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by: Brady Spinka


Marketplace > University of Texas at Austin > Chemistry > CH 369 > FUNDAMENTALS OF BIOCHEMISTRY
Brady Spinka
GPA 3.98

Karen Browning

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Karen Browning
Class Notes
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This 15 page Class Notes was uploaded by Brady Spinka on Monday September 7, 2015. The Class Notes belongs to CH 369 at University of Texas at Austin taught by Karen Browning in Fall. Since its upload, it has received 19 views. For similar materials see /class/181866/ch-369-university-of-texas-at-austin in Chemistry at University of Texas at Austin.




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Date Created: 09/07/15
Water Chapter 2 Water The solvent of life Earth is unique in this solar system because of water Water sculpted Earth39s surface Water apparently also sculpted Mars surface but disappeared from the planet Water39s unusual properties allowed the evolution of life Water as a solvent allows the complex interactions of biomolecules Water comprises 70 of all organisms What is Water water contains one 0 and 2 H atoms each linked by single covalent bonds O is more electronegative pulls electrons from hydrogen so that the electrons are not shared equally in the bondpolar the bond angle in water is bent at 1045 degreesquotVquot shape methaneelectrons are shared more equally nonpolar carbon dioxidepolar but linear attraction of electrons by oxygen is equal on both sides Hydrogen Bonding of Water most unique propertyprobably essential for evolution of life forms H bonds with itself and other molecules N H OH SH may form hydrogen bonds with water or with other functional groups eg in DNA H bonds responsible for the thermal properties of water Hydrogen Bonds type of electrostatice force each water molecule can bond with 4 other water molecules water is both a H bond acceptor and donor water is actually a quotnetquot of molecules reason for the high boiling point and heat of vaporization energy needed to break the H bonds freezing causes the quotnetquot to become a rigid tetrahedral lattice leClConve1terInpuUBa7TJRqe4yhtml272010 60318 PM ET Water lattice breaks molecules move back closer together makes liquid water denser than solid ice oats allows aquatic life to survive in freezing temperatures Other Types of Biological Bonds Electrostatic or chargecharge interactions between oppositely charged molecules type of interaction van der Waalsshort range interactions in a protein or nucleic acid attraction of nuclei from one atom are attracted to electrons of another vice versa but if too close then repulsion occurs from electrons These types of interaction are important for macromolecular interactions proteins nucleic acids etc where many small interactions contribute to make a signi cant effect Why do I care about hydrogen bonds and other types of interactions between molecules Closer look 2A Did you take Tylenol or an antibotic recently All drugs target a protein and by inhibiting that protein s function we will learn much more about this later may reduce pain Tylenol or inhibit bacteria from growing a A biochemist probably found an inhibitor for that protein and then E designed many similar molecules with various functional groups e g F until finding one that interacts better with the protein a New functional groups often make small molecule bind tighter so it will work at a lower concentration with less side effects Example prozac contains a F group that changes the properties of the moleculetarget is a membrane transporter 5HT involved in serotonin transport Water is the Ultimate Solvent leClConverterInpuUBa7TJRqe4yhtm1272010 603 18 PM hydrogen bonds and dipole character make it the ideal solvent high dielectric contstantmeasure of ability to diminish electrostatic interactions substances that dissolve in water are hydrophilic quotwater lovingquot molecules that can form H bonds are water solubleamines esters etc molecules that are charged are water solubleNaCl KCl etc water surrounds the ions and align charges with opposite chargethe interaction with water is stronger than with ions so salt dissolves interactions between water and polar molecules are very strong some organic molecules that are non ionic but have polar properties are water solublephenols esters alcohols E Water biomolecules with groups that are ionized or form hydrogen bonds are easily dissolved e g sugars concentration of glucose in blood is 5 mM and water is 555 M 10000 water molecules for every glucose Molecules inside of a cell make a very dense solution that water molecules not shown intersperse in between the molecules to ll the spaces and