Introduction to Biochemistry BIOC\*2580 Fall 2024 Lecture Slides PDF

Welcome to Introduction to Biochemistry BIOC*2580 Fall 2024 How familiar are you with organic structures, bonding and functional groups? A. Not at all B. Weak C. Average D. Very familiar E. Don’t know An Organic Chemistry primer...

Welcome to Introduction to Biochemistry BIOC*2580 Fall 2024 How familiar are you with organic structures, bonding and functional groups? A. Not at all B. Weak C. Average D. Very familiar E. Don’t know An Organic Chemistry primer: Complete this quiz to review the commonly found organic chemistry functional groups https://forms.office.com/r/g2CSBy8wfB OR  Molecules we study in biochemistry ▪ __________________ ▪ sugars, amino acids, nucleotides, carboxylic acid derivatives ▪ act as building blocks for macromolecules ▪ __________________ ▪ Proteins – chains of amino acids ▪ Polysaccharides – chains of simple sugars ▪ Nucleic acids – chains of nucleotides A macromolecule myoglobin is a protein that stores O2 in muscle tissue C N O S  How large is a protein molecule? ▪ Most proteins: 10,000 to 100,000 g mol-1 ▪ Protein size is expressed in _______________ 1 Dalton (Da) = 1 g mol-1 (mass of H atom) 1 kDa = 1000 g mol-1 ▪ Myoglobin is 16.5 kDa – small protein ▪ P-glycoprotein is 170 kDa – large protein The building block principle of macromolecular structure building blocks repeated simple bonds Understand the building blocks Understand the macromolecules Proteins are made of amino acids amino acids amide bonds Understand amino acids Understand proteins  Proteins are made of amino acids ▪ linear chains of amino acids ▪ linked by peptide bonds (type of amide bond) ▪ Each protein has: ▪ unique sequence of different amino acids ▪ a well defined size and structure ▪ Proteins have diverse functions including: ▪ catalyzing reactions (enzymes) ▪ forming complex subcellular structures  Basic amino acid structure ▪ Each amino acid has an _________ group and a ____________ group ▪ Each amino acid has a different ____________ ▪ 20 different amino acids are found in proteins (see Lehninger)  The amino acid residues in proteins are L- stereoisomers The central carbon atom of all amino acids except one (glycine) is ______ COO- COO- Hence, there are two possible + + NH3 C H H C NH3 stereoisomers. They are designated as D and L CH3 CH3 versions and are mirror images L-Alanine D-Alanine (_______________) of each other The amino acids in proteins are found in the L configuration  Peptide bonds ▪ _______________ involves removal of H2O from the units being linked ▪ ______________ regenerates the original carboxylic acid and amino group ▪ The C=O group of the amide is the point of weakness allowing H2O attack  Large numbers of amino acids can be linked together to form a peptide chain ▪ The combination of different side chains R1, R2, R3, etc gives each protein its unique properties ▪ there are 153 amino acids in myoglobin (16.5 kDa) ______________ – a chain with many amino acids, usually a complete protein Greek poly = many ______________– a chain with a few amino acids, usually a fragment Greek oligo = a few  The number and composition of amino acids in proteins is variable Since the elements of water is removed from amino acids when forming peptide bonds, the amino acids in a protein chain are known as amino acid “______________”. The average molecular weight of amino acids in proteins = ~128 Molecule of water removed to form peptide bond = 18 Therefore, the average molecular weight of an amino acid residue in a protein is: 128 – 18 = _______ The number of amino acid residues in a protein can be estimated by dividing the molecular weight of the protein by 110  Amino acid side chain structure ▪ Carbon atoms of the amino acid core are H 2 1 identified by Greek letters H3N+ C COO- ▪ The  is the central backbone atom 3 b CH2 ▪ The is the first atom of the side 4  CH2 chain, the -carbon is the second, etc ▪ Functional groups may be linked to different 5 d CH2 core atoms: 6  CH2 ▪ -amino NH3+ ▪ -amino  The 20 natural amino acids ▪ Amino acids share a common backbone, but differ in the side chain ▪ You need to be able to reproduce the structures of the amino acids and know their 1- and 3-letter codes ▪ You need to be able to associate particular properties with each amino acid ▪ ______________ ▪ ______________ ▪ _________________________ ▪ Amino acids can be grouped according to structures or by similar properties  6 with very non-polar side chains 5 with moderately non-polar side chains 4 with polar but uncharged side chains 3 with positively charged side chains (very polar) 2 with negatively charged side chains “inverted pyramid” 65432 (very polar) 6 with very non-polar side chains 5 with moderately non-polar side chains 4 with polar but uncharged side chains 3 with positively charged side chains (very polar) 2 with negatively charged side chains (very polar)  Try the Amino Acids quiz ▪ See how many you can identify. Try as many times as you like. ▪ Click the link ▪ https://forms.office.com/r/sRtjRXTKFs ▪ Or scan the QR code  Test your knowledge of the Single Letter Codes of amino acids Click the link below or scan the QR code Enter your answers using UPPER CASE Letters ▪ https://forms.office.com/r/NB8EcQnws7  Amino acids with very non-polar side chains Ala, Val, Leu, Ile, Met, Phe ▪ The side chains are dominated by hydrocarbon, and consist only of C-C and C- H bonds ▪ Hydrocarbon is _____________ and _______________ (or water avoiding) Ala (A) Val (V) Leu (L) Ile (I) Phe (F) Met (M) (Fenylalanine)  Polar and non-polar properties ▪ Polarity is a consequence of atoms having different electronegativity or tendency to hold bonding electrons O>N>S>CH ▪ Atoms with similar ________________ share bonding electrons equally, H : e.g. C-C, C-H, and are non-polar H : C :C : ▪ Pairs of atoms with different electronegativity distribute bonding H electrons unequally – more electronegative atoms such as O or N get greater than 50% share, and this leads to _________________________and polar bonds _ δ- O : H δ+ + : : C O: C O: : δ- N : H δ+  Moderately non-polar: Gly, Cys, Pro, Tyr, Trp ▪ Glycine has single H atom as side chain, not enough to be very non-polar ▪ Hydrophobicity is related to the number of Gly (G) CH, CH2 or CH3 groups present Pro (P) ▪ Cysteine contains the slightly polar SH Cys (C) group ▪ Proline is unique because the side chain links to -N as well as to -C. The polar N moderates the non-polar hydrocarbon. ▪ Tyrosine has a single polar group that partly offsets the very non-polar benzene ring. Tryptophan behaves similarly. Trp (W) Tyr (Y) (tWiptophan)  Amino acids with polar uncharged side chains Ser, Thr, Asn, Gln ▪ Serine and threonine have side chains that include the polar hydroxyl group - OH (simple alcohol) ▪ Asn and Gln both contain the polar amide group ▪ These side chain groups do not gain or lose H+ in aqueous solution at pH 7, so they are uncharged ▪ All four side chains act as good ________________donors or acceptors Ser (S) Asn (N) Gln (Q) Thr (T) (AsparagiNe) (Qlutamine)  Hydrogen Bonds ▪ Hydrogen bonds are electrostatic attractions between a H- bond __________ and an ____________ Donor = Highly polar R1O-H - - :OR2 Acceptor = an –OH or –NH groups d− d+ electronegative atom with an are good H-bond available lone pair of donors R1O-H - - :NR2 electrons, such as O or N R1N-H - - :NR2 R1N-H - - :OR2 ▪ The hydrogen bond ( - - ) is about 5-10% as strong as a covalent bond, enough to make molecule R1 stick loosely to R2 but not to form a permanent link ▪ H-bonds are directional - stronger if donor and acceptor line up with one another  Positively charged side chains His, Lys and Arg ▪ These side chains contain weak bases that gain H+ (become _____________) and so are positively charged in aqueous solution at neutral pH ▪ Charge makes them ______________, overriding the non-polar hydrocarbon chain His (H) Lys (K) Arg (R) (K-The letter before L) (aRginine)  Negatively charged side chains Asp and Glu ▪ Side chains have carboxylic acid groups R-COOH that lose H+ (become deprotonated) at neutral pH ▪ When deprotonated these are described as carboxylate groups R- COO- ▪ Carboxylate groups are negative and also very polar ▪ Asp side chain: -CH2-COO- ▪ Glu side chain: -CH2-CH2-COO- Can you identify how Asp and Asn, and Glu and Gln are related to one another? Asp (D) Glu(E) (asparDate) glutamatE Homework questions 1. Shown below are the amino acids Ala and Ser. Join them together through a peptide bond to form the dipeptide alanylserine (Ala-Ser). H H +H 3N C COO- +H 3N C COO- CH3 CH2 OH Homework Questions ▪ Complete these questions to test your understanding of amino acids and their