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- study of the molecular basis of life
- first and foremost a chemical science - interdisciplinary science: organic chemistry, physical chemistry, physics, biology, medicine, microbiology, physiology
Three Principal Areas of Biochemistry 1. structural chemistry of the components of living matter
2. metabolism or the chemical reactions that occur in living matter
3. the chemistry of processes and substances that store and transmit biological information; molecular genetics
AMINO ACIDS -are the basic structural units of proteins To the -carbon of every amino acid is attached a hydrogen atom, a carboxylic acid, and a side chain.
.All -amino acids (excluding proline) consist of a carboxylic acid (-COOH). a H atom. The side chain (R-group) differentiates one amino acid from the other. an amino (-NH2). and a distinctive R group bonded to the -carbon.
Alanine Glycine Only the L-enantiomers are found in proteins. except glycine.All 20 amino acids. . contain an asymmetric carbon and have L and D enantiomers (exhibit chirality).
Ten Essential Amino Acids - the amino acids that human body can’t synthesize in adequate amounts
1. Arginine 2. Histidine 3. Isoleucine 4. Leucine 5. Lysine 6. Methionine 7. Phenylalanine 8. Threonine 9. Tryptophan 10. Valine
Limiting Amino Acids in Some Foods: • Wheat and grains – lysine, threonine • Peas, beans, legumes – methionine, tryptophan • Nuts, seeds – lysine • Leafy green vegetables - methionine
Classes of -Amino Acids:
1. Amino acid with aliphatic side chains (Gly, Ala, Val, Leu, Ile) - hydrophobicity of the amino acid increases as R group gets bigger - pro has also an aliphatic chain but its side chain is bonded to both the N and the -C atoms; has a 2o rather than a 1o amino group
. although met is more hydrophobic.2. Met) .Cys side chain can ionize at moderately high pH and oxidation can occur between the pairs of cys side chains to form a disulfide bond. Cys. Amino acid with hydroxyl. Thr.more hydrophilic than their aliphatic analogs.or sulfurcontaining side chain (Ser. .
Glu. Gln) . Asn.3. both contain a terminal amide in place of a carboxylate . usually called aspartate and glutamate because they are negatively charges at physiological pH . Acidic amino acids and their amides (Asp.asp and glu contains acidic side chains.asn and gln are the uncharged derivatives of aspartate and glutamate.
have very polar side chains which render them highly hydrophilic . Basic amino acids (His. depending on its environment .4. Lys.lys and arg are positively charged at neutral pH while his can be uncharged or positively charged. Arg) .
amino acids with aromatic side chains.phe and trp are highly hydrophobic.5. Tyr. Aromatic amino acids (Phe. Trp) . the aromatic ring of tyr that contains an OH group makes it less hydrophobic than phe . the delocalized pi-electron in the aromatic ring enable them to interact with other pi-systems and to transfer electrons .
Amino acid with aliphatic side chains Gly Ala Leu Val Ile .
Amino acid with hydroxyl.or sulfurcontaining side chain Ser Thr Cys Met .
Acidic amino acids and their amides Asp Asn Glu Gln .
Basic amino acids His Lys Arg .
Aromatic amino acids Phe Tyr Trp .
Hydrophobic Amino Acids . therefore.tend to repel the aqueous environment and.do not ionize nor participate in the formation of H-bonds . reside predominantly in the interior of proteins .
are often involved in the formation of H-bonds and are predominantly found on the exterior surfaces proteins or in the reactive centers of enzymes .Hydrophilic Amino Acids .tend to interact with the aqueous environment .
derived from the common amino acids and are synthesized by modification of the parent amino acid in a process called posttranslational modification HO NH2CH2CHCH2 OH -hydroxylysine (lysine) 4-hydroxyproline (proline) thyroxine (tyrosine) .Uncommon Amino Acids .
They contain both a basic amino group and an acidic carboxyl group.COOH R .Acid-Base Properties of Amino Acids Amino acids are difunctional. NH2 H .C .
+ H+ R-NH3+ ⇄ R-NH2 + H+ . R-COOH ⇄ R-COO.The -COOH and -NH2 groups in amino acids are capable of ionizing in aqueous environment (as well as the acidic and basic R-groups).