coat molecules with a layer of water that prevents van der Waals contact and keeps a uid state in the cell What Water Cannot Solubilize nonpolar nonionic molecules e g hydrocarbons hydrophobic molecules tend to cluster hydrophobic interactions 0 oil slick in the Gulf 0 olive oil in your dressing H ydrophobicity is important too thermodynamically unfavorable to dissolve non polar substance in water water orients around molecule reduces the entropy of system water cannot formreform as many H bonds due to constraint nonpolar molecules quotclumpquot although thermodynamically unfavorable the quotreleasequot of the water molecules increases over all entropy hydrophobic effect is very powerful force in cell particularly for protein structurenonpolar amino acids form hydrophobic core excluding water from interior of protein 3 leClConve1terInpuUBa7TJRqe4yhtml272010 60318 PM Water Amphipathic Moleculeswant it both ways one end is hydrophilic one end is hydrophobic form particular types of structures in aqueous solutions micelles the ends orient together chemical basis for membranes in cells fatty acids sodium oleate also can form bilayers orienting nonpolar tails in to form hydrophobic layer heads to aqueous solvent membranes hydrophobic effect important in membrane structure keeps polar molecules from diffusing in out helps maintain ionic concentrationsvery different environmentes in vs outside of cell Extracellular Na is much higher Intracellular K is higherthe bilayer prevents diffusion of polar substancesbut we can have specific transport via membrane proteins What Else Can Water D0 Water can be both an acid and a base 0 it can transfer a proton to another water molecule 0 H20 H20 gtH3O OH39 Since water forms clusters it is more likely to be H5024r or H7 O34r species for simplification we can write the equation as o H20 gtH OH39 0 hydronium ion hydroxide ion REMEMBER protons never really exist as free ions in aqueous solutions protons are always in association with other water molecules and appear to jump from one molecule to anotherhigher mobility for protons than other ions leClConve1terInpuUBa7TJRqe4yhtm1272010 603 18 PM Dissociation of water from the law of mass action KaH OH1 H20 H20 lOOOgliter x l liter18 gliter 555 M KaX 555 M LHOH KW KW measured by conductivity at various temperatures 18 X 103916 18 x1016x 555 M HOH1X1039 14 K W Since H OH10 x 10397 M Water Pure Water is Neutral when the H OH are equal the solution is neutral when the H gt OH the solution is acidic when the H lt OH the solution is basic E Biochemical reactions are sensitive to pH biochemical reactions are catalyzed by enzymes enzyme are proteins most proteins lose their activity at extreme pH acid or base proteins are composed of amino acids amino acids have side chains that ionize at various pHs Next chapter you will learn about those aa 3 the pH of a weak acid or base at the mid leClConve1terInpuUBa7TJRqe4yhtml272010 603 18 PM REMEMBER THIS pH scale convenient method to express H ion concentration introduced by SPL Sorenson in 1909 to measure the acidity of water in brewing beer pH scale uses a logarithmic function Here is a link to my tutorial on and another link about pH logH neutrality pH 7 pure water values less than 7 are acidic values greater than 7 are basic biological reactions primarily occur within the range of pH 65 to 8 known as quotphysiologicalquot pH and logs can be confusing but try to understand the mathematical basis of the pH scale and what it means not just push the buttons on your calculator every pH unit represents a 10fold dilTerent in H Maintenance of constant pH in the cell is important bu ersuweak acids and bases do not completely dissociate in contrast to strong acids ie HCl remember acid is a hydrogen ion donor base is a hydrogen ion acceptor conjugate base equilibrium forms between the acid and conjugate base o HAltgtH A39 0 this equilibrium or tendancy to dissociate has a dissociation constant called Ka Ka Hll A391 HA this equation was described by Henderson in 1908 He discovered buffering power of C02 and applied the law of mass action subsequently Hasselbalch in 1916 applied Sorensen s terminology to Henderson s equation in logarithmic form pH pKa logA39 HA called the Henderson Hasselbalch equation the Ka of weak acids can be determined experimentally by several methods titration being the most common Water point in titration pKa note the reasonably at area of the titration curve around the midpointcalled the in ection point this is known as buffering capacity a buffer allows the addition of additional W or OH39 without a great change in pH buffering capacity maintains the pH within cells at a constant level during metabolism a variety of intermediates