properties ▪ https://forms.office.com/r/HR2JzXFSWP Answer for question 1 on the previous slide H O H H3N+ C C N C COO- CH3 H CH2 OH See also next slide Alanylserine: The condensation reaction H O H H H3N+ C C OH :N C COO- CH3 H CH2 OH H2O H O H H3N+ C C N C COO- CH3 H CH2 OH See also slide #10 “Peptide Bond”  Amino Acids can act as acids and bases ▪ The __________ and _______________ groups of amino acids and the side chains of _________ amino acids can act as acids and bases ▪ These groups will gain (protonate) or lose (deprotonate) H+ depending on availability of H+ in solution ▪ Normal biochemical processes occur close to pH 7 (physiological pH is 7.0-7.4) ▪ pH expresses availability of H+: pH = - log10[H+] at pH = 7, [H+] = 10-7 M ▪ The Henderson-Hasselbalch equation relates pH, pKa and the state of ionization of a given group [deprotonated] pH = pKa + log * Know this formula [protonated] 35 The ionization state of the α-carboxylate group at pH 7 (ie. Is the α- carboxylate protonated (COOH) or deprotonated (COO-) at pH 7?) Typical pKa = 2.4 ± 0.5 for amino acid α-carboxylate. At pH 7: Dep Dep pH = pK a + log = 10(7.0 – 2.4) Pro The vast majority of molecules exist Pro Dep at pH 7 as carboxylate __________, Dep = 40,000 NOT as carboxylic acid __________. pH − pKa = log Pro Pro Dep [COO− ] 40,000 10pH −pKa = = Pro [COOH] 1 36  The ionization state of the α-amino group at pH 7 (α-amino when deprotonated = NH2; when protonated = NH3+) Typical pKa = 9.6 for amino acid -amino groups. At pH 7.0: Dep Dep pH = pK a + log = 10(7.0 – 9.6) Pro Pro = 10-2.6 The vast majority of molecules Dep pH − pKa = log Dep exist as protonated ________ Pro = 0.0025 Pro rather than as _______ Dep [NH2 ] 0.0025 10pH −pKa = = Pro [NH3+ ] 1 37  Charged state of amino acids at neutral pH ▪ The correct structure to represent an individual amino acid at neutral pH is A dipolar ion (Zwitterion) ▪ But when the amino acid is part of a peptide chain, the -amino groups and -carboxylate groups are linked as ______________________ (except for the ones at the N and C termini, which are also NH3+ and COO- at pH 7) 38 Which amino acids have ionizable _______________? ▪ There are __________ amino acids with side chains that have acid base properties. ▪ Similar to α-amino and α-carboxylate groups, these too will gain (protonate) or lose (deprotonate) a H+ based on the pH Aspartate Glutamate H H H H + + H3 N C COO- H3 N C COO- + H+ + H3 N C COO- + H3 N C COO- + H+ Charge on side CH2 CH2 CH2 CH2 chain: COOH COO- CH2 CH2 Protonated: _____ H Tyrosine H COOH COO- + + Deprotonated: ____ H3 N C COO- H3 N C COO- + H+ H Cysteine H CH2 CH2 + H3N C COO- + H3N C COO- + H+ **Know these CH2 CH2 structures and SH charges OH O- S- The side chains of Arg, His, and Lys are __________ when deprotonated and ______ when protonated H Histidine H + H3N C COO- + H3N C COO- + H+ H Arginine H CH2 CH2 + - H3 N C COO + H3 N C COO- + H+ C NH C NH CH2 CH2 C NH+ C N CH2 CH2 CH2 CH2 H Lysine H + NH H3N C COO- + H3N C COO- + H + NH C NH2+ CH2 CH2 C NH NH2 CH2 CH2 NH2 CH2 CH2 **Know these structures and charges CH2 CH2 NH3+ NH2 The value of pKa tells you where in the pH scale a group undergoes deprotonation e.g. glutamate H ▪ A molecule can have several -amino + −carboxylate NH3 C COO- ionizable groups pKa = 9.5 pKa = 2.1 CH2 ▪ Each group has its own pKa value CH2 ▪ Value of a pKa depends on its COO- −carboxylate chemical context pKa = 4.1 ▪ an amino acid will have a slightly different pKa when it is part of a NH CH C NH CH C NH CH C peptide chain O CH2 O O CH2 COO- −carboxylate pKa = 5  Ionization of glutamic acid as pH increases from 0 to 14 ▪ Starting with glutamic acid at very low pH, all three functional groups are fully protonated. ▪ As we raise the pH, [H+] becomes less available, so deprotonation is more likely to occur. ▪ Each group undergoes a transition as pH shifts from 0 to 14, starting ~1 pKa = 2.1 pKa = 4.1 Physiological pH unit below its pKa and almost complete by ~1 unit above its pKa. 2.1 ▪ When pH = pKa, 50% of the group is in the protonated form and 50% is in 9.5 9.5 the deprotonated form ▪ Note that once a group deprotonates, 4.1 4.1 it remains that way for the rest of the pH range The ionization state of a group at a given pH depends on its pKa value. Amino Acid pKa (Amino acid in a polypeptide) Aspartate 4.0 Glutamate 5.0 Histidine 