B: Proteins Flashcards
Catabolic Reactions
Breakdown reactions
Anabolic reactions
Synthesis reactions
Hydrolysis
Where molecules are broken down by the reaction of dilute acid/ alkali in high temps (reflux)
- enzyme reactions 37C
- Proteins –> amino acids
- Starch –> simple sugars
Condensation Polymerization
Anabolism
monomers combine together to form polymers and water is eliminated
Functions of Proteins
- Enzymes - biological catalysts
- Transport proteins - haemoglobin
- Immunoproteins - antibodies
- Hormones - chemical messengers
- structural proteins - collagen
- Energy source - when fats and carbohydrates become scarce
Amino Acids
Functional Groups and chemical properties
- COOH carboxy
- can be considered as weak acids as they are partially dissociated aqueous solution
- NH2 Amine
- behaves as base because they accept protons
Amino Acids
Zwitterions
- Amino acids exist as zwitterions in solid or aqueous solution
- Isoelectric point - pH wehre Amino acid becomes zwitterion
Dipeptide
2-Aminoacids will combine together in a condensation reaction to form a dipeptide –> anabolic reaction, energy required
compounds will have an amide/ peptide link
Thin Layer Chromatography
Alumina Al2O3 + Silica SiO2
Retardation factor x/y –> used to identitfy the components as long as external conditions remain the same
Gel Electrophoresis
Buffer solution - zwitter ion used to control the degree of ionization
- Polyacylamide gel (PAGE)
- locating agent: Ninhydrin
Gel electrophoresis - Amino Acid separation
Amino Acids will separate according to
- their Mr
- The overall charge on the molecule
- potential difference applied
Primary Structure
- Sequence of Amino Acids in the polypeptide chain
- bond responsible: peptide bond/ amide link
Secondary Structure
The folding of the polypeptide chain as a result of hydrogen bonding
Proteins
Polymers composed of amino acids (monomers)
Secondary Structure
alpha-helix
alpha-helix
H bond between the C=O of 1 peptide bond and the NH of the peptide bond 4 amino acids down
Secondary Structure
Beta-pleated Sheets
ß-pleated sheet
consists of 2 or more stretches of amino acids in which the polypeptide chain is almost fully extended
H bonds form between a C=O on one strand and an NH on an adjacent strand
Tertiary Structure
The twisting and folding of the Secondary structure to form a specific 3D shape
Bonds involved
- LDF - between nonpolar side chains
- Hydrogen - between polar side chains
- Ionic - between polar side chains
- Disulfide bridges (covalent) - between the side chains of cysteine which contain CH2-SH
Quaternary Structure
- Interactions between polypeptide chains (sub-units)
- sub-units held together by various intermolecular forces
- Dimers - 2 sub-units
- Trimers - 3 sub-units
- Tetramers - 4 sub-units
- (sub units may be identical or different)
Biological catalysts - Enzymes
- increases rate of reaction by lowering the activation energy of the forward and backward reactions equally
- remains unchanged in mass and composition
Enzymes
E + S <—> ES —> E + P
- Enzyme binds to substrate @ active side
- hydrophobic pocket, nonpolar
- Active complex
- E –> unchanged in mass and composition ; P –> products
Induced Fit theory
Lock and Key theory
Enzyme Kinetics
- Molecules must have Ea and a certain orientation to react successfully
- As conc. of substrate increases, rate of reaction increases proportionally
- because of sufficient active sites to bind to
- As the reaction progresses, the active sites are becoming occupied –> fewer are available and rate of reaction decreases
- Eventually, a maximum value is reached, after which the rate no longer increases
Enzymes - Effect of Temperature
- collisions with E > Ea, orientation allows for the ES activated complex to form, frequency of collisions is high
- optimum temperature at which the rate is a maximum
