from skimmed milk (done by Lydia Sloof) Determination of protein concentration (Bradford) Visualization of casein with SDS-PAGE 37 38 Casein Dalgleish 2011 39 Casein Bovine milk: casein and serum (whey) proteins. Casein is 80% (m/m) of the total protein in bovine milk Bovine casein: αS1 (~38%) αS2 (~10%) β (~34%) κ (~15%) Food and non-food applications 40 Casein workflow milk 41 Casein precipitation Calcium caseinate Isoelectric point (PI) pH Aggregation + precipitation > precipitate contains the most protein 42 Casein - isolation 43 11-11-2024 Agarose gel electrophoresis 44 11-11-2024 SDS-PAGE 45 11-11-2024 SDS-PAGE - practice Type of casein Mol. wt. (kDa) αs1 26 αs2 25 β 25 K 15 46 SDS-PAGE - practice Samples: P1 (30 mg/mL) P2 (20 mg/mL) SN2 Standards: a-casein k-casein Bovine serum albumin (BSA) Bio-Rad All Blue protein standard 47 Bradford assay Colorimetric protein assay Coomassie Brilliant Blue G-250 dye Standard curve (BSA) Measure at 595 nm Incubate before measuring! https://www.bioagilytix.com/blog/utilizing-bradford-assay-for- protein-concentration-calculation/