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Biochemistry F12 notes.docx

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University of Guelph
BIOC 2580

Biochemistry F’12 Notes Molecules we study in biochemistry Small molecules  sugars, amino acids, nucleotides, carboxylic acid derivatives  act as building blocks for macromolecules Macromolecules  Proteins – chains of amino acids  Polysaccharides – chains of simple sugars  Nucleic acids – chains of nucleotides How large is a protein molecule?  Most proteins: 10,000 to 100,000 g/mol  Protein size is expressed in kiloDaltons (kDa) o 1 Dalton (Da) = 1 g/mol (mass of H atom) o 1 kDa = 1000 g/mol  Myoglobin is 16.5 kDa – small protein  P-glycoprotein is 170 kDa – large protein Proteins are made of amino acids  linear chains of amino acids  linked by peptide bonds (type of amide bond)  Each protein has: o unique sequence of different amino acids o a well-defined size and structure  Proteins have diverse functions including: o catalyzing reactions (enzymes) o forming complex subcellular structures Basic amino acid structure Figure 1Basic structure of an amino acid  Each amino acid has an amino group and a carboxylate group  Each amino acid has a different side chain R  20 different amino acids are found in proteins Peptide bonds  Condensation involves removal of H2O from the units being linked  Hydrolysis 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 Figure 2 Condensation reaction to form peptide bond 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)  Polypeptide – a chain with many amino acids, usually a complete protein o Greek poly = many  Oligopeptide – a chain with a few amino acids, usually a fragment o Greek oligo = a few Amino acid side chain structure  Carbon atoms of the amino acid core are identified by Greek Letters  The α-carbon is the central backbone atom  The β-carbon is the first atom of the side chain, the γ- carbon is the second, etc Figure 3 Identification of carbon atoms with greek letters  Functional groups may be linked to different core atoms: o α-amino o ε-amino 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 nonpolar and hydrophobic (or water avoiding) Polar and non-polar properties  Polarity is a consequence of atoms having different electronegativity or tendency to hold bonding electrons o O> N > S > C≈ H  Atoms with similar electronegativity share bonding electrons equally, e.g. C- C, C-H, and are non-polar  Pairs of atoms with different electronegativity distribute bonding electrons unequally – more electronegative atoms such as O or N get greater than 50% share, and this leads to unbalanced charges and polar bonds Moderately non-polar: Gly, Cys, Pro, Tyr, Trp  Glycine has single H atom as side chain, not enough to be very non-polar o Hydrophobicity is related to the number of CH, CH2 or CH3 groups present  Cysteine contains the slightly polar SH group  Proline is unique because the side chain linkα -N as well as tα-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. 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, so they are uncharged  All four side chains act as good hydrogen bond donors or acceptors Hydrogen bonds are electrostatic attractions between an H-bond donor and an acceptor  Highly polar –OH or –NH groups are good H-bond donors  An acceptor is an electronegative atom with an available lone pair of electrons, such as O or N  The hydrogen bond (- -) is about 5-10% as strong as a covalent bond, enough to make molecule R 1tick loosely to 2 but not to form a permanent link. Positively charged side chains His, Lys and Arg  These side chains contain weak bases that gain H+ (become protonated) and so are positively charged in aqueous solution at neutral pH  For example, the lysine side chain   Charge makes them very polar, overriding the non-polar hydrocarbon chain Negatively charged side chains Asp and Glu  Side chains have carboxylic acid groups R-COOH that lose H+ (become deprotonat-d) at neutral pH o When deprotonated these are described as carboxylate groups R-COO o Carboxylate groups are negative and also very polar -  Asp side chain: -CH 2COO -  Glu side chain: -CH 2CH2-COO Free amino acids are weak electrolytes due to their amino and carboxylate groups  Normal biochemical processes occur close to pH 7 (physiological pH is 7.0-7.4)  Groups such as carboxylate and amino groups gain or lose H+ depending on availability of H+ in solution  pH expresses availability of H+:  The Henderson-Hasselbalch equation relates pH, pKa and the state of ionization of a given group Figure 4 The Henderson Hasselback Equation Charged state of amino acids at neutral pH  The correct structure to represent an individual amino acid at neutral pH is NH3-CHR-COO -  But when the amino