At physiological pH (around 7.4) the carboxyl group will be unprotonated and the amino group will be protonated. The carboxyl group (-COOH) is a far stronger acid than the amino group (-NH2). .
would be electrically neutral at pH 7.An amino acid with no ionizable Rgroup. This species is termed as zwitterion.g. glycine.4. . e.
have high melting points .Amino acids have many properties associated with inorganic salts: .are crystalline .are soluble in water but insoluble in hydrocarbons .
H+. Acts as the acid and donates the proton. Acts as the base and accepts the proton. in base solution. in acid solution. .Amino acids are amphoteric: can act either as acids or as bases. H+.
an amino acid zwitterion accepts a proton to yield a cation: H + H3O+ ⇄ + H2O .In aqueous acid solution.
In aqueous basic solution. an amino acid zwitterion loses a proton to yield an anion: H + OH.⇄ H + H2O .
2 equiv.Exercises: 1. Write an equation for the reaction of aspartic acid with: a. NaOH 2. Product of (a) + 2 equiv HCl . Phenylalanine + 1 equiv NaOH b. NaOH b. Complete and write the structural formulas for the following equations: a. 3. 1 equiv. Draw phenylalanine in its zwitterionic form. Product of (a) + 1 equiv HCl c.
.Isoelectric Points. pI .pI is the pH when the algebraic sum of all the charged groups present in an amino acid or protein is zero.pI of an amino acid depends on its structure . .pI values are not necessarily neutral (pH 7) because the –COOH groups are stronger acids in aqueous solution than the basic –NH2 groups.
5. in the pH range 5. .Amino acids with neutral side chains have pI values near neutrality.06.
Amino acids with acidic side chains have pI values at lower pH. Amino acid with basic side chains have pI values at higher pH. the pI is the average of the pKa's of the two most similar acids. . which suppresses dissociation of extra COOH. which suppresses protonation of the extra amino group.If additional acidic or basic groups are present as side-chain functions.
Example: Aspartic acid The similar acids are the alpha-carboxyl function (pKa = 2.0 .9)/2 = 3.9) pI = (2.1) and the side-chain carboxyl function (pKa = 3.1 + 3.
5 + 9.5) and the alpha-ammonium function (pKa = 9.0) pI = (12.Example: Arginine The similar acids are the guanidinium species on the side-chain (pKa = 12.0)/2 = 10.75 .
77 2.13 9.10 -amino 9.02 2.76 8.32 10.86 12.Amino Acid pKa Values Amino Acid Alanine Arginine Asparagine Aspartic Acid -carboxylic acid 2.18 9.17 2.47 9.80 9.00 4.25 9.07 6.78 9.01 2.87 9.04 8.35 1.48 Side chain Cysteine Glutamic Acid Glutamine Glycine Histidine Isoleucine 2.82 3.10 2.35 2.10 .05 2.
24 10.38 2.33 2.58 2.15 Threonine Tryptophan Tyrosine 2.11 10.29 9.74 8.72 .00 2.60 9.18 2.53 Phenylalanine Proline Serine 2.Amino Acid -carboxylic acid -amino Side chain Leucine Lysine Methionine 2.95 9.21 9.28 9.10 9.20 9.21 10.07 Valine 2.09 2.39 9.
0 b.0 2.Lys at pH 11.Cys d. His b.0 d. Draw the structure of the predominant form of each of the following: a.0 c. Gln .Exercises: 1. Ala at pH 3. Lys at pH 2. Asp at pH 6. Thr c. Give the pI values of the following amino acids: a.
Water is eliminated in the process. .Peptide Bonds Amino acids can be linked linearly by formation of covalent bond via a dehydration synthesis reaction between the -carboxyl group of the first amino acid with the amino group of the second amino acid.
.Formation of Peptide Bonds The bond formed is called peptide bond.
Peptide bond is metastable. boiling in strong mineral acid (6 M HCl) or when catalysts/ enzymes are present. . Hydrolysis is rapid only under extreme conditions e.g. 2.Stability of Peptide Bond 1. hydrolysis at physiological pH and temperature is exceedingly slow.
Dipeptide .example is aspartame or NutraSweetTM (L-aspartyl-Lphenylalanine). a sugar substitute Peptides -are also called amides (Why?) .a linkage of two amino acids .
the side chain – distinct variable part .Polypeptide – a linkage of amino acids from three to several dozen units Polypeptides are made of: 1. the main chain (backbone) – regularly repeating part 2.