6.5 Lysine 10.2 Arginine 12.5 Cysteine 8.5 Tyrosine 10.0 N-Terminus (α-Amino) 9.5 (9.6 -in a free amino acid) C-Terminus (α-carboxylate) 2.5 (2.4- in a free amino acid) DO NOT HAVE TO MEMORIZE pKa VALUES  How to assess the state of ionization of a functional group ▪ If pH is one unit or more below the pKa, the group is fully ________________ ▪ If pH is one unit or more higher than pKa, the group is fully _______________ ▪ If pH is equal to pKa, the group is 50% deprotonated and 50% protonated ▪ If pH is less than one unit away from pKa, a calculation may be needed to determine the exact state pKa Protonated Deprotonated Calculate pH 1unit 1unit  Charge of an ionized group depends on the functional group ▪ The relationship of pH and pKa tells you whether a group is _____________ or _________________, NOT whether it is __________ or ___________ ▪ Groups that ionize on O or S atoms are neutral when protonated, and negative when deprotonated (e.g., side chains of Asp, Glu) -OH -O- + H+ -SH -S- + H+ ▪ Groups that ionize on N are positive when protonated, and neutral when deprotonated (e.g., side chains of Arg, Lys, His) -NH3+ -NH2 + H+ ▪ There is no group that goes from positive to negative when it becomes deprotonated!! 45  Homework questions Test your understanding of the charge of different ionized groups https://forms.office.com/r/CQ6ZFpiUxx  Calculating the exact state of ionization (and therefore the charge) of a group when pH is less than 1 unit away from pKa ▪ Histidine side chain has pKa = 6.5 ▪ At pH = 7, the major form will be deprotonated His, but some protonated His [HisH+] is present STEP I: Calculate the ratio of [Dep]/[Pro] [His] pH = pKa + log [HisH+ ] [His] 7.0 = 6.5 + log [HisH+ ]  [His] 0.5 + = 10 = 3.2 Protonated HisH+ Deprotonated His  [HisH ] (Charge = +1) (Charge = 0)  [Dep] If = 3.2, what fraction of histidine is [Dep] & what fraction is [Pro]? [Pro] The number of Histidine molecules = 1 Let fraction deprotonated (Degree of deprotonation) = α ∴ The fraction protonated = 1 - α [Dep] α ∴ The ratio = = 3.2 STEP II: Calculate [Dep] and [Pro] [Pro] 1−α fractions Then, solve for α: α = 3.2 (1 – α) α = 3.2 – 3.2 α α + 3.2α = 3.2 4.2α = 3.2 3.2 α= = 0.76 4.2 ∴ Fraction of histidine in [Dep] = 0.76 ∴ Fraction of histidine in [Pro] = 1- α = 1 - 0.76 = 0.24 How do we determine the charge of histidine at pH 7.0? ▪ To determine the charge of histidine side chain at pH 7.0, we multiply each fraction with its associated charge (histidine side chain has a charge of 0 when deprotonated and +1 when protonated) ________________________________ The charge on the side chain of histidine at pH 7 = ________ STEP III: use the charges associated with each fraction to find out the overall charge. How can a functional group have a partial charge? At pH 7, histidine is 76% deprotonated and 24% protonated Molecules exchange H+ millions of times per second A given His molecule is protonated 24% of the time (charge of +1) and deprotonated 76% of the time (neutral) Averaged over time, charge on His at pH 7 is: (0.24 x +1) + (0.76 x 0) = +0.24  Summary: Calculating partial charges FOLLOW THESE 3 STEPS TO FIND THE PARTIAL CHARGE OF ANY IONIZABLE GROUP. IT IS IMPORTANT THAT YOU KNOW THE CHARGES ASSOCIATED WITH EACH FUNCTIONAL GROUP IN THEIR PROTONATED AND DEPROTONATED STATES (E.G. GLU SIDE CHAIN IS 0 WHEN [PRO] AND -1 WHEN [DEP]; HISTIDINE SIDE CHAIN +1 WHEN [PRO] AND 0 WHEN [DEP] ETC. Homework questions https://forms.office.com/r/F2J1ibLNG3 OR  Amino acid analysis ▪ Amino acid analysis helps to determine protein structure ▪ Analysis involves two processes: 1. ____________ of a mixture into components 2. ___________ of the components of interest ▪ can be qualitative (tells you what is present) ▪ can be quantitative (tells you how much is present) ▪ can be preparative (separated components can be recovered for further experiments) Partition chromatography is an important method for separating components of a mixture ▪ Particles of solid are chosen with a specific property, e.g. silica gel has HO-Si-OH groups that can hydrogen-bond to polar amino acids ▪ _____________________ P P P ▪ Liquid solvent or buffer flows past the particles and is non-polar ▪ _____________________ N Column N Chromatography ▪ Amino acids exchange (partition) between phases N ▪ polar amino acids P spend more of their time