Soto Biochem 9/8, 9/10
Soto Biochem 9/8, 9/10 CHEM 351
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This 9 page Class Notes was uploaded by Kayli Antos on Friday September 11, 2015. The Class Notes belongs to CHEM 351 at Towson University taught by Ana Soto in Fall 2015. Since its upload, it has received 54 views. For similar materials see Biochemistry in Chemistry at Towson University.
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Date Created: 09/11/15
Biochem Soto Fall 2015 Amino Acidsz Peptides And Proteins can t 2 0 Polar Uncharged R Groups quotIL These R groups are hydrophilic due to their functional groups which are able to have hydrogen bonds with water it Cys has a modest polarity but it is also a weak acid which enables it to make weak hydrogen bonds 1 Asn and Gln are amides of two other amino acids aspartate and glutamate quotlt Cys can be oxidized to cysteine which is a dimeric amino acid in which two Cys are connected Via a disulfide bond which is covalent and highly hydrophobic Name 3 Letter Abbreviation 1 Letter Symbol Structure Serine Ser S 00 H3Nt C H Threonine Thr T 00 H3N4 H Cysteine Cys C Asparagine Asn N COO Glutamine Gln OO o Positively Charged R Groups His is the only one of the twenty common amino acids With a side chain Whose pKa is near 7 This is Why His can be a proton donor or accepter in many enzyme catalyzed reactions Name 3 Letter Abbreviation 1 Letter Symbol Structure Lysine Lys K 00 H3NLCH Arginine Arg R H3N Histidine His o Negatively Charged Groups Asp and Glu have second carboxyl groups in their R groups Name 3 Letter Abbreviation 1 Letter Symbol Structure Aspartate Asp D COO H3N H Glutamate Glu E o Uncommon Amino Acids quotIL Some other amino acids are formed by a modification after protein synthesis There are others that are not a part of proteins and have other functions 0 Amino Acids As Acids And Bases L The 0c amino and CL carboxyl act as weak acids and bases as well as some of the R groups quotIL A simple amino acid one Whose R group does not act as an acid or base is a diprotic acid When fully protonated and can donate a proton from its amino group NH3 and carboxylic acid COOH it When one of these simple amino acids is dissolved in water it exists as a zwitterion with a pH of 7 with the amino group being protonated and the carboxyl group being deprotonated as they are in these notes 0 Amino Acid Titrations it it The four steps for working an amino acid titration problem are 1 Draw the molecule in its fully protonated form 2 Deprotonate it in pKa order the carboxyl group will lose its proton first because it has a lower pKa than the amino group 3 Write the pKas on the equilibrium arrows 4 Write the charges and OH equivalents that correspond with each stage of deprotonation The titration curve for a simple amino acid looks like a typical diprotic acid titration curve The carboxyl group has a lower pKa than the amino group and will lose its proton first usually around a pH of 2 The amino group will lose its proton around a pH of 9 The pH where the net charge is zero where only the zwitterion eXists in solution is called the isoelectric point pI At lower pHs there is a net positive charge At higher pHs there is a net negative charge To find the pI average the two pKa values that the zwitterion eXists between To fully deprotonate a simple amino acid two equivalents of OH are needed 0 More Complicated Amino Acid Titrations it it When an amino acid has an ionizable R group the titration curve is more compleX and has three stages related to three pKas The nature of the R group acidic or basic is re ected in the isoelectric point It When there are three pKas choose the two that bracket where the amino acid becomes neutral a zwitterion and average those to find the pI Peptides And Proteins quotll The covalent bond that links amino acids is called a peptide bond L Proteins can be comprised of a single chain or multiple chains of amino acids bonded covalently or non covalently it Many proteins have other chemical components called prosthetic groups IL A peptide bond is formed when one amino acid loses a hydroxide ion from its carboxyl group and the other amino acid loses a hydronium ion from its amino group and the carbon from the carboxyl bonds to the nitrogen from the amino quotIL H3Nt CIH 1TI JH COO39 R1 H R2 it The yellow molecule lost a hydroxide ion and the green molecule lost a hydronium ion The blue bond is the peptide bond L The reaction to form a peptide is not favorable The hydrolysis of a peptide is favorable Ionization Behavior quotIL Peptides only have one free 0L amino group and one free 0L carboxyl group These along with some R groups contribute to acidbase behavior quotll The pKas of acidicbasic R groups in a peptide can be different than the pKas of the free amino acid because the R groups in the chain can in uence each other s pKas The pKas of the CL amino groups and 0t carboxyl groups can also change due to the separation between them Protein Puri cation it There are many ways to separate proteins by using their many different properties such as size charge and binding properties quotll The proteins of interest is usually in tissue or microbial cells so the first step of separating the proteins is to break the cells open it After opening the