are produced that are acidiceg lactic acid during muscle exertion from glycolysis Criteria for a good buffer buffer capacity in desired range ie pKa high purity of compound water soluble stable to enzymes and water ie not destroyed nontoxic to enzymes form complexes with cations that are soluble not absorb light in the visible or UV range Important physiological buffers Intracelular phosphate and phosphocompounds H2PO439 lt gtH HPO4239 pKa682 these are not physiological pHs PO4339 El Blood Buffering System bicarbonate in blood and interstitial uid of vertebrates CO2 H20 ltgtH2CO3 lt gtH HCO339 leClConve1terInpuUBa7TJRqe4yhtml272010 603 18 PM phosphate also has two other pKa 214 and 124 but H3PO4 lt gt H H2PO439 lt gt H HPO4239 lt gtH Eli Non physiological buffers used in biochemistry Tris pKa 808 Hepes pKa 75 many other types with various pKas these are used in in vitro assays or tissue culture to maintain proper pH Other physiological buffers proteins can act as buffers e g hemoglobin histidine has a pKa near pH 7 the buffering capacity of hemoglobin is due to histidine more on this in Chapter 4 0 need a ratio of HCO339 C02 20 Water the dissociation of carbonic acid is very fast rewrite as C02 H20 ltgtH HCO339 carbonic anhydrase accelerates this process in vivo over a million fold Table 61 pKa 61 this would not appear to be a suitable buffer to maintain blood pH of 74 more than 1 pH unit on either side of the pKa Why is the bicarbonate system good for blood buffering H may be buffered by recombining with HCO3 and then C02 eliminated in lungs H HCO339 lt gt C02 H20 leClConve1terInpuUBa7TJRqe4yhtm1272010 603 18 PM HINT do this calculation for yourself using the HH equation Several medical conditions can result in lowering of blood pHmetabolic acidosis resulting in rapid breathing to remove H2CO3 in the form of C02quot treated by giving bicarbonate to soak up excess acid and aid in release as C02 Chapter 4 AMINO ACIDS clicker question 1 R can be any of 20 different groups pKa of the COOH group is N225 pKa of the NH2 group is N9lO at pH 7 in cell carboxyl is in conjugate base form COO39 amino is in conjugate acid form NH3 amphotericboth acid and base character a at pH 7 have equal and charges called zwitterions when electrically neutral R groups give each amino acid its unique properties acidic basic hydrophobic hydrophilic aromatic etc Amino Acid Structure the acarbon is asymmetric also called chiral carbonsee closer Non39PrOtem ammo auds look 4A p 91chirality can make a big difference in biological actrvr y acarbon has 4 different groups attached except for glycine whose R group is H there are two possible arrangements in space stereoisomersmolecules that differ only in their arrangement in space Disomers or Lisomers nonsuperimposable mirror imagesenantiomers biological molecules exist either as D or amino acids in proteins are in the L form Classes of amino acids aliphatic sulfur containing hydroxyl containing arom atic basic acidic amp amides eg Dalanine is found in bacterial cell walls neurotransmitters o dopamine serotonin and melatonin tryptophan precursor adrenalin phenylalanine precursor a glutamate Lthyroxine is a thyroid hormonetyrosine precursor Lornithine is an intermediate in metabolism histamine is one of the causes of allergy symptoms histidine precursor Aliphatic 0r Alkane Sulfur containing amino acids Met Cys Cysteine is weakly polar methionine is hydrophobic Met almost always is first amino acid to be translated in a protein AUG codonMet sulfur group very reactive important in enzymatic reaction Cys side chain has pKa 83 carries a net negative charge at basic pH Cys can form covalent disulfide bond by oxidation covalent bondimportant in tertiary structure of proteins Hydroxyl containing amino acids Ser Thr more hydrophilic waterlovin g R groups tend to be on surface of protein in contact with er are weakly polar due to OH group R h htrn1 1 Gly Ala Val Leu lle Pro Nonpolar R group not charged Hydrophobicas the side chain gets longer the more hyrophobic the amino acid becomes Tend to be buried within protein structure away from water Hydrophobic AA tend to be attracted to each otherinterface for proteinprotein interactions Important in protein struc Pro gets put in here by default it is different from most amino acr s Pro has a cyclic structure but aliphatic in character the ring makes it rigid affects tertiary structure of proteins Aromatic Amino Acids Phe Tyr Trp Phe is most hydrophobic along with Val Leu lle Nonpolar R group not charged Absorb light at 280 nm UV absorption used as a measure of protein concentration WarburgChristian method Chapter 4 OH group can be phosphorylated phosphorylation of proteins is very important mechanism