Chem Flashcards
proteins and polypeptides
are polymers of aa linked together by peptide bonds thanks to nucleophilic attack by amino group of one aa to carboxyl of other aa
reaction not spontaneous
- to make it spontaneous;
1. aa activated by tRNA
2. forms aminoacyl-tRNA
3. energy needed provided by hydrolysis of ATP
4. peptide bond formed is spontaneous
peptides can be divided in
oligopeptides - few aa residues
polypeptides - many residues
proteins - more than 10,000
peptides - negative and positive
negative - c terminal carboxyl
positive - aa group
how to degrade protein quickly
- using enzymes
- spontaneous + thermodynamically favored BUT kinetically unfavored
- peptide bond - stable, broken by enzymes
mutation of proteins
carboxyl group is modified
- methylated
- amidated
- carboxyl groups converted to amide group
- negative charge lost
carbohydrates
biggest source of energy
building blocks for molecules
made of C, H and O and are also called saccharides
types:
monosaccharides - simplest
disaccharides - two mono.
polysaccharides - many mono.
hydrolysis
divided 2 mono. using a molecule of water
dehydration
the union/combination of two mono. with the loss of H2O
monosaccharide groups
many hydroxyl groups attached by chains of 3-8 C atoms
hydroxyl groups are attached to C except carbonyl C
aldehyde groups - aldoses
ketone groups - ketoses
fisher projections - aldehydes
top - most oxidized
center - OH and H
bottom - chiral atom
if OH is bonded on left - L isomer (L - left)
if OH is bonded on right - D isomer (D - destra)
most important 6Cs + 5C
D-glucose
- aldohexose
- building block for disaccharides
D-galactose
- aldohexose
- important in the cellular membranes of the brain
D-fructose
- ketohexose
- sweetest and only has 3 chiral centers
- obtained from sucrose
- important for the production of energy
D-ribose
- aldopentose (5C)
- sugar of RNA
hypoglycemia and hyperglycemia
hypoglycemia - the level of sugar in the blood is less than 40 because there is an overproduction of insulin
hyperglycemia - can be caused by diabetes [when the pancreas cannot produce enough insulin (diabetes 1) or when cells cannot respond to insulin (diabetes 2)]
normal blood sugar level - 70/90
haworth structure - ketones
either:
alpha - OH under anomeric carbon
beta - OH over anomeric carbon
oxidation of mono.
they have an aldehyde group and OH
OH can be oxidized to form carboxylic acid
reduction of mono.
reduce carbonyl group convert aldehyde into alcohol producing alditol (sugar alcohol)
disaccharides
formed by 2 mono. bonded by glycosidic bond thanks to dehydration reaction
types of disaccharides
maltose
- made from 2 D-glucose (either alpha or beta)
- alpha contains hemiacetal group and therefore can interact with alcohols –> acetal group (bond on C4)
lactose
- made from D-galactose and alpha/beta D-glucose
- bond formed between C1-C4
sucrose
- made from alpha-D-glucose and beta-D-fructose
- bond formed between C1 and C2
- cannot form an open chain or be ox.