.The linked amino acid moieties are called amino acid residues. The -yl ending indicates that the residue is an acyl unit (a structure that lacks the hydroxyl of the carboxyl group). Ex. glu-gly-ala-lys glutamyl-glycyl-alanyl-lysine. The amino acid residues are named by replacing the ending -ine or -ate to -yl.
Polypeptide sequences are written left to right from the N.to the C-terminus.) The amino end (N-terminus) is the beginning of a polypeptide chain while carboxylic end (C-terminus) is the end of the chain. glu-gly-ala-lys or E-G-A-K (Glu contains the N-terminus while lys contains the C-terminus. Ex. .
. a naturally occuring analgesic) Indicate where the peptide bonds are.Exercise: Give the structure of leucine enkephalin (Y-G-G-F-L.
Examples: hormones and neurotransmitters.Oligopeptides (<10 amino acids) and Polypeptides (long peptides) . Most natural polypeptide chains contain between 50 and 2000 amino acid residues.are formed by polymerization of amino acids. antibiotics and antitumor agents .
000 Since one dalton is equal to one atomic mass unit. a protein with a molecular weight of 50.000 has a mass of 50.The average molecular weight of an amino acid residue is about 110 so the molecular weights of most polypeptides are between 5500 – 220.000 daltons or 50 kd (kilodaltons) .
have partial double bond character due to delocalization of the pi electron orbitals .Peptide Bonds .the resonance structures makes the amide group planar .the peptide bond can be written as a resonance hybrid of two structures .
O=C-N-H. Hence. known as the peptide plane. Due to the specific electronic structure of the peptide bond. . are fixed on the same plane. the atoms on its two ends cannot rotate around the bond.The bond between the -C atom and the carbonylC atom is a pure single bond. likewise the bond between the -C atom and the peptide nitrogen is a pure single bond. the atoms of the group.
. Cα is the carbon atom connected to the R group.Rotation can and does occur on either side of the rigid peptide bond. O H H Ψ H R O The whole plane may rotate around the N-Cα bond (Φ angle) or C-Cα bond (Ψ angle).
Polypeptide chains can fold into regular structures: the helix and the pleated sheets. helix pleated sheets .
helices are commonly made up of hydrophobic amino acids. and the side chains extend out and away from the helix. The polypeptide main chain makes up the central structure. because H bonds (the strongest attraction) are possible between such amino acids. .An helix is a tight helix formed out of the polypeptide chain.
-Helical Structure .
Hydrogen Bonding in Helix In an helix. Every CO and NH group of the backbone is hydrogen bonded. the CO group of one amino acid is hydrogen bonded to the NH group. .
Hydrogen Bonding in Helix The CO group of one amino acid (n) is hydrogen bonded to the NH group of the amino acid four residues away (n+4). .
. pleated conformation consists of pairs of chains lying side-by-side and stabilized by H bonds between the carbonyl oxygen on one chain and the amide hydrogens on the adjacent chain.
pleated conformation .
. Parallel pleated sheets – run in the same N. Antiparallel pleated sheets – run in opposite N.to C-terminal direction.to C-terminal direction. pleated sheets can be either parallel or anti-parallel.
Parallel pleated sheets .
Anti-parallel -pleated sheets .
possibly because the hydrogen bonds are distorted in the parallel arrangement. pleated sheets may contain 2 to 15 strands. with an average of 6 residues per strand. .Parallel pleated sheets are less stable than antiparallel sheets.
are polypeptides of defined amino acid sequence . . The amino acid sequences of proteins are genetically determined. alterations in amino acid sequence can produce abnormal function and disease.PROTEINS .
Biologically active proteins are linked by covalent peptide bonds. Some proteins are held together by noncovalent or covalent forces. .
Some Biological Functions of Proteins • Enzymes - act as biological catalyst; chymotrypsin • Hormones - regulate body processes; insulin • Protective Proteins - fight infection; antibodies
• Storage Proteins - casein stores nutrients; myoglobin stores oxygen in muscles • Structural Proteins - form the structure of the body; keratin, elastin, collagen • Transport Proteins - transport oxygen and other substances through the body; hemoglobin
Conjugated Proteins 1.Glycoproteins – proteins bonded to carbohydrates; cell membranes have glycoprotein coating 2.Lipoproteins – proteins bonded to fats and oils (lipids); these proteins transport cholesterol and other fats through the body
an enzyme necessary for biological production 4. which stores nutrients for growing embryos . example is cytochrome oxidase.Metalloproteins – proteins bonded to a metal ion. found in cell ribosomes 5.Nucleoproteins – proteins bonded to RNA (ribonucleic acids). example is milk casein.Phosphoproteins – proteins bonded to a phosphate group.3.