for regulation of activit OH group Ser Thr site for attachment of carbohydrates Basic Amino Acids Lys Arg His Polar or Charged Hydrophilic on protein surfaces basic amino acids form ion pairs with acidic amino acids affect protein structure Lys Arg pKa NlO39 positive charge at physiological pH39 important in catalytic activity of enzymes His is the least basic pKa N7 0 functions as buffer His important in enzyme catalysis involving proton transfer because of its side chains p Important in protein structure Tyr side chain has pKa lOl The OH of Tyr can also be phosphorylatedregulation of enzyme activity particularly of certain types of cell receptors Acidic Amino Acids and Their Amides Asp Glu Asn Gln Hydrophilic on protein surfaces polar Glu Asp have acidic side chainsnegative charge at neutral pH the acidic amino acids tend to be on the surface of proteins Asn Gln amides forms of Asp Glu uncharged side chains but are polar and hydrophilic also found on protein surfaces Go to this site and pick the amino acids game for target practice clicker questions 234 The peptide bond the peptide bond is an amide linkage between acarboxyl and aam ino group by the elimination of water side chains occur on opposite sides of the polypeptide bond trans form is favored due to less steric interference peptide bond is rigid and at planar is two resonate formspartial double bond character because of this 13 of the bonds of a peptide backbone are unable to rotate freelym aj or role in influencing overall protein conformation rotational freedom limited to the acarbon and the C2 or Nl bond clicker question 5 6 78 Peptides are Important Messengers linked amino acids in a polypeptide are called residues 2 amino acidsdipeptide I 3 amino acidstripeptide etc Nterminusfree amino group Cterminusfree carboxyl group note the middle amino acids do not have amino and carboxyl to contribute to charge anymore hormones insulin signals feed statestore carbohydrate and fatty acids vassopressin antidiuretic to keep blood pressure constant oxytocin induction of labor and lactation HGH hum an grow 0111 one neurotransmitters enkephalins pain relieffound in brainopiates mimic structure and bind to receptors antioxidant glutathionereversible formation of a disulfide bond to scavenge oxidizing agents a oxidants are thought to cause dam age to cellular components DNA proteins leading to cancer Peptides can be sweet aspartame is a dipeptide of of aspartic acid and the methyl ester R h htrn1 Peptides can be antibiotics gramicidin A 2 molecules forms helix forces its way into a membrane contains N50 hydrophobic amino acids Chapter 4 of phenylalanine form a pore in memebrane transports ions into bacterial cells it is considered to be a w food additive for normal metabolism Disrupts the ion concentration and kill cells maybe harmful to persons with phenylkentonuria similar mechanism for other peptide antibiotics including the LaspartylL phenylalanine methyl ester defensins Magainin is a antibiotic peptide isolated from frogjkin Biological Functions of Proteins Proteins Biological Function depends on conformation enzymescatalysts for reactions nutrientstorage proteinscasein in milk gluten in wheat transport of molelculeshemoglobin39 lipoproteins contractilemotionmyosin actin structuralcollagen39 keratin defenseantibodies horm onesinsulin toxinsdiptheria39 enterotoxins bad E coli Globular Proteins water soluble compact hydrophobic interior hydrophilic surface enzymes receptors carriers hormones etc dynamic agents Fibrous Proteins water insoluble structural roles extended structure collagen tendons bone akeratin hair nails etc Nstatic agents clicker question 9 Erythropoitin protein hormone 165 aa glycoprotein produced in kidneys to stimulate bone marrow to make red blood cells increases amount of oxygen carried increases stamina for these patients cloned and manufactured in cultured cellsrecombinantbillion dollar product for Amgenalso subject of a nasty patent fight that Am en won used to treat anemia in chemo patients and dialysis patients second generation product now available that is partially modified with carbohydrate groupssupposed to be more active abused by athletes to increase perfomance by carrying more oxygen in blood due to more blood cellscan be very dangerous blood becomes more viscous especially if dehydrated test determines recombinant vs natural based on amount and type of carbohydrate present test is not as precise as other tests for steroids etc and may have a higher rate of false positive Levels of protein structure primary structurecovalent peptide bonds linear sequence of the amino acids secondary structurelocal conformations of backbone maintained by hydrogen bon s a two major types alpha