polysaccharides (types)
starch
- formed from 2 poly. amylase and amylopectin
amylose
- made of molecules of alpha-D-glucose bonded by alpha (1-4) glycosidic bond
- form a coiled structure
amylopectin
- contains same bonds as amylose + alpha (1-6) bonds that allow the protein to be packed in a more condense way
glycogen
- a polymer of glucose
- in the liver and muscles and sometimes lungs
- bonds are the same as amylopectin except bonds 1-6 happen every 10-15 units of glucose
cellulose
- structural unit of wood and plants
- formed by linear beta-(1-4) glycosidic bonds (opposite of amylose)
- OH group cannot react with water thus molecule is insoluble and rigid
lipids
organic compounds from biological origins
soluble in organic solvents but insoluble in water thus most of them apolar
functions of lipids
energy storage
central components of membrane
produce energy for metabolism
signal molecules
fatty acids
long acyl chains with thermal carboxylic function
the longer the chain, the lower the solubility
can have two isomers - cis and trains
- if H groups are on the same side (cis)
- if H groups are on the opposite sides (trans)
prostaglandins - eicosanoids
derive from arachidonic acid
any of these acids can transform in a prostaglandin through activity of cyclooxyrgenase enzyme - responsible for inhibition and synthesis of prostaglandins
waxes
have vegetal or animal origin
esters that form through saturated Fas and long chain alcohols - hydrophobic
protective function
triglycerides
ester molecules with OH
glycerol if esterified –> 3Fas with formation of 3 ester bonds
- Fas can be sat. or unsat. or both
used as storage
triglyceride reactions
- addition of H2 to double bond –> hydrogenated fats
- addition of water –> alcohol
- hydrolysis of ester bond –> acid and alcohol
- oxidation –> aldehyde and acids
- saponification –> create soaps
glycerophospholipids
similar to triglycerides BUT have an ester linkage to phosphoric acid (thus forms phosphoester bond) in position 3 with an alcohol or aa alcohol
the aa alcohol can be:
- choline
- serine
- ethanol amine
sphingolipids
composed of sphingosine (present in brain and nervous tissue)
ceramides - amide bond between amino group that is present in sphingosine and acyl chain
sphingomyelin - amino bond to fatty acid mol. and through ester bond to phosphoric ester of amino alcohol choline
glycosphingolipids
contains sugar and are divided in:
- cerebrosides - glycosidic linkage between alcohol function of terminal part of sphingosine and alpha anomeric C of either galactose or glucose (cellular recognition)
- gangliosides - 2 or more saccharodic units attached to sphingosine, characterized by silica acid presence (receptors)
steroids
common core structure composed by three 6-membered rings and one 5
cholesterol - basic core structure with double bond, OH group at position 3 and branched acyl chain at position 17
- maintains fluidity (HDL)
bile salt - derived from cholesterol and secreted in gall bladder
- helps us digest FAs (works as soap)
steroid hormones - synthesized by cholesterol and used to transport chemical messages in our body
cortisone - increases glycemia and synthesis in liver of aa
protein purification - chromatography
produce protein
isolation and purification
extraction
purification
protein purification - column chromatography
used to separate proteins
- stationary (solid porous matrix) and mobile (eluent) phase
- eluent poured in stationary phase
- protein present in sample is separated (depending on type of stationary phase and interaction)
protein purification - size or gel filtration chromatography
stationary phase - porous polymer bead
filtration based on size exclusion technique
- each bead that is hydrophilic has different size and molecule may enter or not
- also based on function of hydrodynamic radius, size and molecular weight
- small molecules take longer to be eluted
- after filtration we can attach spectrophotometer to measure absorbance
protein purification - ion exchange chromatography
stationary phase - chemically modified in order to have positive or negative charge
method based on interaction of protein with stationary phase
- beads are negatively charged when sample is loading proteins (+)
- proteins (+) bind with column while (-) will be eluted
- to detach + from column, change pH of eluent = change of charge of protein
protein purification - affinity chromatography
stationary phase - beads chemically modified with ligand of protein
based on interaction of protein with stationary phase by bio-selective non-covalent binding
- when proteins eluted, protein that can be bio-selective, eluent will bind to column
- impurities washed out
- to detach protein from column change pH or ionic strength or use denaturing agent
protein purification - hydrophobic interaction chromatography
stationary phase - beads chemically changed in order to have surface some hydrophobic group
technique based on interaction of hydrophobic regions of a protein with surface of beads
- protein with hydrophobic regions will bind to beads
- increasing length of hydrophobic group, increase hydrophobicity
- protein detached by decreasing salt conc.