acid is part of a peptide chain, theα -amino groups and α -carboxylate groups are linked as uncharged amide bonds x-NH-CHR-CO-x The value of pKa tells you where in the pH scale a group undergoes deprotonation  A molecule can have several ionizable groups  Each group has its own pKa value  Value of a pKa depends on its chemical context  an amino acid will have a slightly different pKa when it is part of a peptide chain 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 unit below its pKa and is almost complete by ~1 unit above its pKa How to assess the state of ionization of a functional group  If pH is one unit or more higher than pKa, the group is fully deprotonated. - o Carboxylate groups with pKa=2.4 exist as –COO not –COOH at pH 7  If pH is equal to pKa, the group is 50% deprotonated and 50% protonated  If pH is one unit or more below the pKa, the group is fully protonated + o Amino groups with pKa= 9.6 exist as -NH ,3not -NH at2pH 7  If pH is less than one unit away from pKa, a calculation may be needed to determine the exact state  The relationship of pH and pKa tells you whether a group is protonated or deprotonated, NOT whether it is positive or negative  Groups that ionize on O or S atoms are neutral when protonated, and negative when deprotonated  Groups that ionize on N are positive when protonated and neutral when deprotonated  There is no group that goes from positive to negative when it becomes deprotonated!! Calculating the exact state of ionization of a group at a given pH  Histidine side chain has pKa= 6.5  At pH = 7, the major form will be deprotonated His, but some HisH is present If ratio [His] / [HisH+] = 3.2, what percentage of the total histidine is deprotonated? We define α, degree of deprotonation, as the fraction of histidine molecules that are deprotonated, i.e. [His]/[total] The fraction protonated is 1 – α The ratio [deprotonated] / [protonated] 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 Averaged over time, charge on His at pH 7 is 0.24 x (+1) = +0.24 Amino acid analysis  Amino acid analysis helps to determine protein structure  Analysis involves two processes: o Separation of a mixture into components o Detection 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) 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 o Stationary phase  Liquid solvent or buffer flows past the particles and is non-polar o Mobile phase  Amino acids exchange between phases o polar amino acids P spend more of their time hydrogen bonded to silica and move slowly o non-polar amino acids N spend more time in solvent, and move almost as fast as solvent Thin layer chromatography 1. Silica gel is spread in a thin layer on a plastic sheet 2. Samples are applied near the lower edge 3. The lower edge is placed in solvent 4. 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 solvent front  Each amino acid can be identified by its characteristic relative mobility RF  Very polar amino acids have low RF(A) ,non-polar amino acids have high RF(C) Another common format: column chromatography Volume of buffer needed to move a compound through the column is the elution volume. Compounds can be identified by their characteristic elution volume. How are amino acids detected?  Amino acid-6are c-10urless, and samples may be 10 to 10 moles  They can be detected by adding ninhydrin which reacts with primary and secondary Figure 5 Picture representation of column chromatography 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 (1010moles detectable) Different types of chromatography  Ion exchange chromatography separates on the basis of charge o Uses charged resins as stationary phase o Cation exchanger resins contain negative groups, which bind positive molecules (cations) o Anion exchanger resins contain positive groups, which bind negative molecules (anions) o 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 3.5, α-amino groups exist as NH3 while α-carboxylate groups exist mainly as COO -  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  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 proteins from complex mixtures  Proteins are derived from natural sources such as microbial cultures, plants, or animal tissues such as liver  Extracts may contain thousands of different proteins  Separation by ion exchange is based on charge differences among proteins o depends on the relative number of Asp + Glu (negative) versus His + Lys + Arg (positive) in each protein, and on pH o ~65% of all proteins are negatively charged at pH 7 Charge differences among peptides and proteins  Peptides and proteins can show large differences in charge  Ion exchange is frequently used to separate protein mixtures Metal affinity chr
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