Tertiary Structure 4.Quaternary Structure .Secondary Structure 3.Structures of Proteins 1.Primary Structure 2.
Structures of Proteins .
if any.Structures of Proteins Primary Structure – the amino acid sequence or order in which the amino acids are linked together and the location of disulfides. that make up a protein .
Protein primary sequences can be written with a 3-letter code for the or with a 1-letter code. Example: bovine insulin A-Chain: GIVEQCCTSICSLYQLENYCN B-Chain: FVNQHLCGSHLVEALYLVCGERGF FYTPKT .
Secondary Structure -refers to the way in which segments of the peptide bonds orient into a regular pattern. Examples: alpha helix and the beta sheet .
Helices and sheets could be in the same proteins .
including weak bonds .Tertiary Structure -refers to the way in which the entire protein molecule coils into an overall three-dimensional shape -final shapes of proteins are determined and stabilized by chemical bonds.
and turns. beta sheets. contribute to the ribonuclease tertiary structure .Ribonuclease Alpha helices.
the arrangement of the individual subunit of a protein with multiple polypeptide subunits (ex. hemoglobin has two alpha and two beta subunits) .refers to the spatial arrangement of polypeptide chains (subunits) and the nature of their contacts .Quaternary Structure .
Hemoglobin Hemoglobin. The red patches are the heme group. . two alpha globins and two beta globins. a protein with four polypeptides.
plays a major role. because subtle changes in the sequence can easily change the secondary and tertiary structures of proteins 2. Amino acid sequence .Folding is a thermodynamically favored process. Thermodynamic Factors .Factors that Determines Secondary and Tertiary Structure 1. .
3. Disulfide Bonds .Bonds between cysteine residues in a protein help to stabilize it once it has folded. can only be denatured at 100oC in very acid solutions. is one of the stablest proteins known. which has 3 disulfide bonds. . Bovine pancreatic trypsin inhibitor (BPTI).
BPTI S-S bonds: Cys5-Cys55. Cys14-Cys38 and Cys30-Cys51 RPDFC LEPPY TGPCK ARIIR YFYNA KAGLC 5 14 30 QTFVY GGCRA KRNNF KSAEDCMRTCGGA 38 51 55 .
Ribonuclease (RNase A) .
Protein noncovalent interactions help stabilize tertiary structure of protiens Noncovalent interactions are individually weak but collectively strong. .
Hydrophobic interactions 3. Ionic interactions 2.Three principal kinds of noncovalent forces: 1. Hydrogen bonds .
As the pH drops. .Ionic Interaction Ionic interactions are highly sensitive to pH changes and salt concentration. neutralizing their negative charge. and H+ bind to the unoccupied pair of electrons on the N atom of the amino (NH2 ) groups of Lys and Arg giving them a positive charge. H+ binds to the carboxyl groups (COO-) of Asp and Glu.
The strength of hydrophobic interactions is not appreciably affected by changes in pH or in salt concentration.Hydrophobic Interaction The side chain-R groups such as phe and leu are nonpolar and thus interact poorly with polar molecules like water. Nonpolar residues in proteins are directed toward the interior of the molecule and have hydrophobic interactions. .
. O and N) approaches a hydrogen atom which is covalently attached to a second strongly electronegative atom.g.Hydrogen Bonds Hydrogen bonds can form when a strongly electronegative atom (e.
Types of Hydrogen Bonds in Proteins: Hydroxyl-hydroxyl Hydroxyl-carbonyl Amide-carbonyl Amide-hydroxyl Amide-imidazole N .
e.Forces responsible for protein folding: 1. hydrogen bonding 2.ionic attraction 3. disulfide bridges .g. salt bridges . crosslinking . hydrophobic effect 4.
include the major proteins of skin and connective tissues and of animal fibers like hair and silk .are tough and insoluble in water .favors the secondary structure . Fibrous proteins – consist of polypeptide chains arranged side by side in long filaments such as collagen and keratin .structural materials of animal cells and tissues .Two major classes of proteins 1.
tendons. feathers. silk.Some Common Fibrous Proteins • Collagens: animal hides. wool. nails • Myosins: muscle tissues . ligaments • Fibrinogen: necessary for blood clotting • Keratins: skin. connective tissues • Elastins: blood vessels. hooves.
play important structural roles in the nuclei. doesn’t stretch due to crosslinking of disulfide bonds . cytoplasm. helical-keratins are the major proteins of hair and fingernails and compose a major fraction of animal skin. and surfaces of many cell types.
