Biochemie
Algemene informatie [2024-2025]
1e sem
2e zittijd mogelijk
Jan Steyaert (titularis)
Onderdelen en contacturen
39 contacturen Hoorcollege
39 contacturen Werkcolleges, practica en oefeningen
Inhoud
→De cursus biochemie geeft een inleiding tot de moleculaire design van het leven. De moleculaire structuur van nucleïnezuren, eiwitten en hun onderlinge relaties
(replicatie, transcriptie, translatie) worden eerst beschouwd. Hierop volgt een korte inleiding tot de biologische membranen.
→In een tweede cursusonderdeel wordt dieper ingegaan op de struktuur/funktie relatie van eiwitten. Enzymefunctie (Lysozyme, RNase A, Chymotrypsine, ...),
Coöperativiteit (Hemoglobine en ATCase), enzymeregulatie en protein-folding en -design worden in detail behandeld.
→Een inleiding tot het metabolisme vormt het laatste hoofdstuk van deze cursus. Hierin worden de belangrijkste catabolische en enkele anabolische patways
toegelicht (glycolyse, Krebbs cyclus, oxidatieve fosforylatie, glycogeen metabolisme, vetzuur metabolisme, pentosefosfaat patway, gluconeogenese).
[Voor de bio-ingenieurs behoort ook het metabolisme van aminozuren en nucleinezuren tot de leerstof]
Studiemateriaal
Handboek (Vereist) : Biochemistry, Jeremy M. Berg, John L. Tymoczko and Lubert Stryer, 10de, Freeman / Macmillan, 9781319498504, 2023
Biochemistry, L. Stryer, Editie 10
Verplichte leerstof: Hoofstukken 1 tot 25 exclusief, 6, 13 & 14.
Doelstellingen
De studenten een conceptueel inzicht verschaffen in de moleculaire grondslagen van het leven. Waar mogelijk worden deze concepten bijgebracht aan de hand van
experimentele gegevens om de studenten te confronteren met de proefondervindelijke methodiek van de moleculaire bioloog/enzymoloog/geneticus, ...
Eindtermen
De studenten moeten in staat zijn:
- De covalente structuur van de belangrijkste biologische moleculen neer te schrijven
- De moleculaire krachten die de biochemische spelregels bepalen te beschrijven.
- De stroom van informatie (DNA replicatie, transcriptie en translatie) conceptueel uit te leggen.
- De structuur-functie relatie van biologische macromoleculen (DNA, eiwitten,...) aan de hand van concrete voorbeelden uit te leggen.
- De belangrijkste catabolische en anabolische wegen te beschrijven en deze in het globaal metabolisch netwerk te situeren.
1
,Biochemie
Beoordelingsinformatie
De beoordeling bestaat uit volgende opdrachtcategorieën:
Examen Mondeling bepaalt 100% van het eindcijfer
Toelichting: Het examen wordt schriftelijke voorbereid en mondeling afgenomen.
Voorbeelden van examenvragen:
- Schrijf de covalente structuur van het peptide Lys Ala His Val. Wat is de netto lading van dit peptide bij pH5 ?
- Verklaar de coöperatieve eigenschappen van Hemoglobine.
- Beschrijf het proces van transcriptie
- Beschrijf het oxidatief deel van de oxidatieve fosforylatie
H1-2p
H2-4p
H3-6p
H4-4p
H5-4p
H6-3p
H7-4p
H8-8p
H9-8p
H10-6p
H11-5p
H12A-5p
H12B-6p
H13-10p
H14-6p
H15A-6p
H15B-6p
H16-6p
H17-6p
H18-6p
2
, Biochemie
CHAPTER 1: AN EVOLVING SCIENCE DNA Illustrates the Interplay between Form and Concepts from Chemistry Explain the
Biochemie= chemistry of life Function Properties of Biological Molecules
➢ DNA is constructed from four building blocks ➢ The formation of the DNA double helix as a key example
Unity Underlies Biological Diversity DesoxyriboNucleinAcid= sugar-fosfate backbone + monomer
*alle organismen hebben dezelfde bouwstenen= zelfde Desoxyribose + fosfate group + bases
biochemie
→ resultaat= veel verschillende functies, vormen,…
2 classes molecules
➢ Biological macromolecules: large molecules such as
proteins and nucleic acids
➢ Metabolites: Low-molecular-weight molecules, that are
chemically transformed in biological processes ➢ Covalent and noncovalent bonds are important for the
structure and stability of biological molecules
Covalente binding Niet-covalente binding
-sharing a pair of electrons -Electrostatic interactions:
between adjacent atoms *Coulombs low, E = (k*q1*q2)/D*r
-C-C, 1.54Å, 85 kcal mol-1 *D = 80 (di-elektrische constante water)
~common, with minor variations, to all living things -C=O, 175 kcal mol *1.4 kcal mol-1
Eg. DNA, Proteins have the same 20 amino-acids, proteins ➢ Two single strands of DNA combine to form a double helix -quasi irreversibel
that play similar roles in different organisms often have very James Watson, Francis Creek, Rosalind Franklin -For many molecules,
similar threedimensional structures →double helix more than one pattern of - Van der Waals interactions
covalent bonds can be
➢ One common ancestor written (resonantievormen)
=Chemical reactions entail
complemantairy bases: hydrogen bonds the breaking and
forming of covalent bonds
*0.5 to 1 kcal mol-1
Water = solvent of life (55molair
- Hydrogen bonds
(H2O: polarity, cohesive properties,
hydrophobic affect)
*Electrostatic repulsion
disfavors the formation
➢ DNA structure explains heredity and the storage of of a double helix
information *Solvent properties!