helix and beta sheet tertiary structurefolded and compact structure the sum of the secondary structuresdomains active conformation a stabilized by hydrogen bonds hydrophobic effect chargecharge interactions sometimes covalent disulfide 0 regions that are far apart in the primary structure may be brought close together quaternary structureassociation of two or more protein chains to form a complex a chains may be identical or not a interactions are primarily in uenced by hydrogen bonds hydrophobic effect chargecharge and sometimes by disulfide bonds Denaturation Partial to complete unfolding of native conformation Denatured Protein Protein that has lost its native conformation El This image is rom the UKMS site created by Dr George Helmkamp Jr Primary Structure Amino Acid Compositionthe type and amouts of amino acids that occur in a protein a This is important for proper nutrition to obtain essential amino acids that hum ans cannot synthesize a must balance protein sources particularly plant sources of protein which tend to be low in certain essential amino acids Amino Acid Sequencethe order in which the amino acids occur in the N terminal to C terminal direction Secondary Structure the bulkier side groups eg trp have fewer allowed rotations glycine no side group will have fewer constraints proline has a very restricted bond angletends to disrupt secondary structure R h htrn1 Chapter 4 must have 45 residues with the N same bond angles to generate a regular secondary structure such as an alpha helix or beta sheet Linus Pauling and Robert Corey predicted for proteins based on Xray diffraction data derived from amino acids and peptides that the two atoms cannot be closer than van der Waals forces allow amide group must be planar and in trans configuration rotation only about the acarbon non covalent bonding was necessary to stabilize the structure Two proposed structures fit these predictions ahelix bsheet These are the 2 most common secondary structures in proteins The aHelix rigid rodlike structure righthanded coiled quotspringquot R groups extend away from the helix 0 helices may be amphipathicside chains on one side of helix may be hydrophobic in character while the other side is hydrophilic N 436 residues per tum of the helix Hbonds between amino and carboxyl of every 4th residue some R groups promote or interfere with helix M TWO types 0f b39SheetS formation glycine is too smallmakes it too flexible tends to 39 Parallelbah Strands g0 N I C disrupt helix formation antiparallelone strand is N ID C one is C ID Nthese are the most stable prolinerigid ring prevents rotation to form helix and cannot form intrachain Hbonds Proline is rarely found in ahelix but often at the end or beginning of ahelix mmLementbfbackbone clicker question 11 The Globular ProteinsStructure and Stability hydrophobic AA generally on the insidehydrophobic core hydrophilic AA generally on the outside in contact with water may have ionic interactions with other AA Mixtures of ahelix and bsheet Additional structures turn loops 0 loopsturns provide quotbridgequot between regions of ahelix and b sheet Domainsupersecondary structures recognizable combinations of ahelix andor bsheet in a compact A protein may have one or more domains A domain frequently can be removed from a protein and still perform its function Calcium binding nucleotide binding etc a Zn finger u Leucine zipper domains are often associated with a particular function R h htrn1 J Chapter 4 clicker question 13 Protein F oldingSeries of events each in uencing the next cooperativity of folding nucleationform ation of some alpha helix and beta sheets no actiVity structural consolidationfolding of helixes and sheets Tertiary Structure togethersome activity final rearrangements if needed modifications such as 3D arrangement of all the atoms to give it its functional form diSUI de b0nd5fuu activity domains helices sheets metal ions hydrophobic interactions disulfide bonds hydrogen bonding ionic attractions odifications to proteins that affect structure andor function include disulfide bonds sugars lipids phosphates binding prosthetic group eg heme NAD FAD Protein folding helpers enzymes to help with disulfide bond formationdisulfide isomerase enzymes to help with folding itselfuchaperones Sometimes denaturation reverSibleremove the offending substance or condition and protein refolds Chaperonms Example ribonucleasecatalyzes the hydrolysis of RNA into nucleotides 0 Heat denature it even autoclave and slow cool reforms the proper structure and is active quotchaperonesquot the protein chain to the proper conformation preventing folding from occurring too soonmembrane proteins or exported proteins Th quotkn H h V f I I preventing