hydrogen bonded to silica and move slowly ▪ non-polar amino acids N spend more time in solvent, and move almost as fast as solvent Amino acids are identified by the volume of buffer needed to move each through the column- Elution Volume Concentration of amino acids Fraction N N N P P Collection is measured in each test tube and the results are graphed To elute more polar amino 12 acids, progressively polar 10 mobile phase is run through 8 [Concentration] the column 6 Compounds can be identified 4 by their characteristic 2 _____________ 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Elution Volume (ml) 58  Another format: Thin layer chromatography ▪ Silica gel is spread in a thin layer on a plastic sheet ▪ Samples are applied near the lower edge ▪ The lower edge is placed in solvent ▪ As solvent soaks up the sheet, different components of sample move with the solvent at different rates ▪ The highest point reached by solvent is the ______________ ▪ Each amino acid can be identified by its characteristic ______________RF ▪ Very polar amino acids have low RF, non-polar amino acids have high RF  How are amino acids detected? ▪ Amino acids are colourless, and samples may be 10-6 to 10-10 moles ▪ They can be detected by adding ninhydrin which reacts with primary and secondary amines ▪ Gives intense purple colour (10-8 moles detectable), or yellow colour for proline ▪ Spray ninhydrin onto TLC plates, or add to amino acid solution, and heat ▪ Colour intensity is proportional to quantity of amino acid, and can be measured ▪ Alternative is fluorescamine, giving yellow fluorescence under UV light (10-10 moles detectable)  Different types of chromatography ▪ Ion exchange chromatography separates on the basis of _________ ▪ Uses charged resins as stationary phase ▪ Cation exchanger resins contain negative groups, which bind positive molecules ( _______ ) - Cation Exchange Chromatography ▪ Anion exchanger resins contain positive groups, which bind negative molecules ( _______) – Anion Exchange Chromatography ▪ Elution is by: ▪ Competition with a high ion concentration (usually NaCl), which displaces the amino acid from the resin ▪ Changing the pH to alter the charge on the amino acid, so it no longer binds to the resin  At pH 2.5, a-amino groups exist as NH3+ while a- pH 2.5 pH 2.5 carboxylate groups exist as 50% COO- & 50% COOH giving the amino acid an overall positive charge -- - -- - Side chains can also contribute to the charge - -- - -- - - - - The exact value of overall charge depends on specific --- -- - pKa values of the various groups in each amino acid - -- - -- - - - - -- - -- - Size of net charge determines how tightly each amino - -- - -- acid binds - - - - -- - -- - High Na+ present in elution buffer first displaces weakly - -- - -- bound amino acids. As [Na+] is increased, more - - - - tightly bound amino acids are progressively displaced Alternatively, pH may be increased to eliminate the positive charge on the amino acid, so it no longer binds to the resin  Separation of amino acids by ion exchange Amino acids are detected and their concentration measured in buffer coming out of the column The volume of buffer needed to move a given amino acid from the top to the bottom of the column is the elution volume Elution volumes are often compared relative to a common standard, such as Ala or Leu Elution volumes are characteristic for each amino acid, and allow them to be identified Separation of ________ from complex mixtures ▪ Proteins are derived from natural sources such as microbial cultures, plants, or animal tissues such as liver ▪ The cells are broken open to release the proteins into a solution – ______________ (Fig 3-17a) ▪ Extracts may contain thousands of Cation Exchange Chromatography different proteins ▪ Separation by ion exchange is based on charge differences among proteins depends on the relative number of Asp + Glu (negative) versus His + Lys + Arg (positive) in each protein, and on pH ~65% of all proteins are negatively charged at pH 7  Charge differences among peptides and proteins e.g. at pH 7 Ala–Asp–Leu–Gly–His–Gln–Tyr–Cys–Ile–Glu–Lys–Ser-Thr –1 0.24 –1 +1 due to side chains +1 --------------------------------due to N- and C- terminal --------------------------------------- –1 Net charge = +1 – 1 +0.24 – 1 + 1 – 1 = –0.76 Peptides and proteins can show