-efficiently storage of genetic information: base sequence *Dielectric constant of water
-accurate replication: specific base-pairing between *Na+ or Mg++ ions
complementary strands -Hydrophobic interactions
* Nonpolar molecules cannot
participate in hydrogen bonding or ionic
interactions with the solvent
3
, Biochemie
➢ The double helix is an expression of the rules of chemistry ➢ Heat is released in the formation of the double helix -Low pH Effects: Below pH 5, protonation of base-pair acceptors
-Chemical interactions drive the formation of the double helix =thermodynamics disrupts hydrogen bonding, also destabilizing the double helix
*ionic interactions
*hydrogen bonds -Phosporic acid is an important buffer in biological systems
*van der Waals forces (biological pH=generally 7.5)
*hydrophobic effect
-Specific binding through complementarity:
*Hydrogen bonding: complementary bases (A-T, G-C),
follows chemical rules of donor-acceptor interactions ➢ Buffers regulate pH in organisms and in the laboratory
-buffers= solutions that
➢ The laws of thermodynamics govern the behavior of resist changes in pH when
biochemical systems small amounts of acids or
Nature= relatively organised= strange? *2 sole complementary DNA strings will automatically/ bases are added. They work
Thermodynamics= direction of a system (past, future)= spontaniously combine to double helix (more order?) by neutralizing added H⁺
explains order out of choas *S lowers BUT H increases (hyrdogen-bonds)->surroundings heat (hydrogen ions) or OH⁻
up->surrounding S increases → total G<0 (hydroxide ions), helping to
→First Law of Thermodynamics: The total energy of a system maintain a stable pH in a
and its surroundings remains constant. ➢ Acid–base reactions are central in many solution
biochemical processes
→Second Law of Thermodynamics: The total entropy S =ionisation (loss or take up of protons) The Genomic Revolution is Transforming
(disorder) of a system and its surroundings always increases. -Covalent Bonds & Acid-Base Reactions: Biochemistry, Medicine, and Other Fields
(hydrophobic effect:releases water molecules->increases S ) forming/cleaving covalent bonds (via H+ transfer) ➢ Genome sequencing has transformed biochemistry and
- pH = -log[H+], where H+ exists as H3O+ in solution. other fields
*Water Dissociation: H2O ⇌ H+ + OH−, -Advancements in sequencing technology→ cheaper and faster
with constant Kw = [H+][OH−] = 1.0 x 10^(-14) -learning about human phylogenie:man migrations supported by
*pH & OH− Relationship: pH and [OH−] are inversely related; at DNA sequence comparisons
pH 7 (neutral), [H+] = [OH−]. ➢ Environmental factors influence human biochemistry
The human microbiome
➢ Acid–base reactions can disrupt the double helix Proper health depends on a balanced nutrition to provide an
-DNA Stability at pH 7: Double helix remains stable at neutral pH. optimal mix of biologicals
-Effect of High pH (Base Addition): As pH rises towards 9-10, DNA ➢ Genome sequences encode proteins and patterns of
strands dissociate due to deprotonation of guanine's N-1 expression
H=enthalpie (heat) nitrogen. Molecular representations
-pKa of Guanine: Proton dissociation occurs around pKa 9.7, Space filling/ Ball-and-stick /Sceletal
→Gibbs Free Energy: destabilizing hydrogen bonds and causing helix separation.
= energy term, described by system-> takes both the entropy of
the system and the entropy of the surroundings into account
Gibbs free energy (ΔG)<0→spontanous
= overall increase in entropy of the universe.
*e.g. guanine (NH2 carries proton-> when pH >9.7 ->proton loss) Color code
DNA carries the protonated form of guanine and is not compatible
Carbon (C) black
with unprotonated form (bc it has no H to form hydrogen bond)
Hydrogen (H) white
Oxygen (O) red
Nitrogen (N) blue
Sulfur (S) yellow
Phosphorus (P) purple 4
Chlorine (Cl) Green
, Biochemie
CHAPTER 2: PROTEIN COMPOSITION ➢ Hydrophobic amino acids Primary Structure: Amino Acids Are Linked by
nonpolar R groups
AND STRUCTURE Peptide Bonds to Form Polypeptide Chains
Ala (A), Val (V), Leu (L), Ile (I), Met (M), Phe (F), Trp (W), Pro (P), Gly (G)
-Proteins are linear polymers of amino acids linked by peptide
➢ Polar amino acids
bonds
neutral R groups but the charge is not evenly distributed
-Peptide bonds= reaction between acid-group and amine groep
Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q)
formed with the loss of water, and require energy for synthesis
➢ Positively charged amino acids
R groups that have a positive charge at physiological pH
→
Lys (K), Arg (R), His (H)
➢ Negatively charged amino acids
R groups that have a negative charge at physiological pH
Asp (D), Glu (E) -Polypeptide chains have directionality, with an amino-terminal
-Protein structure dictates function
-Many proteins act in complexes (N-terminal=START) and a carboxyl-terminal (C-terminal=END)
-Seven amino acids have ionizable side chains
-Some proteins are quite rigid, whereas others display flexibility end, and the sequence determines their chemical properties
= greater importance than zwitterions of whole molecule →lineair structures
+ influenced by pH of enviroment
Proteins Are Built from a Repertoire of 20 (Tyr (Y), Cys (C), Lys (K), Arg (R), His (H), Asp (D), Glu (E)) -Peptide bonds are rigid due to partial double-bond character,
Amino Acids but rotation around other bonds allows proteins to fold into
Amino-acids *central C
specific three-dimensional structures.
*4substituants (always 1 H, 1 NH2, 1 acid COO-, 1R)
*2 amino-acids: free e- pair-> delocalisation (resonance)
BUT in double bond: no free rotation→ rigid molecule
-A constant backbone and variable side chains
*Protein: 50 to 2000 amino acids
2 enantiomers= in nature only L-isomers (D is synthetic)
*Peptide: <50 amino acids
-average molecular weight= 100Dalton
*1 amino acid 110 daltons
→Twenty different side chains (Size, Shape, Charge, Hydrogen
-linear polypeptide chain can
bond capacity, Hydrophobic character, Chemical reactivity)
be cross-linked= Cysteine
→Free amino acids are zwitterions at neutral pH (total molecul
(Cys, C)- sulfurbridge (no
by neutral pH= neutral (1+,1-))
rules, equilibrium: sometimes
formed sometimes not)
[Bovine insuline, Sanger 1953]
➢ Proteins have unique amino acid sequences specified by genes
➢ Polypeptide chains are flexible yet conformationally restricted
-double bond implies no rotational energy: partial double (resonance)
-Peptide bonds are planar: 6 atoms in one plane
-humans: 10-11 amino-acids can be self-synthesized, rest is -Why these 20? →makeable in primitive enviroment ( Size, Shape,
taken in via nutrition Charge, Hydrogen bond capacity, Hydrophobic character, Chemical
-micro-organism (bacteries): able to self-synthesize all 20 reactivity)
5
, Biochemie
-Cis →Trans (trans peptides are strongly favored) Secondary Structure: Polypeptide Chain Can ➢ Beta sheets are stabilized by hydrogen bonding between
*interference/ hindrance of sidechains in cis conformation polypeptide strands
Fold into Regular Structures Such As the
-sheets are build from extended β-strands (2 or more polypeptide chains)
Alpha Helix, the Beta Sheet, and Turns and -β-strands= almost fully extended (not tightly coiled as in the α-helix)