premature association with other proteinslarge 6 Fromm 0W5 W at 1F 5 Proper con OI39mathH IS complexes and assumes that conformation Sometimes irreversiblestays denatured and inactive t t d t f ld ATP d d t preven s aggrega ion an ass1s s in 0 ing epen en process a choker questlon 10 Demm ation of Folding is not a random process Heatincrease molecular vibrations disrupt structure pHualters some charges to disrupt Structure ribonuclease has countless conformations poss1ble I 1 M 1 u ts 1 1 1 and 1 if would take years to try all conformations detergent is charged SDSr takes less than a minute for a protein to fold urea guanidineform hydrogen bonds with protein disrupting other Hbonds also disrupt hydrophobic mercaptoethanolreduces disulfide bonds combined with urea to facilitate access to disulfide bonds Blueprint for folding must be contained within the primary sequencewe just do not fully understand it yet R h htrn1 Chapter 4 When folding goes wrong mad cows and prions TS E transmissible spongiform encephalopathies fatal neurodegenerative diseases incubation period is months to decades symptoms include dementia and loss of coordination described in 18th century in sheep scrapie the infected sheep would scrap off their wool shown to be transmissible in 1936 by injection of infected spinal cord material into a healthy sheep BSE madcow disease bovine spongiform encephalophypeaked in 1991 in England Chronic wasting disease CWD is endemic in deer and elk in the US and Canada How is it infectious What is the function of PrP Can it cross species lines How is it transmitted no known nucleic acid has been found associated with the infectious materia lead to theory by Stanley Preusner that the infectious particle is actually a proteinprion found that in infectious material is a protein present in the brain and other tissues PrPcnormal form soluble alpha helical protease sensitive attaches at cell surface lipid anchored glycoprotein PrPSCabnormal form insoluble beta sheet protease resistant accumulates intracellularly forming quotamyloid plaques Infectivity may be a small section of the protein that recruits other proteins to misfoldquotrecognition element may also provide species specificity unknown transgenic mice that have PrP knockout appear to be normal and are resistant to prion disease not known Hum an T SE Kuru quottrembling diseasequotdescribed in 1959 by Hadlow among the Fore people of New Guinea 6 ritualistic ingestion of deceased brain tissue Crutzfeldt Jacob diseasefamilial germline mutation fCJD infected hum an growth hormone spontaneous mutation infection from contaminated animal products these types of diseases are reported to occur in all mammals cat mink deer etc How does conversion take place from PrPc to PrPSC some how the structure of one prion is inducing a change in conformation of other protein molecules why is brain and central nervous system targeted if PrP is a non yes with difficulty It takes a very high dose to induce disease but once induced it is easily transmitted within that species some species specificity some prions may have more than one quotrecognitionquot element What can be done not knownmust be possible from diet or other methods genetic predisposition to the conformational change distinct mutants are known BSE onset Quaternary structure two or more polypeptide chains defined stoichiometry symmetry may be the same or different polypeptide chain R h htrn1 J essential protein Other protein misfolding diseases Alzheimer39samyloid beta Parkinson39s alpha synuc le in increase in beta sheet that lead to amyloid deposits drugs may be targeted to quotrecognitionquot elements early detectionW300 genes were identified in mice associated with may have blood test to look for these markers before symptoms appear and if new drugs are available may slow down progression of disease Why do globular proteins assume this type of association oligomers are more stable active sites may be formed between subunits Chapter 4 o sam e usually dimers or tetramers that would not exist otherwise a differenteach chain has a different function multienzyme complex minimize errors jn synthesis of long protein so contains several polypeptides than each catalyze a different reaction make more short ones chains held together by mostly noncovalent interactions principally hydrophobic allosteryquotquotcommunicationquot between protejn CffCCt bUt CleCiIOStath may play a T015 subunits by subtle changes in conformation Modifications of proteins Proteins have more than one conformation chemical modifications more in chapter 22 1p1d proteins are dynamic carbohydrate may have multiple conformations pH oxidation state binding phosphorylation partners quotf39 39 I J 39 A A