large differences in charge Ion exchange is frequently used to separate protein mixtures pKa values chart: ----------------------------------------------------- Side chain pKa values of amino acids: Asp 4.0 Glu 5.0 His 6.5 Cys 8.5 Tyr 10.0 Lys 10.2 Arg 12.5 α-amino group = 9.6 α-carboxylate group = 2.4 Average N-terminal amino group has pKa = 9.5 Average C-terminal carboxylic acid group has pKa = 2.5 ----------------------------------------------------------------------------------------- pKa values will be given at the exam. You are not required to memorize them. 66  Affinity Chromatography A chemical group known as a ________ is covalently attached to the beads in the column Proteins that have an affinity towards the ligand bind tightly to it Other proteins move faster down the column and are eluted from the column The bound proteins are eluted by the addition of high concentration of salt (weakens the binding between the ligand and the proteins) or ligand (competes with attached ligands) e.g., an ATP binding protein can be isolated by attaching a molecule that resembles ATP to the beads Genetic engineering can fuse a “Tag” to the target protein allowing purification by affinity chromatography Tag = A peptide or protein that binds a ligand with high affinity and specificity that is fused to the gene encoding target protein Table 9-3 Commonly Used Protein Tags Tag protein/peptide Molecular mass Immobilized ligand (kDa) Protein A 59 Fc portion of IgG (His)6 0.8 Ni2+ Glutathione-S- 26 Glutathione transferase (GST) Maltose-binding 41 Maltose protein β-Galactosidase 116 р-Aminophenyl-β-D- thiogalactoside (TPEG) Chitin-binding domain 5.7 Chitin Metal Affinity Chromatography Clusters of His in a protein bind tightly to Ni2+ or Co2+ Ni2+Ni2+ Ni2+ His Ni2+ tagged 6-8 extra His residues are fused to the target protein at N- or C- Ni2+Ni2+ protein Ni2+ terminus (___________) Column is made up of chelating resin containing Ni2+ His-tagged proteins binds tightly to the Ni2+ resin The bound protein is eluted by adding _________ (structure similar Imidazole to His side chain) to the buffer Imidazole out-competes His-tag, and protein no longer binds to the Ni2+Ni2+ Ni2+ Ni2+ column Ni2+Ni2+ Ni2+ High degree of purification in one step May affect the properties of the protein The tag can be removed once the protein is isolated Separating proteins on the basis of size ▪ Different proteins or peptides can vary widely in size (number of amino acids) ▪ Gel filtration or molecular exclusion chromatography allows separation of proteins on the basis of ________ ▪ Beads of polymeric gel - a loose network of polymer with many water-filled pores. ▪ Protein molecules can enter the pores if they fit ▪ Larger proteins are excluded from the pores ▪ Proteins of intermediate size may enter some of the pores ▪ Proteins separate by size; larger proteins elute first, smaller proteins elute later  Gel filtration can be used to measure the molar mass of proteins -Sample is compared to proteins of known size ▪ Measure elution volume of proteins of known mass ▪ Elution volume Ve is the volume of buffer needed to move a protein from top to bottom of column ▪ Elution volume is a linear function of 4 log molar mass (negative slope) log molar mass (kDa) Thyroglobulin read log 3 692 kDa ▪ Then measure elution volume of molar 2 serum albumin unknown protein and project back to the mass 66 kDa log mass axis 1 ▪ Find antilog to determine the molar mass elution volume measure Ve for of unknown (Ve) (ml) unknown protein ▪ Alternately, find Mr by using coordinates Problem set 2 to derive equation for straight line y=mx+b Proteins can be separated and characterized by electrophoresis - movement of charged molecules in an electric field Does not contribute to purification as structure is commonly affected by electrophoresis Can visualize and characterize purified proteins can be used to estimate: number of different proteins in a mixture degree of __________ of a mixture isoelectric point approximate molecular weight Rate of movement depends on size, _________ and charge Polyacrylamide gel is easily prepared in lab, has right porosity for proteins 10 kDa -1000 kDa Electrophoresis of proteins is generally carried out in gels made up of the polymer polyacrylamide A typical gel is 5-15% polymer and 90- 95% water with conductive buffer