cells the next step is salting out which involves selectively precipitating some proteins by adding salt to the solution It can be isolated by centrifuging separating and redissolving The protein can then be resuspended dialyzed and then purified via column chromatography 0 Column Chromatography it A column contained a porous solid material that has the necessary chemical properties A buffered solution is poured through it it Ion Exchange Chromatography gt The column contains positively or negatively charged beads depending on the charge of the protein The affinity of the protein for the charged beads depends on pH and salt concertation gt After the solution runs through the column the beads can then be washed with a salt solution to remove the desired protein if Size Exclusion Chromatography gt The column contains beads with pores that will trap the protein as it runs through while larger molecules will not be able to fit in the pores and will elute faster gt The protein does not get stuck in the pores it will elute without an intervention The protein will just elute after the rest of the solution it Affinity Chromatography gt The column contains beads which contain a chemical group ligand which binds the protein and prevents it from eluting from the column gt To remove the protein from the beads there are a few options One way is changing the pH so the protein will change shape and be unable to bind to the ligand Another way is to add a solution of abundant ligand which the protein will bind with and wash out of the column 0 Electrophoresis Is used not for purifying proteins but for analyzing whether or not the purification process was a success How far the protein travels through the gel depends on its size charge and shape The ratio of a proteins velocity and electrical potential can be eXpressed as u the electrophoretic mobility This value can also be eXpressed as the ratio of the proteins charge Z and shape which is related to f the frictional coefficient 11 g SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SDS is a detergent which binds to proteins in an amount that is proportional to their molecular weight Since SDS is charged this binding infers charges as well as mass It also works to unfold the protein PAGE is the specific gel type that is used After the gel is run the proteins are dyed so they can easily be seen The most common dye used it called coomassie blue Some other electrophoretic procedures are gt Isoelectric focusing proteins migrate across a gel which has a pH gradient The protein stops when it hits the pH that matches its p1 gt 2D electrophoresis combines isoelectric focusing and SDS electrophoresis Protein Structure it it it There are four levels of a proteins structure The primary structure of a protein is the sequence of amino acids The secondary structure of a protein is the repetitive arrangement of amino acids into reoccurring structural patterns like 0L helixes and 3 sheets These structures are stabilized by hydrogen bonds The tertiary structure of a protein is the 3D folding of polypeptides The quaternary structure of a protein is the arrangement in space of the polypeptide subunits This is only for proteins with multiple subunits o Sequencing Proteins il The way that we currently determine the amino acid sequence of a protein is by deriving it indirectly from the DNA The traditional method still proves useful for other purposes L A summary of the traditional method is gt Determine the terminal amino group and react it with FDNB 1 uoro 24 dinitrobenzene gt Use acid hydrolysis to determine the amino acid content gt Remove the disulfide bridge and cleave the chain into smaller polypeptides with enzymes gt Sequence proteins by using Edman degradation gt Use the fragments created by reacting the original protein fragment with two different enzymes to determine the sequence 0 Edman Degradation L L il Removes only the amino acid on the amino terminal Under basic conditions the peptide reacts with phenylisothiocyanate PTC by the PTC binding to the amino terminal amino acid The nonaromatic carbon of PTC forms a double bond with the amino nitrogen The sulfur of PTC then attacks the carboxylic carbon of the terminal amino acid breaking it off from the rest of the peptide and forming a ring with the PTC and terminal amino acid This derivatized amino acid is then extracted stabilized and identified This process can be repeated up to about 40 times Any fragments larger than that should be cleaved into smaller fragments before this process 0 Divide Proteins Into Smaller Peptides il Some proteases and chemical reagents are used to cut peptide bonds at known amino acid sequences Specific enzymes can be chosen for particular proteins based on their amino acid composition 0 Remove Disulfide Bonds il There are two ways to irreversibly remove disulfide bonds by reducing cystine il OXidation with performic acid which creates a sulfur trioxide group sulfur double bonded to two oxygens and then single bonded to a third oxygen which has a negative charge il Reduction with dithiothreitol DTT which adds a hydrogen to each sulfur This can then by carboxymethylated by iodoacetate which removes the hydrogen on the sulfur and adds CH2 COO instead
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