Loops
➢ The alpha helix is a coiled structure stabilized by
intrachain hydrogen bonds
-Right-handed
-3.6 residues per turn
*prolines in bond-> more change of cis (X-Pro linkages) -Translation 1.5 Å (100°)
-Pitch 5.4 Å -antiparallel/parallel/ mixed
*both possible with hydrogen-bonds
-The other main chain bonds are pure single bonds
(can rotate-> many different conformations for a molecule)
-Two adjacent rigid peptide units rotate about Φ and ψ
*many Φ/ψ combinations are forbidden because of steric
collisions between atoms
-compatible with Ramachandran diagram
-Schematic representation: ball and stick (above)- schematic model (below)
-main chain=innerside, side chains/R-groups outer parts
-hydrogen-bonds between carbonyls and amines: 90°
-Ramachandran diagram -Sheets can be twisted or even fully closed barrels
= visualizes the allowed angles (phi, ψ) between amino acids in *connectivities can be complex
protein structures, showing favored and disallowed ➢ Polypeptide chains can change direction by making
conformations due to steric hindrance. -helix is compatible with Ramachandran diagram reverse turns and loops
-PDB= protein data base-> software with all known protein -Polypeptide chains change direction by reverse turns
structures (120 000) and loops (almost no regularity)
-Schematic representations of a -helix
➢ Fibrous proteins provide structural support for cells and
tissues
Different forms than α-helix or β-sheets for secundairy structures
-Coiled coil proteins
e.g. keratin: two right handed a-helixes →forming a left handed superhelix
3.5 residues per turn! (Leu-Leu in wool→ Cys-Cys in horn
(sulfurbridges= covalent bonding)
e.g. collagen: three intertwined helical polypeptide chains→superhelical
cable stabilized by H-bonds between strands
6
, Biochemie
Tertiary Structure: Water-Soluble Proteins -Some polypeptides fold into multiple domains More info about the experiment
Fold into Compact Structures with Nonpolar
Cores
-Proteins fold into compact structures
e.g. myoglobin via X-ray crystallographic and nuclear magnetic
resonance (NMR)→ schematic model+ space filling model *still 3 main factors->each segment
*globular (dense, hydrophobe inside, hydrophile inside)
*compact
*Backbone atoms pack
Quaternary Structure: Polypeptide Chains
tightly together
*Side chains pack the Can Assemble into Multisubunit Structures
-Urea and guanidinium chloride disrupt the protein’s noncovalent bonds
intervening space -Arrangement and interaction of multiple polypeptide chains
*Van der Waal distance (subunits), such as in dimers or complex structures like
between atoms hemoglobin (α2β2 tetramer)
dimeren, tetrameren, ….
-Water-soluble proteins have nonpolar cores e.g.Cro is a dimer of
*blue= hydrophile →outside identical subunits
*yellow= hydrophobe →inside -Viruses make the most from
*Globular proteins hide a limited amount of information
their hydrophobic e.g. Human rhinovirus uses 60 copies of each of 4 subunits
side chains from solvent (left= hemoglobine, right=rhinovirus)
*Charged atoms rarely → disulphide bonds can be reduced reversibly
occur on the interior of a
protein
*Charged atoms prefer the
protein surface
-Inside-out architecture of membrane proteins
The Amino Acid Sequence of a Protein
Determines Its Three-Dimensional Structure
➢ Amino acids have different propensities for forming a 1. adding of urea and beta-mercaptoethanol (BME)
helices, b sheets, and turns *denaturation to random coil
Experiment Christian Anfinsen in 1950s.
Bovine pancreatic ribonuclease denatured (with urea and β-
mercaptoethanol) and then renatured.
*Shows proteins can refold into functional conformations
after denaturation
→Protein structure is determined by its amino acid
sequence, crucial for function.
2. renaturation: removal of urea and beta-mercaptoethanol
*dialysis: Denatured ribonuclease->Regains enzymatic activit->
-Certain combinations of secondary structure are common to Sulfhydryl groups oxidized-> Spontaneous refolding into native
many proteins (patterns)
structure (trial and error)
*helix-turn-helix (often for interactions with DNA)
*helix-b-sheet configuration (NAD+ interactions)
7
, Biochemie
➢ Protein folding is a highly cooperative process Prediction of three-dimensional structure from sequence ▪ Parkinson's Disease
-All or nothing: there is no ‘partially folded’ protein at significant remains a great challenge Neurodegenerative disorder linked to the accumulation of alpha-
concentrations e.g. Predictions of secondary structure adopted by a stretch synuclein aggregates (Lewy bodies).
of 6 or 7 AA. Have proved to be 60 to 70 % accurate Misfolded alpha-synuclein proteins form fibrillar aggregates that
both VDLLKN-> different structure impair neuronal function.
Toxicity may arise from soluble oligomers disrupting cellular
processes and membranes, leading to neuron death.
➢ Some proteins are inherently unstructured and can exist
in multiple conformations
➢ Proteins fold by progressive stabilization of intermediates -Lymphotactin exists in two conformations that are in equilibrium
rather than by random search ➢ Protein modification and cleavage confer new capabilities
-Proteins do not fold by random search -Covalent Modifications for Enhanced Functionality
-Folding problem leventhal paradox: -Attachment of Special Groups
*Structure of most proteins are unknown (even if we know -Activation through Cleavage
sequence: no laws, no rules) e.g.
*why not random trial and error? Would take YEARS for a protein to form *Green fluorescent protein (GFP) has a unique structure.
100 residues ->Fluorescence is produced by the rearrangement and oxidation
3 conformations/residue= 3^100 = 5 x 10^47 structures of the amino acid sequence Ser-Tyr-Gly within the protein.
5 x 10^47 x 10^-13 seconds = 5 x 10^34 seconds = 1.6 x 10^27 years ➢ Protein misfolding and aggregation are associated with *GFP Mutants and Light Emission:
-Proteins fold by progressive some neurological diseases Various GFP mutants have been engineered to emit light across
stabilization of intermediates: -structurally changed proteins can become toxic to the brain
Cumulative selection → trial-and- environment
error: correct tries are being ▪ e.g. Mad cow disease ->Creutzfeldt-Jacob= Prion disease
remember/ kept= intermediate form
*e.g. folding pathway of chymotrypsin
inhibitor
MODELS
Space-filling model Ball-and-stick model Backbone model
*Infectious neurodegenerative diseases caused by misfolded
prion proteins (e.g., PrP^SC)->amyloid fibers
-BUT: suggests pathways= is not the case (not the best theory) • How: Abnormal prions aggregate and induce normal prion
*energetical levels= Folding funnel proteins to misfold, creating nucleation sites for further
Folding funnel
aggregation.
▪ Alzheimer's Disease Riboon diagram Ribbon diagram w highlights
*Neurodegenerative disorder characterized by the formation of
amyloid plaques. The amyloid-beta (Aβ) peptide aggregates to
form insoluble fibrils and plaques in the brain, derived from the
amyloid precursor protein (APP).
->Smaller aggregates may disrupt cell membranes, leading to
cellular toxicity and neurodegeneration.
