Proteins Flashcards
name 3 globular proteins
Myoglobulin, Haemoglobin, enzymes
what are the 3 main classes of proteins
fibrous, membranes and globular
globular structure of a protein is induced by?
they are soluble because
the proteins’ tertiary structure
the nonpolar A.A. are buried in interior hydrophobic region
the polar - on its surface and reacts with hydrophilic medium - dipole-dipole interactions occur - soluble
Function of globular proteins x5
- structural roles
- transporters - other molecules through membrane
- enzymes - catalyse organic reactions
- regulatory role
- messengers - transmit messages to control biological processes i.e. insulin
primitive animals transport x in their blood but x to be adequate for most active animals
oxygen - but O2 solubility is far too low
Myoglobin is x colour and found in what is its function
red, in striated muscles, function - oxygen store used during intense muscular activity when O2 consumption> O2 production
Hemoglobulin function? is it a big amount cells of dry content % and % of total, Hemoglobulin structure-, what is the major type of haemog in adults, this is made up of? notation what do they form?
describe what a polypeptide chain consists of in a haemoglobin and myoglobulin? what’s inside hydrophobic crevice
oxygen carrier - big component dry- 97%, total (cell + water)- 35%
structure - 4 polypeptide chains
HbA- 2 alpha chains, 2 beta chains (α2β2)- different types of polypeptides
tightly packed spherical structure held together by noncovalent interactions
8 alpha helices each - haem prosthetic group in interior aids to reversible O2 binding
myoglobin- size, entire structure consists of?
small protein -153 A.A.
single polypeptide chain - 8 alpha helices each
how is the haem p. group bonded to the myoglobulin needed for?
noncovalently - haem p is a non-polypeptide- biological activity O2 binding
where are the highest concentrations of myoglobulin found + why
skeletal and cardiac muscle - during contraction they need lots of energy and therefore lots of O2
compare haemoglobin to myoglobin
myoglobin- simple
haemoglobin- more complex and sophisticated
Haem is a x x x with x side chains on 3 sides and z group on fourth side, what colour is haem and this is due to ?
ferrous iron-tetrapyrrole, hydrophobic, hydrophilic carboxyl - intense, red-conjugated double bond system
what 6 bonds can haem make
4 bonds- pyrrole nitrogen atoms
5th - Histidine residue
final- reversibly attached to oxygen
O2 saturation Curve
The myoglobin curve is x M is almost fully saturated at x found in this means? therefore its x carrier in blood
hyperbolic- low O2 pressure - capillaries high affinity and will not release O2 at this low O2 pressure - only release O2 when muscular activity further lowers press- reverse store of O2 in time of need- its unsuitable O2 carrier
give another name for RBC WBC and platelets
erythrocytes
leukocytes
thrombocytes
the haemoglobin curve is ? higher O2 concentrations is required for? x affinity comment about O2 and lungs, capillaries
sigmoidal - 50% saturation i.e. so it releases it easily- lower affinity - readily picks up O2 in lungs + readily releases O2 in capillaries - steep at O2 pressure in lungs means it can become saturated O2, but low at capillary O2 pressure - so releases
Haemoglobin is an allosteric protein means? binding O2 is said to be cx - what is contrast to this
when 1 subunit binds to O2 induces conformational changes to other subunits- they a higher affinity for O2 and bind more to O2- cooperative
single binding of O2 to single polypeptides is said to be noncoperative
what haemoglobin is found in the foetus how is it different - comment on binding HbF binds X x than HbA
HbF - consist of 2 alpha and 2 y chains - higher affinity allows for optimised transfer of O2 from maternal to foetal circulation through placenta
binds 2,3 bisphosphoglycerate more strongly
what happens near birth
synthesis for y chain is switched off while synthesis of B chain is switched on
characterised abnormal haemoglobin has been characterized as? what is the most common? what type of haemoglobin does this have describe shape of this RBC, what gives rise to this mutation - what gives rise to crescent shape. what happens when there is high concentration of deoxy-Hbs why does it happen
haemoglobinopathies - sickle cell anaemia- sickle cell haemoglobin- HbS -crescent shaped haemoglobin- change = glutamic acid residue is changed for a valine residue in beta chain so hydrophilic residue is substituted instead for hydrophobic residue- sticky, in corner between E and F helices theres deoxy-Hbs complementary to sticky patch- this part binds to sticky patch - crescent shape. long fibres form- oxy-HbS is masked
how is sickle cell anaemia transmitted, describe RBC in this condition
genetically transmitted- more fragile so lyse easier and have shorter lifespan - leads to severe anaemia
patients with sickle cell anaemia effects?