Structure of typical -keratin in hair -keratin is built on a coiled-helical structure .
that twist into a regularly repeating triple helix and give tendons and skin their great tensile strength .usually contain three very long polypeptide chains. each with about 1000 amino acids. tendons. the most abundant protein found in vertebrates . and cartilage. skin.Collagens .make up bones.
Collagen Structure .
their chains are shortened to form gelatin.Collagens When long collagen fibrils are denatured by boiling. .
Fibroin -a sheet protein -almost half of its residues are gly and between them lie either ala or ser residues this allows the sheets to fit together and pack on top of one another which results in a strong and relatively inextensible fiber. .
Structure of Silk Fibroin .
.The covalently bonded chains are stretched to nearly their maximum possible length.Bonding between the sheets involves van der Waals force of attraction which provide little resistance to bending .Fibroin .
Silk Fibroin .
with little secondary structure . and is very flexible and easily extended -its conformation approximates that of a random coil. alanine.Elastin -forms elastic fibers found in ligaments and blood vessels -rich in glycine. and valine.
causing the fibers to snap back when tension is removed . alanine. which can be involved in cross-links -these cross-links prevent the elastin fibers from being extended indefinitely.Elastin -the glycine. and valine sequence also contains frequent lysine side chains.
filamentous forms of the fibrous proteins .Globular Proteins -far outnumber fibrous proteins -perform most of the chemical "work" of the cell: synthesis.are generally soluble in water and are mobile within cells . and catabolism -are folded into compact structures. unlike the extended. nearly spherical shapes. transport.
examples are egg albumin. and many enzymes .Globular Proteins . pleated. serum globulins in blood.there may be single. insulin.have no systematic structures but are relatively spherical in shape . the chains could be helical. myoglobin. hemoglobin. or completely random structures . two or more chains.
helical (blue) sheet (orange) structures Examples of globular protein structures .
Common Globular Protein • Hemoglobin: involved in oxygen transport • Immunoglobulins: involved in immune system • Insulin: hormone for controlling glucose metabolism • Ribonuclease: enzyme controlling RNA synsthesis .
.General rules that have been observed in globular proteins: 1. Folds favor orientation of amino acid residues in specific ways that pack hydrophobic amino acid residues on the inside of the protein (away from water) and hydrophilic amino acid residues on the outside of the protein.
sheets are usually twisted. immunoglobulin and prealbumin . or wrapped into barrel structures e.2.g.
.3. Turns (interruptions between secondary structures) can occur in a number of ways: occur at the surface of proteins via hydrogen bonding between residues 1 and 4 ( turns).
A tighter turn. . can also occur in only 3 amino residues. called the g turn (or hairpin loop).
the heme which binds the oxygen -has eight -helical regions -H-bond stabilizes the -helical region -present in skeletal muscles as an extra storage protein to enable muscle cells to have a readily available supply of O2 .Myoglobin (Mb) -consists of a single polypeptide chain of 153 amino acid residues and includes a prosthetic group.
.heme Each myoglobin molecule contains one heme prosthetic group inserted into a hydrophobic cleft in the protein. Each heme residue contains one bound iron atom that is normally in the Fe2+oxidation state.
At the center of the heme group is the Fe2+.Heme is made of a series of nitrogen cyclic rings and joined to each other by more rings. The N atoms bind to Fe2+ through coordinate covalent bonds. .
Protoporphyrin IX Skeletal structure Molecular structure .
the -chains and -chains of hemoglobin and the myoglobin are homologous.Hemoglobin (Hb) .consists of four polypeptide chains.both . two -chains and two -chains . . residues while the -chain has 146. the -chain has 141 a.and -chains are very similar to myoglobin. the protoporphyrin IX and Fe (II) complex. each subunit contains a heme.a.
Comparison of Myoglobin and Hemoglobin Structures Myoglobin Hemoglobin .
function in human blood is oxygen transport from the lungs to the tissues of the body .coordination of Fe (II) in a porphyrin within a hydrophobic globin pocket allows O2 binding without iron oxidation .each hemoglobin molecule can bind to a total of four oxygen molecules .Hemoglobin .