dialysis
use for protein purification
methods used for separation of proteins from salts
electrophoretic techniques
used for characterization of protein while chromatography for preparative functions
electrophoretic techniques - polyacrylamide gel electrophoresis
based on migration of charged proteins in electric field
- proteins start to migrate in electric field and 2. migration depends on charge, size and molecular shape
electrophoretic techniques - isoelectricfocusing
separate proteins as a function of isoelectric point
- put a gel into a tube
- after an electric current is applied, ampholytes migrate and create different pH along tube generating pH gradient
- put proteins at top of sample and protein migrates through gel
- migrate until they find their pH corresponding to isoelectric point
purification and characterization of proteins - determination of aa sequence
edman degradation methods
- determine to allow aa composition
- need original protein intact
- need phenylisothiocynate (PITC)
we can label a protein of 50 aa so if protein is longer it needs to be cut and analyzed fragment by fragment
protein denaturing and folding
denaturing = unfolding a protein = loss of function
denaturing agents:
- heat
- pH
- solvent
- urea
- detergents
monitoring - with fluorescent tryptophan
renaturing protein
anfinsen’s experiment - renaturing is a spontaneous process
levinthal’s paradox - describe folding of a protein with Molten globule model
- protein start from disordered structure
- becomes ordered
folding can be assisted by molecular chaperones
- both need to use ATP
ligand
molecule that can bind a protein in a reversible way
it can bind to binding site of protein
protein functions
protein that binds to O2
- heme group is prostetic group (permanently bonded to protein)
- O2 is bonded to heme group thanks to iron in it
hemoglobin
multivalent allosteric homotropic
protein that undergo a conformational change when O2 binds to it
multivalent
one mol. can bind more than one ligand
allosteric
binding to O2 at one site can modify structures of others
homotropic
one mol. of O2 can modify binding of another mol. of O2
sequence and structure of aa
determine the structure and function of a protein
general formula and structure of aa
an alpha C attached to:
- amino group NH2
- carboxylic group COOH
- side chain R
- hydrogen H
non-polar –> hydrophobic
tends to stabilize proteins’ structure using hydrophobic interaction
- glycine –> only non chiral –> R group and H
- alanine –> R group –> methyl group
- valine –> R group –> isopropyl group CH-(CH3)2
- leucine –> more hydrophobic than valine –> R group –> isobutyl group (CH3)2-CH-CH2
- methionine –> one of the 2 sulfur containing aa –> R group –> aliphatic side chain with 4C
aromatic
relative non-polar (hydrophobic) = can participate in hydrophobic interaction
- phenylalanine –> R group –> methyl + phenyl
- tyrosine –> R group –> phenyl group + OH
- tryptophan –> R group –> indoxyl group C8H7N (cyclic)
polar uncharged
more soluble in water and contain functional group that can form H bond
- serine –> R group –> OH and amido group
- theorize –> R group –> OH
- cysteine –> R group –> thiol group SH
- aspartate –> R group –> carboxyl COOH
- glutamate –> R group –> C3H5O2
- proline –> R group –> amino group C-N
positively charged (basic)
- lysine –> second primary amino group on fourth C in aliphatic chain
- arginine –> R group –> guanidine group CH5N3
- histidine –> R group –> imidazole group C3H4N2
negative charged - acidic
aspartate and glutamate
- both have second COOH but with OH not amino
non standard aa
aa found in proteins
once protein have been produced in cell, can undergo any modification
formation of S-S bond between cysteine residues
cysteine has thiol group that undergoes ox. to from S-S with another cysteine molecule in present of oxidant
aa are amphoteric
can behave as either acid or a base
zwitterionic form - with charges
enzyme inhibitor (reversibile inhibitors) - competitive inhibitor
molecule that competes against substrate to reach enzyme’s active site
increases Km
does not affect Vmax
enzyme inhibitor (reversibile inhibitors) - uncompetitive inhibitor
only block processes beyond ES formation
binding site of inhibitor is different from substrate one and can bind to enzyme-substrate complex
both Vmax and Km are changed
enzyme inhibitor (reversibile inhibitors) - mixed inhibitor
combination of competitive and uncompetitive inhibitor
the binding site of inhibitor is different from substrate one