8
, Biochemie
CHAPTER 3: EXPLORING PROTEINS AND ➢ Proteins must be released from the cell to be purified Ion-exchange chromatography Separation based on el. charge
-Differential centrifugation: denser materials will form a *Positive Proteins:
PROTEOMES
pellet at lower centrifugal force Bind to negatively charged beads
➢ The proteome is the functional representation of the genome
homogenate forms ->supernatant -> cytoplasm (e.g., carboxymethyl cellulose)
➢ Proteins can be purified according to solubility, size, charge, in cation-exchange chromatography.
and binding affinity *Negative Proteins:
-Purification Indicator: Increased specific activity per step. Bind to positively charged
Sample Complexity: Fewer different proteins at each purification step beads (e.g., DEAE-cellulose)
Homogenate: Obtained by centrifugation in anion-exchange chromatography.
Salt fractionation Separation based on solubility
Standard technique: adding of ammoniumsulfate (reversible way of How to harvest protein back
-Protein encoding genes are just a list of ‘the parts of a car’ making a protein insoluble) from column (now its captured by
-The proteome is the functional representation of the genome the beads)?
-Only a subset of the gene products is actually expressed in a given -add something with higher
biological context, i.e. the proteome is not static concentration which competes
-Proteome is derived from proteins expressed by the genome *rough but cheap with your proteins for the beads
-Because the proteome is the functional expression of the genome, it *quick volume reducing Increasing salt concentration releases bound proteins by displacing
varies with cell type, developmental stage and environmental Dialysis to remove salts them with sodium ions, allowing proteins to elute in order of their
conditions -placing the protein mixture in charge strength. (in fractions: lineair gradient)
a dialysis bag made of a Affinity chromatography Separation based on functional
The Purificatin of Proteins Is an Essential First semipermeable membrane properties
with pores.
Step in Understanding Their Function ->Larger molecules, such as
➢ The assay: How do we recognize the protein that we are proteins, remain inside the bag,
looking for? while smaller molecules and
-Importance of Protein Purity: ions diffuse through the pores
*accurate amino acid sequencing into an external buffer solution
*remove contaminants + concentrate protein → Dialysis effectively removes small molecules (e.g., salts) but cannot
-Purification Process: isolation from complex mixtures through differentiate between different proteins
multiple separation techniques based on physical properties (e.g.,
Gel filtration chromatography Separation by size -Covalently attach target molecule (X) or its derivative to the column.
size and charge) to achieve a sample with only the target protein
-column packed with porous beads made of hydrated polymers (e.g., -Pass a protein mixture through the column and wash with buffer to
-Assays and Specific Activity: identifies the target protein by
dextran, agarose, or polyacrylamide). remove unbound proteins.
measuring unique properties, like enzyme activity.
*Large molecules: Cannot enter the pores -> remain in the solution -Elute the desired protein by adding a soluble form of X or altering
e.g. test for lactate dehydrogenase (NADH is fluorescent at 340nm->
between beads-> exiting the column first. conditions to reduce binding affinity
so testing fluorescent properties)
*Medium molecules: Sometimes enter the pores ->intermediate exit HPL-chromatography Separation based on size and charge
-Protein purification is to maximize the specific activity
time. =high-pressure-liquid chromatography
*specific activity will rise as the purification proceeds and the protein
*Small molecules: Enter the pores fully-> longer path and exiting last. -more advanced way of Column Techniques
mixture being assayed consists to a greater extend of your target
→ Larger molecules flow faster through the column, separation based on -Finer particles provide more interaction sites,
protein: pure protein has a constant and high specific activity
molecular size. (fractions) leading to greater resolving power
-Larger surfaces, More interaction sites
Smaller columns, More resolving power, Higher pressure
Faster separations
Higher peak=
Higher resolution
9
, Biochemie
➢ Proteins can be separated by gel electrophoresis and -Separation Basis: Primarily based on protein mass (the more amino- ➢ A protein purification scheme can be quantitatively evaluated
displayed acids on a protein-> the bigger the protein-> the more negatively =calculating how pure the protein is at the end of the procedure
charged but all relatively equal to mass so smaller migrates faster *Activity of Protein: how well the protein works at each stage
trough medium) *Gel Test (SDS-PAGE): Creates a "protein fingerprint" that shows if unwanted
Detection: Coomassie blue, silver staining, or autoradiography for proteins are decreasing.
visualization. → Fewer bands = fewer unwanted proteins.
Reduction in protein bands across steps; one prominent band for
target protei *Total Protein: #total protein we have in each sample.
*Total Activity: # work the protein can do in each sample.
*Specific Activity: activity (or "pure") of target protein, compared to others.
*Yield: # of the original protein’s function we still have after each step.
-Gel Electrophoresis *Purification Level: # purer the protein is after each step.
*Principle: Charged molecules move in an electric field Our target protein’s band becomes more visible as it’s purified.
(electrophoresis). (smaller molecules fast, bigger slow (friction))
*Velocity Factors: Electric field strength, protein charge, and frictional →Good purification keeps enough of the target protein (high yield) but
coefficient. reduces contaminants.
V = Ez/f (snelheid evenredig met elektrisch veld, lading partikel en →Low yield with high purification means less target protein; high yield with
omgekeerd evenredig met frictie quotient) low purification means more contaminants
f = 6πήr (frictiequetient bevat straal van partikel)
➢ Ultracentrifugation is valuable for separating biomolecules
-Matrix: Polyacrylamide gel, acting as a molecular sieve and determining their masses
In a buffer acrylamide: radical polymerizes spontaneously to long *without SDS-page= no denaturation so by natural charge of protein
chains (fibers) +by adding bisacryl we link the chains Iso-electric focusing (no standard technique)
->viscous medium (bc of polymers) *iso-electric point= point on the molecule where total charge is 0
*proteins move to pH matching their pI
Two-dimensional electrophoresis
Combines isoelectric focusing (horizontal) with SDS-PAGE (vertical) *ultra-ctfgt.: high speed, separates small particles (DNA, RNA,
-1. Isoelectric focusing (separation by pI) viruses…)
-2. Second step: SDS-PAGE (separation by mass) *differential tcfgt.: stepswise, based on size and density through
-High resolution: Separates thousands of proteins (e.g., over 1000 in E. increasing speeds
coli) *zonal ctfgt.: based on density gradient (e.g., sucrose) in a
→ Protein spots on gel can be analyzed by mass spectrometry centrifuge tube to separate particles based on their sedimentation
*SDS-PAGE: Sodiumdodecylsulfate (detergent) denatures proteins, rate
adds uniform negative charge (per 2 amino-acids, 1 negatively *equilibrium ctfgt: particles move to
charged SDS: overall negative charge for protein) the point in a density gradient that
matches their own density and remain
in equilibrium there
10
Algemene informatie [2024-2025]
1e sem
2e zittijd mogelijk
Jan Steyaert (titularis)
Onderdelen en contacturen
39 contacturen Hoorcollege
39 contacturen Werkcolleges, practica en oefeningen
Inhoud
→De cursus biochemie geeft een inleiding tot de moleculaire design van het leven. De moleculaire structuur van nucleïnezuren, eiwitten en hun onderlinge relaties
(replicatie, transcriptie, translatie) worden eerst beschouwd. Hierop volgt een korte inleiding tot de biologische membranen.