O2 binding of Hb and Mb Oxygenation – the reversible binding of O2 to Hb or Mb Oxymyoglobin – oxygen-bearing myoglobin Deoxymyoglobin – oxygen-free myoglobin .
Fe(II) Coordination in Oxymyoglobin Heme pocket showing the proximal (F8) and distal (E7) histidine side chains (His 93 and 64 resp.). His 64 forms H bond with O2 and His 93 is complexed to Fe2+. .
O2-binding site in oxymyoglobin
Six ligands are coordinated to Fe2+, with the ligands in octahedral geometry around iron. Four of the ligands are the N atoms of the tetrapyrrole ring system, the fifth is an imidazole ring from His-93 (proximal) and the sixth is the O2 bound between the Fe and the imidazole side chain of His-64 (distal).
Fe2+ N N
Deoxygenated heme group
The heme group is nonplanar when it is not bound to oxygen; the iron atom is pulled out of the plane of the porphyrin, toward the histidine residue to which it is attached. This nonplanar configuration is characteristic of the deoxygenated heme group, and is commonly referred to as a "domed" shape.
" . it is easier for the other three heme groups to become oxygenated. In the new shape. the binding of one molecule of O2 to hemoglobin enhances the ability of hemoglobin to bind more O2 molecules. Thus. This property of hemoglobin is known as "cooperative binding. the whole protein changes its shape.When a single heme group in the hemoglobin protein becomes oxygenated.
mm Hg .Comparison of the O2 binding properties of Mb and Hb Mb Hb hyperbolic Saturation sigmoidal O2 partial pressure.
is a positive homotropic effector.The curve of oxygen binding to hemoglobin is sigmoidal typical of allosteric proteins in which the substrate. In contrast the oxygen binding curve for myoglobin is hyperbolic in character indicating the absence of allosteric interactions in this process. . in this case oxygen.
Normal pO2 (40 mm Hg) in the capillaries in resting tissues. This is constant and does not change under normal circumstances. .Hemoglobin (Hb) Saturation Curve Normal pO2 (100 mm Hg) in the capillaries in the lungs.
. The tissues that perform the most metabolic activity produce large quantities of CO2 and H+.The Effect of CO2 and H+ on O2 Binding Increased concentrations of CO2 and H+ promote the release of O2 from Hb in the blood (Bohr Effect). CO2 and H+ are produced from metabolic activities of the body. facilitating the release of O2 from the blood.
+ H+ This is catalyzed by carbonic anhydrase. A drop in pH in tissues and in venous blood signals a demand in more oxygen. . Accumulation of CO2 lowers the pH in erythrocytes (red blood cells) through the bicarbonate reaction: CO2 + H2O ⇌ HCO3. CO2 is produced.Bohr Effect As O2 is utilized in tissues.
Hb∙4O2 + nH+ ⇌ Hb∙nH+ + 4O2 where n is about > 2. The response of hemoglobin to changes in pH is called the Bohr effect. The overall reaction may be written. .Bohr Effect A pH drop in the blood in the capillaries lowers the oxygen affinity of hemoglobin and allows more efficient release of oxygen.
Effect of pH on O2 Binding .
Bohr Effect The reverse reaction occurs in the lungs or gills where there is high O2 conc. The free CO2 can then be exhaled. This releases H+ by shifting the equilibrium to the left. This favors oxygenation (oxy state). Hb∙4O2 + nH+ ⇌ Hb∙nH+ + 4O2 This tends to release CO2 from the HCO3.by the reversal of the bicarbonate reaction.+ H+ . CO2 + H2O ⇌ HCO3.
+ H+ releasing protons that contribute to the Bohr effect. .Some of the CO2 becomes HCO3 CO2 + H2O ⇌ HCO3.Release of CO2 from respiring tissues lowers the O2 affinity of Hb in two ways: 1. -.
2. A portion reacts directly with Hb. binding to the N-terminal amino groups of the chains to form carbamates (carbamation reaction): NH3+ + HCO3.is transported out of the erythrocytes and is carried dissolved in the blood serum. Some of the HCO3.⇌ -NHCOO.+ H+ + H2O or CO2 + Hb-NH2 ⇌ H+ + Hb-NH-COO- .