can increase affinity of enzyme for substrate - uncompetitive inhibition
can decrease affinity for substrate - competitive inhibition
Vmax will decrease
enzyme inhibitor (reversibile inhibitors) - non- competitive inhibitor
an inhibitor binds to enzyme at location other than active site
Km does not change
Vmax decreases
enzyme inhibitor - irreversible inhibitors
substance that permanently blocks action of an enzyme
inhibitor and enzyme are bonded with covalent bond
enzyme inhibitor - suicide inhibitors
inhibitors bind to enzyme’s active site creating irreversible covalent complex
leads to modification of inhibitor and formation of other molecules that react irreversibly with enzyme too
end of process the enzyme’s molecule is useless
antibodies
are produced by B lymphocytes that differentiate into plasma cells that secrete antibodies into blood
antibodies - immunogen
compound capable of inducing immune response
antibodies - antigen
compound recognized by produce of immune response
antibodies - hapten
compound that can induce an immune response only when couples by carrier protein
BUT after producing antibodies capable to recognize hapten + carrier complex
antibody will be able to bind also to hapten freed from carrier
antibodies - antigenic determinant/epitope
antibody binding site of antigen
antibodies - paratope
antigen binding site of antibody
immune response
response that organism gives against pathogen or foreign compound
cellular response - mediated by cells
humoral response - mediated by secreted antibodies
immunoglobuline (antibody)
divalent molecules made of 4 chains structure not constant arms are flexible interacts with antibody --> change in conformation of paratope (inducing fitting)
CDR
in arms of antibodies there are hyper variable regions that allow extensive range of antibodies
fragment of antibodies
possible to generate fragments by using enzymes
papain claves the S-S so that Fc and one of the two Fab are detached from other Fab - monovalent antibody
pepsin claves C-C so that Fc is removed = 2 Fab - divalent antibody
affinity
strength of interaction between epitope and paratope
avidity
strength of overall binding
monovalent have higher affinity
divalent have higher avidity
epitopes
small part of antigen that binds specific antibody
accessible region
hydrophilic region
flexible region
non=conservated region
antibody classification
IgM - represents first response IgG - replaced IgM + lower avidity but higher affinity and specificity IgA - defense of our mucosa IgE - allergic responses IgD
production of monoclonal antibodies
- inject mouse with antigen
- collect spleen of mouse where B-cells are stimulated to proliferate
- fuse cell with myeloma cell
- hybridomas
- spleen cell dies and myeloma with them
- after few days hybridoma comes back
select hybridomas we want
- separate cell
- all separate cells will duplicate
- immunoassay or acidic fluid
- precipitation with ammonium sulfate
- affinity chroma.
- ion exchange chroma.
- gel filtration
production of antibodies by phage libraries
phage libraries - virus that can infect bacteria
- column with immobilized antigen
- phages glow into column
- all other phages washed away
- gather bond phages and amplify it
polyclonal and monoclonal application
- research
- in vitro diagnosis
- in vivo diagnosis
- therapy
regulatory enzymes - allosteric mechanisms
a ligand binds to one side of mol. resulting in conformational change on other side of mol.
can be positive - increase activity - stabilize active form = curve shift to left
can be negative - decrease activity - stabilize inactive form = curve shift to right
regulatory enzymes - covalently modified enzymes
enzymes can be activated or deactivated using phosphorylation and dephospho rylation reactions
- where phosphoric group attached or detaches to enzymatic protein
phosphorylation
reversible
protein kinases are responsible for catalyzing transfer of terminal phosphoric group from ATP onto hydroxyl-containing residue
methylation
irreversible
remove phosphoric group by using enzyme phosphatase
regulatory enzymes - enzymes affected by regulatory proteins
protein binds to enzymatic protein
thanks to binding, enzyme activity can be regulated through allosteric mechanisms
regulatory enzymes - enzymes activated by proteolytic cleavage
enzymes produced in an inactive form
can be activated through proteolytic cleavage operated by another enzyme
regulated by proteolytic cleavage of enzyme precursor