→In een tweede cursusonderdeel wordt dieper ingegaan op de struktuur/funktie relatie van eiwitten. Enzymefunctie (Lysozyme, RNase A, Chymotrypsine, ...),
Coöperativiteit (Hemoglobine en ATCase), enzymeregulatie en protein-folding en -design worden in detail behandeld.
→Een inleiding tot het metabolisme vormt het laatste hoofdstuk van deze cursus. Hierin worden de belangrijkste catabolische en enkele anabolische patways
toegelicht (glycolyse, Krebbs cyclus, oxidatieve fosforylatie, glycogeen metabolisme, vetzuur metabolisme, pentosefosfaat patway, gluconeogenese).
[Voor de bio-ingenieurs behoort ook het metabolisme van aminozuren en nucleinezuren tot de leerstof]
Studiemateriaal
Handboek (Vereist) : Biochemistry, Jeremy M. Berg, John L. Tymoczko and Lubert Stryer, 10de, Freeman / Macmillan, 9781319498504, 2023
Biochemistry, L. Stryer, Editie 10
Verplichte leerstof: Hoofstukken 1 tot 25 exclusief, 6, 13 & 14.
Doelstellingen
De studenten een conceptueel inzicht verschaffen in de moleculaire grondslagen van het leven. Waar mogelijk worden deze concepten bijgebracht aan de hand van
experimentele gegevens om de studenten te confronteren met de proefondervindelijke methodiek van de moleculaire bioloog/enzymoloog/geneticus, ...
Eindtermen
De studenten moeten in staat zijn:
- De covalente structuur van de belangrijkste biologische moleculen neer te schrijven
- De moleculaire krachten die de biochemische spelregels bepalen te beschrijven.
- De stroom van informatie (DNA replicatie, transcriptie en translatie) conceptueel uit te leggen.
- De structuur-functie relatie van biologische macromoleculen (DNA, eiwitten,...) aan de hand van concrete voorbeelden uit te leggen.
- De belangrijkste catabolische en anabolische wegen te beschrijven en deze in het globaal metabolisch netwerk te situeren.
1
,Biochemie
Beoordelingsinformatie
De beoordeling bestaat uit volgende opdrachtcategorieën:
Examen Mondeling bepaalt 100% van het eindcijfer
Toelichting: Het examen wordt schriftelijke voorbereid en mondeling afgenomen.
Voorbeelden van examenvragen:
- Schrijf de covalente structuur van het peptide Lys Ala His Val. Wat is de netto lading van dit peptide bij pH5 ?
- Verklaar de coöperatieve eigenschappen van Hemoglobine.
- Beschrijf het proces van transcriptie
- Beschrijf het oxidatief deel van de oxidatieve fosforylatie
H1-2p
H2-4p
H3-6p
H4-4p
H5-4p
H6-3p
H7-4p
H8-8p
H9-8p
H10-6p
H11-5p
H12A-5p
H12B-6p
H13-10p
H14-6p
H15A-6p
H15B-6p
H16-6p
H17-6p
H18-6p
2
, Biochemie
CHAPTER 1: AN EVOLVING SCIENCE DNA Illustrates the Interplay between Form and Concepts from Chemistry Explain the
Biochemie= chemistry of life Function Properties of Biological Molecules
➢ DNA is constructed from four building blocks ➢ The formation of the DNA double helix as a key example
Unity Underlies Biological Diversity DesoxyriboNucleinAcid= sugar-fosfate backbone + monomer
*alle organismen hebben dezelfde bouwstenen= zelfde Desoxyribose + fosfate group + bases
biochemie
→ resultaat= veel verschillende functies, vormen,…
2 classes molecules
➢ Biological macromolecules: large molecules such as
proteins and nucleic acids
➢ Metabolites: Low-molecular-weight molecules, that are
chemically transformed in biological processes ➢ Covalent and noncovalent bonds are important for the
structure and stability of biological molecules
Covalente binding Niet-covalente binding
-sharing a pair of electrons -Electrostatic interactions:
between adjacent atoms *Coulombs low, E = (k*q1*q2)/D*r
-C-C, 1.54Å, 85 kcal mol-1 *D = 80 (di-elektrische constante water)
~common, with minor variations, to all living things -C=O, 175 kcal mol *1.4 kcal mol-1
Eg. DNA, Proteins have the same 20 amino-acids, proteins ➢ Two single strands of DNA combine to form a double helix -quasi irreversibel
that play similar roles in different organisms often have very James Watson, Francis Creek, Rosalind Franklin -For many molecules,
similar threedimensional structures →double helix more than one pattern of - Van der Waals interactions
covalent bonds can be
➢ One common ancestor written (resonantievormen)
=Chemical reactions entail
complemantairy bases: hydrogen bonds the breaking and
forming of covalent bonds
*0.5 to 1 kcal mol-1
Water = solvent of life (55molair
- Hydrogen bonds
(H2O: polarity, cohesive properties,
hydrophobic affect)
*Electrostatic repulsion
disfavors the formation
➢ DNA structure explains heredity and the storage of of a double helix
information *Solvent properties!