Carbamation Reaction 1. . Releases H+ which contributes to the Bohr effect 3. Introduces a negatively charged group at the N-terminus of the chains. Aids in the transport of CO2 from tissues to lungs or gills 2. a characteristic of the deoxy state. stabilizing salt bridge formation between the and chains.
oxygenation favors the oxy conformation of hemoglobin which stimulates release of CO2. . As the blood travels via arteries into tissue capillaries. In the lungs or gills. the lower pH and high CO2 content favors the deoxy form. promoting O2 release from hemoglobin and binding of CO2. 2.Summary of the effects of H+ and CO2 in the respiratory cycle: 1.
.Mb and Hb protect the oxygen-binding Fe2+ from irreversible oxidation. How? O2 will normally oxidize Fe2+ to Fe3+ in close contact. The hydrophobic interior of Mb and Hb protects the heme and Fe2+ to become easily oxidized. The heme does not protect Fe2+ since heme if dissolved free in solution is readily oxidized by O2 .
a temporary electron rearrangement occurs.When O2 is bound. the iron remains in the ferrous state. . Mb and Hb provide environments in which binding of O2 is permitted but oxidation is blocked. When O2 is released.
CO ties up O2 binding sites and thereby blocks respiration.Why is CO toxic? The heme pocket can also accept other small molecules like CO which has approximately the same size and electron configuration as O2. . and the binding is not readily reversible. CO is bound with much greater affinity to Mb and Hb than is O2. However.
Protein breakdown: Hydrolysis vs Denaturation Hydrolysis -involves breaking of the peptide bonds through the addition of water -requires high temperatures and either strong acid or strong base -takes place in the cells at body temperature when catalyzed by an enzyme .
Denaturation of Proteins -involves the disruption and possible destruction of both the secondary and tertiary structures of native proteins -disrupts the normal -helix and sheets in a protein and uncoils it into a random shape -results in loss of biological activity .
-Denaturation reactions are not strong enough to break the peptide bonds. . -Most common observation in the denaturation process is the precipitation or coagulation of the protein. the primary structure or the sequence of amino acids remains the same after a denaturation.
Examples: .disrupts hydrogen bonds and nonpolar hydrophobic interactions .occurs because heat increases the kinetic energy and causes the molecules to vibrate so rapidly and violently that the bonds are disrupted.Causes of Protein Denaturation 1.proteins in eggs coagulate upon heating . Heat .sterilization by heating denature proteins in bacteria and thus destroy the bacteria .
Example: 70% alcohol solution is used as a disinfectant on the skin. Alcohol denatures proteins by forming new hydrogen bonds between the alcohol molecule and the protein side chains.2. . Alcohol Alcohol disrupts the hydrogen bonding between amide groups in the 2o structure and those between “side chains” in the 3o structure.
salt bridges result from the neutralization of an acid and amine on side chains .3. Acids and Bases .acids and bases disrupt salt bridges held together by ionic charges.
salt bridge has the effect of straightening an alpha helix. when the acidic gastric juices cause the curdling (coagulating) of milk. denaturation reaction on the salt bridge by the addition of an acid results in a further straightening effect on the protein chain Example: reaction in the digestive system. ..
The reaction of a heavy metal salt with a protein usually leads to an insoluble metal protein salt .4. Heavy Metal Salts -heavy metal salts denature proteins in much the same manner as acids and bases -salts are ionic thus disrupt salt bridges in proteins.
Tl+. Pb2+.Heavy metal salts usually contain Hg2+. is also used in the treatment of nose and throat infections. used to prevent gonorrhea infections in the eyes of new born infants. Cd2+ and other metals with high atomic weights. Ag+ . Heavy metal salts may also disrupt disulfide bonds because of their high affinity and attraction for sulfur. . AgNO3.
. -Different protein chains or loops within a single chain are held together by the strong covalent disulfide bonds. Reducing -Disulfide bonds are formed by oxidation of the sulfhydryl groups on cysteine. -Reducing agents add hydrogen atoms to make the thiol group. -SH.5.
Disulfide bonds in Insulin .