shorter-life span due to renal failure, cardiac failure or thrombosis [clots forming in blood vessel] - red blood cells become trapped in blood vessels
Bohr effect
drop in Ph causes reduces haemoglobin affinity
the amount of light absorbed=, give eg. x6 of spectroscopic methods of protein quantification + detection
=directly proportional to concentration of sample, Absorbance at 280nm- Colorimetric method [prosthetic absorbance group], biuret method, lowry method, Bradford assay, Bicinchoninic acid assay
UV absorption- @? - what A.A. absorbs UV light- what does protein absorb at this wavelength
280nm - tryptophan + tyrosine [more], phenylamine +cysteine [absorb less] - most proteins have multiple aromatic A.A. so absorbs @ 280nm- not absolute as variation in aromatic A.A. content so variation in variable extinction coefficients
Interference of absorbance what can interfere? does it lead to how is this known
DNA absorbs UV light at 260nm - other contaminants absorb UV light. nucleic acid absorbs UV light 10X more than proteins. ratio A280/A260- criteria if not right due to contamination
What are the advantages of detecting+ quantifying protein using spec
? Disadvantages x3
sample-not destroyed
rapid
can be accurate- when comparing different solutions of same protein
Dis- can’t be called accurate as difference of try, tyr, phe, disulphide bridges from protein-protein
standard curve is usually required - with known protein concentrations-compare
Quartz cuvette needed- pretty expensive as glass + plastiic absorb UV light
Give Example of prosthetic molecule- what prosthetic group does it contain what are the 2 forms - how to they differentiate? impact does that have on absorbance
haemoglobin- heme A -oxyhaemoglobin & deoxyhaemoglobin - colour deoxy-darker red
different spectrums
another e.g. of Prosthetic Group? C. function? + contains?
Cytochrome C552 - electron carrier - heme C
Prosthetic group R. found where? function.? contains? /Structure absorbance @ what colour + wavelength - what’s colour, benefit
Rhodopsin - rods of eye - vision in dark -retinal - in 7 transmembrane helices green light @ 495nm - purple so reagent does not need to be added- heme C
uses of these intrinsic proteins x3
Determination of protein presence due to maxima characteristic i.e., oxyhaemoglobin absorbs at 405, determination of protein concent. [as long as you know extinction co-efficient] using Lambards law, spectrum can be used to determine different forms of the protein i.e. oxy + deoxyhaemoglobin, electron bond [reduced cytochrome C] + electron free [oxidized bound]
what 3 spectroscopic methods are used for detection and quantification of proteins
lowry method, biuret method, bradford assay
what is the principle of the Biuret Reaction/ test
proteins react with alkaline copper solution. the Cu2+ forms complex with NH groups provided by the peptide bonds of the protein, and reduced to Cu+ a purple colour is produced as a result of this reaction this colour change is measured using a spec with a maxima of 300-545nm usually 545nm is used as interference occurs at 300nm. the colour is related to protein peptide bonds present greater peptide bonds = deeper colour change
what 3 reagents make up the biuret reagent
copper sulphate, sodium potassium tartrate (prevents Cu2+ ions precipitating out of alkaline conditions and sodium hydroxide provide alkalinity
how to detect concentration of unknown [biuret assay analysis]
prepare standard curve with known BSA protein concentration absorbance values and use graph to detect unknown concentration [take dilutions into account - blue colour produced
Advantages of Biuret reaction x3
- can be scaled down to decrease protein volume req. [limited] 2. simple + reliable 3. can be automated
what disadvantages are associated with Biuret Reaction x3
can consume lots of protein, have limits [upper limit of 2.0 mg/mL and lower limit of 0.5mg/mL where beer lambert law is still obeyed, necessary to make standard curve with known concentrations to allow identification of unknown
Principle of Lowry Method
same in terms of Cu2+ being reduced. but also contains folin ciocalteau which is made up of 2 acids phosphomolybdic and phosphotungstic which are reduced by phenolic amino acids tryp and tyrosine in the Cu-protein complex this produces molybdeum blue and tungsten blue which intensifies blue color
Advantage of Lowry Method
more sensitive than Biuret method 20micrograms/ml - 2mg/mL of protein compared to Biurets 500microgr/mL to 2mg/mL.
disadvantage of Lowry
- calibration curve is necessary 2. depends on tryp and tyros amounts so will vary protein to protein and is subject to interference from K Mg e.g. EDTA and Tris buffer and other phenolic compounds
Bichinoic Acid [Pierce Assay] - what is the principle
modified version of Lowry Method - Cu-protein complex is formed and further combined with bichinoic acid- absorbs@ 562nm - purple colour [microtiter plate]
Advantages
- suffer less from interference than lowry method & - very sensitive
Disadvantages
- original reagents - subject to interference from lipid and detergent [Detergents need to be modified] - reagent is stable only for a short time
Bradford assay [Bio-rad]- principle
a dye binds to protein molecules in solution [acidic solution] wavelength shifts - absorbance @ 595nm - directly related to protein concentration - blue colour