-efficiently storage of genetic information: base sequence *Dielectric constant of water
-accurate replication: specific base-pairing between *Na+ or Mg++ ions
complementary strands -Hydrophobic interactions
* Nonpolar molecules cannot
participate in hydrogen bonding or ionic
interactions with the solvent
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, Biochemie
➢ The double helix is an expression of the rules of chemistry ➢ Heat is released in the formation of the double helix -Low pH Effects: Below pH 5, protonation of base-pair acceptors
-Chemical interactions drive the formation of the double helix =thermodynamics disrupts hydrogen bonding, also destabilizing the double helix
*ionic interactions
*hydrogen bonds -Phosporic acid is an important buffer in biological systems
*van der Waals forces (biological pH=generally 7.5)
*hydrophobic effect
-Specific binding through complementarity:
*Hydrogen bonding: complementary bases (A-T, G-C),
follows chemical rules of donor-acceptor interactions ➢ Buffers regulate pH in organisms and in the laboratory
-buffers= solutions that
➢ The laws of thermodynamics govern the behavior of resist changes in pH when
biochemical systems small amounts of acids or
Nature= relatively organised= strange? *2 sole complementary DNA strings will automatically/ bases are added. They work
Thermodynamics= direction of a system (past, future)= spontaniously combine to double helix (more order?) by neutralizing added H⁺
explains order out of choas *S lowers BUT H increases (hyrdogen-bonds)->surroundings heat (hydrogen ions) or OH⁻
up->surrounding S increases → total G<0 (hydroxide ions), helping to
→First Law of Thermodynamics: The total energy of a system maintain a stable pH in a
and its surroundings remains constant. ➢ Acid–base reactions are central in many solution
biochemical processes
→Second Law of Thermodynamics: The total entropy S =ionisation (loss or take up of protons) The Genomic Revolution is Transforming
(disorder) of a system and its surroundings always increases. -Covalent Bonds & Acid-Base Reactions: Biochemistry, Medicine, and Other Fields
(hydrophobic effect:releases water molecules->increases S ) forming/cleaving covalent bonds (via H+ transfer) ➢ Genome sequencing has transformed biochemistry and
- pH = -log[H+], where H+ exists as H3O+ in solution. other fields
*Water Dissociation: H2O ⇌ H+ + OH−, -Advancements in sequencing technology→ cheaper and faster
with constant Kw = [H+][OH−] = 1.0 x 10^(-14) -learning about human phylogenie:man migrations supported by
*pH & OH− Relationship: pH and [OH−] are inversely related; at DNA sequence comparisons
pH 7 (neutral), [H+] = [OH−]. ➢ Environmental factors influence human biochemistry
The human microbiome
➢ Acid–base reactions can disrupt the double helix Proper health depends on a balanced nutrition to provide an
-DNA Stability at pH 7: Double helix remains stable at neutral pH. optimal mix of biologicals
-Effect of High pH (Base Addition): As pH rises towards 9-10, DNA ➢ Genome sequences encode proteins and patterns of
strands dissociate due to deprotonation of guanine's N-1 expression
H=enthalpie (heat) nitrogen. Molecular representations
-pKa of Guanine: Proton dissociation occurs around pKa 9.7, Space filling/ Ball-and-stick /Sceletal
→Gibbs Free Energy: destabilizing hydrogen bonds and causing helix separation.
= energy term, described by system-> takes both the entropy of
the system and the entropy of the surroundings into account
Gibbs free energy (ΔG)<0→spontanous
= overall increase in entropy of the universe.
*e.g. guanine (NH2 carries proton-> when pH >9.7 ->proton loss) Color code
DNA carries the protonated form of guanine and is not compatible
Carbon (C) black
with unprotonated form (bc it has no H to form hydrogen bond)
Hydrogen (H) white
Oxygen (O) red
Nitrogen (N) blue
Sulfur (S) yellow
Phosphorus (P) purple 4
Chlorine (Cl) Green
, Biochemie
CHAPTER 2: PROTEIN COMPOSITION ➢ Hydrophobic amino acids Primary Structure: Amino Acids Are Linked by
nonpolar R groups
AND STRUCTURE Peptide Bonds to Form Polypeptide Chains
Ala (A), Val (V), Leu (L), Ile (I), Met (M), Phe (F), Trp (W), Pro (P), Gly (G)
-Proteins are linear polymers of amino acids linked by peptide
➢ Polar amino acids
bonds
neutral R groups but the charge is not evenly distributed
-Peptide bonds= reaction between acid-group and amine groep
Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q)
formed with the loss of water, and require energy for synthesis
➢ Positively charged amino acids
R groups that have a positive charge at physiological pH
→
Lys (K), Arg (R), His (H)
➢ Negatively charged amino acids
R groups that have a negative charge at physiological pH
Asp (D), Glu (E) -Polypeptide chains have directionality, with an amino-terminal
-Protein structure dictates function
-Many proteins act in complexes (N-terminal=START) and a carboxyl-terminal (C-terminal=END)
-Seven amino acids have ionizable side chains
-Some proteins are quite rigid, whereas others display flexibility end, and the sequence determines their chemical properties
= greater importance than zwitterions of whole molecule →lineair structures
+ influenced by pH of enviroment
Proteins Are Built from a Repertoire of 20 (Tyr (Y), Cys (C), Lys (K), Arg (R), His (H), Asp (D), Glu (E)) -Peptide bonds are rigid due to partial double-bond character,
Amino Acids but rotation around other bonds allows proteins to fold into
Amino-acids *central C
specific three-dimensional structures.
*4substituants (always 1 H, 1 NH2, 1 acid COO-, 1R)
*2 amino-acids: free e- pair-> delocalisation (resonance)
BUT in double bond: no free rotation→ rigid molecule
-A constant backbone and variable side chains
*Protein: 50 to 2000 amino acids
2 enantiomers= in nature only L-isomers (D is synthetic)
*Peptide: <50 amino acids
-average molecular weight= 100Dalton
*1 amino acid 110 daltons
→Twenty different side chains (Size, Shape, Charge, Hydrogen
-linear polypeptide chain can
bond capacity, Hydrophobic character, Chemical reactivity)
be cross-linked= Cysteine
→Free amino acids are zwitterions at neutral pH (total molecul
(Cys, C)- sulfurbridge (no
by neutral pH= neutral (1+,1-))
rules, equilibrium: sometimes
formed sometimes not)
[Bovine insuline, Sanger 1953]
➢ Proteins have unique amino acid sequences specified by genes
➢ Polypeptide chains are flexible yet conformationally restricted
-double bond implies no rotational energy: partial double (resonance)
-Peptide bonds are planar: 6 atoms in one plane
-humans: 10-11 amino-acids can be self-synthesized, rest is -Why these 20? →makeable in primitive enviroment ( Size, Shape,
taken in via nutrition Charge, Hydrogen bond capacity, Hydrophobic character, Chemical
-micro-organism (bacteries): able to self-synthesize all 20 reactivity)
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-Cis →Trans (trans peptides are strongly favored) Secondary Structure: Polypeptide Chain Can ➢ Beta sheets are stabilized by hydrogen bonding between
*interference/ hindrance of sidechains in cis conformation polypeptide strands
Fold into Regular Structures Such As the
-sheets are build from extended β-strands (2 or more polypeptide chains)
Alpha Helix, the Beta Sheet, and Turns and -β-strands= almost fully extended (not tightly coiled as in the α-helix)