Determining Protein Denaturation 1. Loss of Solubility .one of the oldest methods utilized to follow the course of denaturation was to measure changes in solubility .
most native proteins are quite resistant to the action of proteolytic enzymes. catalyzes hydrolysis of proteins. a protease that functions optimally in acidic environment.2. During digestion. Pepsin. Increased Proteolysis . hydrochloric acid in the stomach denatures proteins. .
may be affected by denaturation of enzyme molecules. specificity of reaction. temperature optimum.. . Loss of Biological Activity -For enzymes. the affinity for substrate.3. etc. denaturation can be defined as the loss of enough structure to render the enzyme inactive. pH optimum. -Changes in the rate of the reaction.
Loss of biological activity of an enzyme .
. Spectroscopic Procedures Both ultraviolet and fluorescence spectroscopy have been utilized to follow changes in the environments of various groups within protein molecules. Such changes in environment reflect changes in protein structure and thus denaturation.4.
Protein Sequencing Is accomplished using several methods performed in combination. Enzymatic – very specific Chemical Instrumental
Specificities of Several Endoproteases
Enzyme Source Specificity peptide bond C-terminal to R, K, H but not if next to P Additional Points
highly specific for positively charged residues
peptide bond C-terminal to F, Y, W but not if next to P
residues, cleaves slowly at N, H, M, L
peptide bond C-terminal to A, G, S, V, but not if next to P
peptide bond N-terminal to I, M, F, W, Y, V, but not if next to P
Bacillus thermoproteolyt icus
prefers small neutral residues, can cleave at A, D, H, T exhibits little specificity, requires low pH
Bovine gastric mucosa
peptide bond N-terminal to L, F, W, Y, but when next to P
carboxypeptidases. exopeptidases. cleaves peptides at the C-terminal residue which can then be analyzed chromatographically and compared to standard amino acids. This class of exopeptidases are called. .Carboxy-Terminal Sequence Determination Enzymes.
Chemical Digestion Cyanogen bromide (CNBr) cleaves specifically at the C-terminal side of M residues. . The number of peptide fragments that result from CNBr cleavage is equivalent to more than the number of M residues in a protein. This method is only used on carboxypeptidase resistant peptides.
2.Sanger's Method Frederick Sanger – determined the complete sequence of insulin .4-dinitrofluorobenzene (DNF) reacts with the N-terminal residue under alkaline conditions. . .The derivatized amino acid can be hydrolyzed and will be labeled with a dinitrobenzene group that imparts a yellow color to the amino acid.
(Sanger's Method) .the modified amino acids (DNPderivative) is separated by electrophoresis and comparison with the migration of DNPderivative standards allows for the identification of the N-terminal amino acid. .
Dansyl chloride reacts with the Nterminal residue under alkaline conditions.Has higher sensitivity than the Sanger method. .Analysis of the modified amino acids is carried out similarly as the Sanger method except that the dansylated amino acids are detected by fluorescence. 5-(dimethylamino)-1naphthalenesulfonyl chloride) . .Dansyl chloride. .
The technique utilizes phenylisothiocyanate (PITC) to react with the Nterminal residue under alkaline conditions. .Edman Degradation Technique Pehr Edman developed a technique that permits removal and identifi-cation of one residue at a time from the Nterminus of a protein. .
(Edman Degradation Technique) -cleaves the N terminal and allows for additional amino acid sequence to be obtained from the N-terminus inward because it leaves the remaining peptide intact -the entire sequence of reactions can be repeated over and over to obtain the sequences of the peptide .
e. 6N HCl) or strong bases or by exhaustive enzymatic digestion. This can be accomplished with strong acids (i. minimum length for the polypeptide will be known.Protein Sequencing Basic strategy to determine the sequence of proteins: 1. . Determine the amino acid composition.
. Break all Disulfide Bonds Disulfide cross-links usually are cleaved by reduction or oxidation before sequence analysis.2.
Use end group. Perform an Initial N-terminal and C-terminal Sequence Determination. aminopeptidase and Edman degradation experiments to determine the N-terminal sequence.3. . and use carboxypeptidase to determine the C-terminal amino acids.
Several cleavage methods are available. each of which have different specificity (i.4. Divide and Conquer Break the polypeptide into fragments by cleaving at specific amino acids. .e. cleave at different amino acids).
5. a unique sequence for the protein or polypeptide of interest can be constructed. Repeat steps 3 and 4 to determine subsequences and create “overlappings” From the overlapping peptides and information gained from the original protein. . The overlaps should be at least two amino acids in length.
. Locate the Disulfide Bonds No primary structure analysis of a cysteine-containing protein can be regarded as complete before the presence and location of disulfide bonds has been established.6.
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