latiuu all require a set of enzymes to carryout the modification Gel Filtration PFOteiIl Puri cation and AnalysisColumn separates on the basis of size not charge chromatography porous beadsthink of golf balls small molecules go into the holes and get trapped temporarily 39 a matrix is in a cylindrical homer large molecules are too large to enter the holes and pass on by buffer OWS through the matrix exclusion sizedepends on the size of the holes fractions are collected separation of biomolecules how long the molecules get trapped determines elution order large out first gt medium gt small out last choose the size of matrix for the separation needed Ionexchange chromatography animation in BB Af nity Chromatography animation in BB proteins have charges due to amino acid side groups column matrix has a ligand that specifically binds a bind to charged column matrix depending on their charge at a particular pH protein anionicnegatively charged phosphocellulose heparin sepharose S o eg ATPagarose sepharose specialty affinity columns for binding recombinant cationicpositively charged DEAEsepharose Qsepharose proteins with certain quottagsquot elute bound proteins from column based on charge and displacement by salt a 6XHis added atN or C terminusbinds Ni or pH column Electrophoresis method of separation of charged molecules SDS sodium dodecyl sulfate gelsan1mat10n in BB movement of charge particles in an electrical field SDS is a datagen amphipathjc has both electrophoretic mobility reflects both charge and sizeshape hydrophobic and hydrophilic characteristics Many kinds of electrophoresis usually have a solid medium e g paper number of SDS molecules bound is proportional to gel TLC plate the number of amino acids a gel electrophoresrs used to separate large molecules DNA RNA 1 molecule of SDS bound per N2 amino acids Fromm SDS overwhelms any inherent charges and a quotgelquot 15 Ilka Very Stlff JELLO effectively turns the protein into a polyelectrolyte acrylamrde forms polymers that can be crosslinked to varying SDS also disrupts tertiary Structure degrees in a chemical process the crosslinking determines the size of quotholesquot that the molecules pass through bmercaptoethanol is also included to reduce disulfide bonds and destroy intra chain crosslinking protein mobility in SDS PAGE is roughly proportional to protein size although abherant mobilities do occur based on amino acid composition larger or smaller than true molecular weight apply electrical current across the medium Anode and Cathode positively charged molecules move to the Cathode negatively charged molecules move to the Anode proteins depending on their charge will move in either direction Primary Sequence Determination Amino Acid Composition amino acid analysis is used to determine the composition of protein but does not determine the ORDER of amino acids R h htrn1 J Chapter 4 a use phenylisothiocyanate PITC to generate amino acid derivativesPITC absorbs in the UV range for easy detection 0 hydrolyze peptide bonds with 6 M HCl 0 se arate amino acid derivatives by HPLC amino acids elute at various times and can be identifiedquantiated complications asn gln converted to asp glu39 trp destroyed ser thr tyr suffer losses Amino Acid Sequencethe order in which the amino acids occur Edman developed in 1950 a method to remove a single aa from the Nterminus must be unblocked of a protein in sequential fashionEdm an degradation 0 PITC phenyl isothiocyanate is used to derivitize the free N terminus trifluoroacetic acid causes cleavage of the N terminal amino acid from the protein 0 acid treatment rearranges derivitized aa to stable PTH amino acid a o the PTH amino acid is separated by chromatography HPLC and identified remaining protein now has a new Nterminus that may be subjected to another round of degradation recovery only approximates 100 so can only get so far from the N terminus before background begins to come up a lucky to get 3050 amino acids most proteins are much larger than this Protein Sequencing Strategiesanimation will be shown in class remove any disulfide bonds by reduction with dithiothreitol or betamercaptoethanol determ ine N terminusCterminus o cyanogen bromide CN cleaves after m cut the protein into smaller pieces for sequencing with chemicals or proteases of different specificities o trypsm cleaves after arg or lys o chymotrypsin cleaves after aromatic amino acids sequence all the fragments using Edm an degradation overlap the sequencesmuch like a giant jigsaw puzzle Mass spectrometry may also be used for determining amino acid sequence Protein structure tutorials Protein Architecture at the Online Museum of Molecules O R h htrn1


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