Loops
➢ The alpha helix is a coiled structure stabilized by
intrachain hydrogen bonds
-Right-handed
-3.6 residues per turn
*prolines in bond-> more change of cis (X-Pro linkages) -Translation 1.5 Å (100°)
-Pitch 5.4 Å -antiparallel/parallel/ mixed
*both possible with hydrogen-bonds
-The other main chain bonds are pure single bonds
(can rotate-> many different conformations for a molecule)
-Two adjacent rigid peptide units rotate about Φ and ψ
*many Φ/ψ combinations are forbidden because of steric
collisions between atoms
-compatible with Ramachandran diagram
-Schematic representation: ball and stick (above)- schematic model (below)
-main chain=innerside, side chains/R-groups outer parts
-hydrogen-bonds between carbonyls and amines: 90°
-Ramachandran diagram -Sheets can be twisted or even fully closed barrels
= visualizes the allowed angles (phi, ψ) between amino acids in *connectivities can be complex
protein structures, showing favored and disallowed ➢ Polypeptide chains can change direction by making
conformations due to steric hindrance. -helix is compatible with Ramachandran diagram reverse turns and loops
-PDB= protein data base-> software with all known protein -Polypeptide chains change direction by reverse turns
structures (120 000) and loops (almost no regularity)
-Schematic representations of a -helix
➢ Fibrous proteins provide structural support for cells and
tissues
Different forms than α-helix or β-sheets for secundairy structures
-Coiled coil proteins
e.g. keratin: two right handed a-helixes →forming a left handed superhelix
3.5 residues per turn! (Leu-Leu in wool→ Cys-Cys in horn
(sulfurbridges= covalent bonding)
e.g. collagen: three intertwined helical polypeptide chains→superhelical
cable stabilized by H-bonds between strands
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, Biochemie
Tertiary Structure: Water-Soluble Proteins -Some polypeptides fold into multiple domains More info about the experiment
Fold into Compact Structures with Nonpolar
Cores
-Proteins fold into compact structures
e.g. myoglobin via X-ray crystallographic and nuclear magnetic
resonance (NMR)→ schematic model+ space filling model *still 3 main factors->each segment
*globular (dense, hydrophobe inside, hydrophile inside)
*compact
*Backbone atoms pack
Quaternary Structure: Polypeptide Chains
tightly together
*Side chains pack the Can Assemble into Multisubunit Structures
-Urea and guanidinium chloride disrupt the protein’s noncovalent bonds
intervening space -Arrangement and interaction of multiple polypeptide chains
*Van der Waal distance (subunits), such as in dimers or complex structures like
between atoms hemoglobin (α2β2 tetramer)
dimeren, tetrameren, ….
-Water-soluble proteins have nonpolar cores e.g.Cro is a dimer of
*blue= hydrophile →outside identical subunits
*yellow= hydrophobe →inside -Viruses make the most from
*Globular proteins hide a limited amount of information
their hydrophobic e.g. Human rhinovirus uses 60 copies of each of 4 subunits
side chains from solvent (left= hemoglobine, right=rhinovirus)
*Charged atoms rarely → disulphide bonds can be reduced reversibly
occur on the interior of a
protein
*Charged atoms prefer the
protein surface
-Inside-out architecture of membrane proteins
The Amino Acid Sequence of a Protein
Determines Its Three-Dimensional Structure
➢ Amino acids have different propensities for forming a 1. adding of urea and beta-mercaptoethanol (BME)
helices, b sheets, and turns *denaturation to random coil
Experiment Christian Anfinsen in 1950s.
Bovine pancreatic ribonuclease denatured (with urea and β-
mercaptoethanol) and then renatured.
*Shows proteins can refold into functional conformations
after denaturation
→Protein structure is determined by its amino acid
sequence, crucial for function.
2. renaturation: removal of urea and beta-mercaptoethanol
*dialysis: Denatured ribonuclease->Regains enzymatic activit->
-Certain combinations of secondary structure are common to Sulfhydryl groups oxidized-> Spontaneous refolding into native
many proteins (patterns)
structure (trial and error)
*helix-turn-helix (often for interactions with DNA)
*helix-b-sheet configuration (NAD+ interactions)
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, Biochemie
➢ Protein folding is a highly cooperative process Prediction of three-dimensional structure from sequence ▪ Parkinson's Disease
-All or nothing: there is no ‘partially folded’ protein at significant remains a great challenge Neurodegenerative disorder linked to the accumulation of alpha-
concentrations e.g. Predictions of secondary structure adopted by a stretch synuclein aggregates (Lewy bodies).
of 6 or 7 AA. Have proved to be 60 to 70 % accurate Misfolded alpha-synuclein proteins form fibrillar aggregates that
both VDLLKN-> different structure impair neuronal function.
Toxicity may arise from soluble oligomers disrupting cellular
processes and membranes, leading to neuron death.
➢ Some proteins are inherently unstructured and can exist
in multiple conformations
➢ Proteins fold by progressive stabilization of intermediates -Lymphotactin exists in two conformations that are in equilibrium
rather than by random search ➢ Protein modification and cleavage confer new capabilities
-Proteins do not fold by random search -Covalent Modifications for Enhanced Functionality
-Folding problem leventhal paradox: -Attachment of Special Groups
*Structure of most proteins are unknown (even if we know -Activation through Cleavage
sequence: no laws, no rules) e.g.
*why not random trial and error? Would take YEARS for a protein to form *Green fluorescent protein (GFP) has a unique structure.
100 residues ->Fluorescence is produced by the rearrangement and oxidation
3 conformations/residue= 3^100 = 5 x 10^47 structures of the amino acid sequence Ser-Tyr-Gly within the protein.
5 x 10^47 x 10^-13 seconds = 5 x 10^34 seconds = 1.6 x 10^27 years ➢ Protein misfolding and aggregation are associated with *GFP Mutants and Light Emission:
-Proteins fold by progressive some neurological diseases Various GFP mutants have been engineered to emit light across
stabilization of intermediates: -structurally changed proteins can become toxic to the brain
Cumulative selection → trial-and- environment
error: correct tries are being ▪ e.g. Mad cow disease ->Creutzfeldt-Jacob= Prion disease
remember/ kept= intermediate form
*e.g. folding pathway of chymotrypsin
inhibitor
MODELS
Space-filling model Ball-and-stick model Backbone model
*Infectious neurodegenerative diseases caused by misfolded
prion proteins (e.g., PrP^SC)->amyloid fibers
-BUT: suggests pathways= is not the case (not the best theory) • How: Abnormal prions aggregate and induce normal prion
*energetical levels= Folding funnel proteins to misfold, creating nucleation sites for further
Folding funnel
aggregation.
▪ Alzheimer's Disease Riboon diagram Ribbon diagram w highlights
*Neurodegenerative disorder characterized by the formation of
amyloid plaques. The amyloid-beta (Aβ) peptide aggregates to
form insoluble fibrils and plaques in the brain, derived from the
amyloid precursor protein (APP).
->Smaller aggregates may disrupt cell membranes, leading to
cellular toxicity and neurodegeneration.