- due to increased vibration of the molecule, the active site’s shape changes and hence fewer substrate molecules can bind –> denature
Enzymes - Effect of pH
Low pH - Protonation of NH2
- changes the shape of the active site –> prevents formation of ES complex
High pH - Denaturisation
- the charge on the R group changes –> responsible for the formation of attractions between the enzyme and substrate –> denaturisation
Enzymes and Heavy Metal Ions
- Pb, Ag, Hg
-
Have a strong affinity for the S-H groups in enzymes. These groups form the quaternery structure of the enzyme
- the shape is altered and the enzyme can no longer function effectively
Fibrous Proteins
- Shape: long and narrow (secondary structures)
- Role: structural (strength and support)
- Solubility: mostly insoluble
- Sequence: repetative amino acid sequence
- Stability: less sensative to changes in pH / temp
- Examples: Collagen, Keratin
Globular Proteins
- Shape: rounded / spherical
- Role: functional (catalyst and transport)
- Solubility: mostly soluble
- Sequence: irregular amino acid sequence
- Stability: more sensative
- Examples: hemoglobin, insulin, catalase
Michaelis-Menten Curve - Vmax
Vmax - the point where all the active sites are bound to substrate (enzyme is saturated)
- High Vmax = fast conversion of substrate per unit tiem
Michaelis-Menten Curve - Km
Km Michaelic Constant - concentration of substrate when the rate of the reaction has 0.5Vmax
- gives indication of how strongly the enzyme is bound to the substrate
- the lower Km the stronger the bonding between the enzyme and substrate
- Km does not depend on substrate concentration
Inhibitors - Temperature
- Low temp - molecules of enzyme will have low values of Kinetic E.
- High temp - denaturing happens, enzymes lose its tertiary / quaternary structure and will no longer function
Inhibitors - pH
- Low pH - protonation of NH2 –> NH3+
- High pH - becomes anion –> ionic bonds in the quaternary structure are disrupted
Inhibitors - Chemical
- React with the S-H bonds preventing the formation of S-S links
- can be removed from H2O supply by host-guest chemistry
Competitive Inhibitors
- reduces enzyme activity
- these substances bind directly and reversibly to the active site without producing products
- they compete with the substrate for the active sites –> reduces the numbre of enzyme molecules available to bind with the substrates
Noncompetitive Inhibitors
- Inhibitor binds reversibly to the enzyme away from the active site at the allosteric site –> changes the shape of the active site
- The enzyme loses its tertiary structure
UV Visible Spectrophotometry - π e-
- Chromophores
- molecules with π electrons ( C=C, C=O, C=-N)
- π e- absorbs energy to excited state; releases energy to return to ground state
- wavelength corresponds to wavelength of UV light
Beer-Lambert Law
A = log10( I0/I ) = Σlc
A = absorbance
l = pathlength (1cm)
c = concentration moldm-3
Σ = molar absorptivity constant
UV spectrophotometry - process
- scan sample to determine the wavelength at which the chromophore absorbs UV
- make a range of standard solutions of known concentration and measure absorbance at the wavelength above
- plot the standard curve
- measure the absorbance of the sample and extrapolate
Buffer Solutions - Acid buffer
weak acid + salt of weak acid
CH3COOH CH3COO-Na+
ethanoic acid sodium ethanoate
CH3COOH <—> CH3COO- + H+
CH3COO-Na+ —-> CH3COO- + Na+
- adding acid: H+ reacts with conjugate base reservoir –> equilibrium shifts to the left
- adding base: OH- reacts with H+ to give H2O –> equilibrium shifts to the right, using up acid
Buffer Solutions - Base buffer
weak base + its salt
NH3 NH4Cl
ammonia ammonium chloride
NH3 + H2O <—> NH4+ + OH-
NH4Cl —> NH4+ + Cl-
- adding acid: H+ reacts with NH3, moves equilibrium right
- adding base: OH- reacts with NH4+, shifts equilibrium left
Henderson-Hasselboch Equation
pH = pKa + log([A-]/[HA])
Ka = [H+][A-] / [HA]
[H+] = Ka[acid] / [salt]