Separated proteins are visualized by adding a dye such as Coomassie blue which binds to proteins. In SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) the protein is pre-treated with the detergent sodium dodecyl sulfate (SDS) ▪ SDS binds to proteins and partially unfolds them – all proteins have a similar rod-like shape ▪ The sulfate moieties of the SDS molecules confer a large net negative charge on the protein swamping out the native charge ▪ All proteins have a uniform ________________________ ▪ separation is based strictly on ______________ ▪ Smaller peptides migrate faster than larger ones ▪ By comparing with the positions to which proteins of known molecular weight (standards) migrates to in the gel, the molecular weight of an unknown protein can be estimated Isoelectric focussing: separation based on isoelectric point of proteins Isoelectric point, pI: pH at which the net charge deprot’d -ve charge on a protein is ________ at high pH, protein is deprotonated, moves toward the + electrode e- as it passes through gradient of decreasing pH, becomes protonated, net -ve charge decreases prot’d when the net charge = 0, protein stops moving +ve charge each protein in a mixture has a different isoelectric point, so they become separated along the pH tube of gel with pH gradient gradient from one end to the other Two-dimensional gels: Separation of complex samples Combines isoelectric focusing and SDS electrophoresis Can separate individual proteins in very complex mixtures Soluble proteins of Schistosoma mansoni  Mass spectrometry provides a way to identify proteins ▪ A protein is vaporized by laser beam, yielding charged protein particles ▪ Particles travel toward the detector ▪ Velocity depends inversely on the mass of the particle (larger = slower) ▪ The _______________ to the detector yields a very accurate mass measurement (5 significant figures of accuracy) ▪ We can compare the mass of the protein with a _____________ of proteins of known mass, allows identification Purification of a specific protein from a crude mixture requires the use of multiple separation methods Method 1 Method 2 Method 3 After each purification step, how do you know how successful the method has been in purifying the specific protein? The functions of proteins (e.g. enzymes) can be used to detect and quantify them in unseparated protein mixtures Enzymes are a common target of protein purification Enzymes are proteins that catalyze chemical reactions Enzyme (Protein) Substrate Product After each method, the amount of the target enzyme present in the mixture can be determined by measuring its ___________________ (moles of substrate converted to product per unit time) Enzyme Activity = The _____________ of enzyme present in a solution 1 enzyme unit is defined as the amount of enzyme that convert 1 μmol of substrate to product per minute (1μmol /min) at 25˚C under optimal conditions of measurement  Specific Activity Specific Activity = The number of enzyme units per milligram of total protein (μmol min-1mg-1) Enzyme Activity Specific Activity = Total Protein Purification of an enzyme from a mixture of proteins increases the specific Activity which reaches a maximum and remains constant once the enzyme is pure. Unpurified After several When pure and impure samples of same enzyme mixture purification are compared, specific activity is a measure of steps enzyme ______________ e.g. If a pure enzyme has a specific activity of 10 µmol min-1 mg-1 sample with a specific activity of 2 µmol min-1 mg-1 is 20% pure. Less pure sample includes impurities making up 80% of its mass Enzyme Activity vs Specific Activity With step-by-step purification of a protein: 1. Enzyme Activity _______________ (since successive separation methods leads to the loss of some of the protein of interest) 2. Total protein ____________ (goal is to remove unwanted proteins) 3. Specific Activity _____________ (loss of unwanted (non-specific) protein is greater than the loss in activity) Once a protein has been successfully isolated… 1. Further purification steps do not lead to an increase in specific Activity 2. Only a single protein species is detected (e.g. SDS- PAGE)  Homework Questions https://forms.office.com/r/hFxNNgmegE OR Worked solutions for some questions on this question set are posted on Courselink under Weeks 1 & 2 content.