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, Biochemie
CHAPTER 3: EXPLORING PROTEINS AND ➢ Proteins must be released from the cell to be purified Ion-exchange chromatography Separation based on el. charge
-Differential centrifugation: denser materials will form a *Positive Proteins:
PROTEOMES
pellet at lower centrifugal force Bind to negatively charged beads
➢ The proteome is the functional representation of the genome
homogenate forms ->supernatant -> cytoplasm (e.g., carboxymethyl cellulose)
➢ Proteins can be purified according to solubility, size, charge, in cation-exchange chromatography.
and binding affinity *Negative Proteins:
-Purification Indicator: Increased specific activity per step. Bind to positively charged
Sample Complexity: Fewer different proteins at each purification step beads (e.g., DEAE-cellulose)
Homogenate: Obtained by centrifugation in anion-exchange chromatography.
Salt fractionation Separation based on solubility
Standard technique: adding of ammoniumsulfate (reversible way of How to harvest protein back
-Protein encoding genes are just a list of ‘the parts of a car’ making a protein insoluble) from column (now its captured by
-The proteome is the functional representation of the genome the beads)?
-Only a subset of the gene products is actually expressed in a given -add something with higher
biological context, i.e. the proteome is not static concentration which competes
-Proteome is derived from proteins expressed by the genome *rough but cheap with your proteins for the beads
-Because the proteome is the functional expression of the genome, it *quick volume reducing Increasing salt concentration releases bound proteins by displacing
varies with cell type, developmental stage and environmental Dialysis to remove salts them with sodium ions, allowing proteins to elute in order of their
conditions -placing the protein mixture in charge strength. (in fractions: lineair gradient)
a dialysis bag made of a Affinity chromatography Separation based on functional
The Purificatin of Proteins Is an Essential First semipermeable membrane properties
with pores.
Step in Understanding Their Function ->Larger molecules, such as
➢ The assay: How do we recognize the protein that we are proteins, remain inside the bag,
looking for? while smaller molecules and
-Importance of Protein Purity: ions diffuse through the pores
*accurate amino acid sequencing into an external buffer solution
*remove contaminants + concentrate protein → Dialysis effectively removes small molecules (e.g., salts) but cannot
-Purification Process: isolation from complex mixtures through differentiate between different proteins
multiple separation techniques based on physical properties (e.g.,
Gel filtration chromatography Separation by size -Covalently attach target molecule (X) or its derivative to the column.
size and charge) to achieve a sample with only the target protein
-column packed with porous beads made of hydrated polymers (e.g., -Pass a protein mixture through the column and wash with buffer to
-Assays and Specific Activity: identifies the target protein by
dextran, agarose, or polyacrylamide). remove unbound proteins.
measuring unique properties, like enzyme activity.
*Large molecules: Cannot enter the pores -> remain in the solution -Elute the desired protein by adding a soluble form of X or altering
e.g. test for lactate dehydrogenase (NADH is fluorescent at 340nm->
between beads-> exiting the column first. conditions to reduce binding affinity
so testing fluorescent properties)
*Medium molecules: Sometimes enter the pores ->intermediate exit HPL-chromatography Separation based on size and charge
-Protein purification is to maximize the specific activity
time. =high-pressure-liquid chromatography
*specific activity will rise as the purification proceeds and the protein
*Small molecules: Enter the pores fully-> longer path and exiting last. -more advanced way of Column Techniques
mixture being assayed consists to a greater extend of your target
→ Larger molecules flow faster through the column, separation based on -Finer particles provide more interaction sites,
protein: pure protein has a constant and high specific activity
molecular size. (fractions) leading to greater resolving power
-Larger surfaces, More interaction sites
Smaller columns, More resolving power, Higher pressure
Faster separations
Higher peak=
Higher resolution
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, Biochemie
➢ Proteins can be separated by gel electrophoresis and -Separation Basis: Primarily based on protein mass (the more amino- ➢ A protein purification scheme can be quantitatively evaluated
displayed acids on a protein-> the bigger the protein-> the more negatively =calculating how pure the protein is at the end of the procedure
charged but all relatively equal to mass so smaller migrates faster *Activity of Protein: how well the protein works at each stage
trough medium) *Gel Test (SDS-PAGE): Creates a "protein fingerprint" that shows if unwanted
Detection: Coomassie blue, silver staining, or autoradiography for proteins are decreasing.
visualization. → Fewer bands = fewer unwanted proteins.
Reduction in protein bands across steps; one prominent band for
target protei *Total Protein: #total protein we have in each sample.
*Total Activity: # work the protein can do in each sample.
*Specific Activity: activity (or "pure") of target protein, compared to others.
*Yield: # of the original protein’s function we still have after each step.
-Gel Electrophoresis *Purification Level: # purer the protein is after each step.
*Principle: Charged molecules move in an electric field Our target protein’s band becomes more visible as it’s purified.
(electrophoresis). (smaller molecules fast, bigger slow (friction))
*Velocity Factors: Electric field strength, protein charge, and frictional →Good purification keeps enough of the target protein (high yield) but
coefficient. reduces contaminants.
V = Ez/f (snelheid evenredig met elektrisch veld, lading partikel en →Low yield with high purification means less target protein; high yield with
omgekeerd evenredig met frictie quotient) low purification means more contaminants
f = 6πήr (frictiequetient bevat straal van partikel)
➢ Ultracentrifugation is valuable for separating biomolecules
-Matrix: Polyacrylamide gel, acting as a molecular sieve and determining their masses
In a buffer acrylamide: radical polymerizes spontaneously to long *without SDS-page= no denaturation so by natural charge of protein
chains (fibers) +by adding bisacryl we link the chains Iso-electric focusing (no standard technique)
->viscous medium (bc of polymers) *iso-electric point= point on the molecule where total charge is 0
*proteins move to pH matching their pI
Two-dimensional electrophoresis
Combines isoelectric focusing (horizontal) with SDS-PAGE (vertical) *ultra-ctfgt.: high speed, separates small particles (DNA, RNA,
-1. Isoelectric focusing (separation by pI) viruses…)
-2. Second step: SDS-PAGE (separation by mass) *differential tcfgt.: stepswise, based on size and density through
-High resolution: Separates thousands of proteins (e.g., over 1000 in E. increasing speeds
coli) *zonal ctfgt.: based on density gradient (e.g., sucrose) in a
→ Protein spots on gel can be analyzed by mass spectrometry centrifuge tube to separate particles based on their sedimentation
*SDS-PAGE: Sodiumdodecylsulfate (detergent) denatures proteins, rate
adds uniform negative charge (per 2 amino-acids, 1 negatively *equilibrium ctfgt: particles move to
charged SDS: overall negative charge for protein) the point in a density gradient that
matches their own density and remain
in equilibrium there
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