Proteins

Question 1

name 3 globular proteins

Answer 1

Myoglobulin, Haemoglobin, enzymes

Question 2

what are the 3 main classes of proteins

Answer 2

fibrous, membranes and globular

Question 3

globular structure of a protein is induced by?
they are soluble because

Answer 3

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

Question 4

Function of globular proteins x5

Answer 4

1. structural roles
2. transporters - other molecules through membrane
3. enzymes - catalyse organic reactions
4. regulatory role
5. messengers - transmit messages to control biological processes i.e. insulin

Question 5

primitive animals transport x in their blood but x to be adequate for most active animals

Answer 5

oxygen - but O2 solubility is far too low

Question 6

Myoglobin is x colour and found in what is its function

Answer 6

red, in striated muscles, function - oxygen store used during intense muscular activity when O2 consumption> O2 production

Question 7

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

Answer 7

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

Question 8

myoglobin- size, entire structure consists of?

Answer 8

small protein -153 A.A.
single polypeptide chain - 8 alpha helices each

Question 9

how is the haem p. group bonded to the myoglobulin needed for?

Answer 9

noncovalently - haem p is a non-polypeptide- biological activity O2 binding

Question 10

where are the highest concentrations of myoglobulin found + why

Answer 10

skeletal and cardiac muscle - during contraction they need lots of energy and therefore lots of O2

Question 11

compare haemoglobin to myoglobin

Answer 11

myoglobin- simple
haemoglobin- more complex and sophisticated

Question 12

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 ?

Answer 12

ferrous iron-tetrapyrrole, hydrophobic, hydrophilic carboxyl - intense, red-conjugated double bond system

Question 13

what 6 bonds can haem make

Answer 13

4 bonds- pyrrole nitrogen atoms
5th - Histidine residue
final- reversibly attached to oxygen

Question 14

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

Answer 14

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

Question 15

give another name for RBC WBC and platelets

Answer 15

erythrocytes
leukocytes
thrombocytes

Question 16

the haemoglobin curve is ? higher O2 concentrations is required for? x affinity comment about O2 and lungs, capillaries

Answer 16

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

Question 17

Haemoglobin is an allosteric protein means? binding O2 is said to be cx - what is contrast to this

Answer 17

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

Question 18

what haemoglobin is found in the foetus how is it different - comment on binding HbF binds X x than HbA

Answer 18

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

Question 19

what happens near birth

Answer 19

synthesis for y chain is switched off while synthesis of B chain is switched on

Question 20

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

Answer 20

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

Question 21

how is sickle cell anaemia transmitted, describe RBC in this condition

Answer 21

genetically transmitted- more fragile so lyse easier and have shorter lifespan - leads to severe anaemia

Question 22

patients with sickle cell anaemia effects?

Answer 22

shorter-life span due to renal failure, cardiac failure or thrombosis [clots forming in blood vessel] - red blood cells become trapped in blood vessels

Question 23

Bohr effect

Answer 23

drop in Ph causes reduces haemoglobin affinity

Question 24

the amount of light absorbed=, give eg. x6 of spectroscopic methods of protein quantification + detection

Answer 24

=directly proportional to concentration of sample, Absorbance at 280nm- Colorimetric method [prosthetic absorbance group], biuret method, lowry method, Bradford assay, Bicinchoninic acid assay

Question 25

UV absorption- @? - what A.A. absorbs UV light- what does protein absorb at this wavelength

Answer 25

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

Question 26

Interference of absorbance what can interfere? does it lead to how is this known

Answer 26

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

Question 27

What are the advantages of detecting+ quantifying protein using spec
? Disadvantages x3

Answer 27

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

Question 28

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

Answer 28

haemoglobin- heme A -oxyhaemoglobin & deoxyhaemoglobin - colour deoxy-darker red
different spectrums

Question 29

another e.g. of Prosthetic Group? C. function? + contains?

Answer 29

Cytochrome C552 - electron carrier - heme C

Question 30

Prosthetic group R. found where? function.? contains? /Structure absorbance @ what colour + wavelength - what’s colour, benefit

Answer 30

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

Question 31

uses of these intrinsic proteins x3

Answer 31

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]

Question 32

what 3 spectroscopic methods are used for detection and quantification of proteins

Answer 32

lowry method, biuret method, bradford assay

Question 33

what is the principle of the Biuret Reaction/ test

Answer 33

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

Question 34

what 3 reagents make up the biuret reagent

Answer 34

copper sulphate, sodium potassium tartrate (prevents Cu2+ ions precipitating out of alkaline conditions and sodium hydroxide provide alkalinity

Question 35

how to detect concentration of unknown [biuret assay analysis]

Answer 35

prepare standard curve with known BSA protein concentration absorbance values and use graph to detect unknown concentration [take dilutions into account - blue colour produced

Question 36

Advantages of Biuret reaction x3

Answer 36

1. can be scaled down to decrease protein volume req. [limited] 2. simple + reliable 3. can be automated

Question 37

what disadvantages are associated with Biuret Reaction x3

Answer 37

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

Question 38

Principle of Lowry Method

Answer 38

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

Question 39

Advantage of Lowry Method

Answer 39

more sensitive than Biuret method 20micrograms/ml - 2mg/mL of protein compared to Biurets 500microgr/mL to 2mg/mL.

Question 40

disadvantage of Lowry

Answer 40

• 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

Question 41

Bichinoic Acid [Pierce Assay] - what is the principle

Answer 41

modified version of Lowry Method - Cu-protein complex is formed and further combined with bichinoic acid- absorbs@ 562nm - purple colour [microtiter plate]

Question 42

Advantages

Answer 42

• suffer less from interference than lowry method & - very sensitive

Question 43

Disadvantages

Answer 43

• original reagents - subject to interference from lipid and detergent [Detergents need to be modified] - reagent is stable only for a short time

Question 44

Bradford assay [Bio-rad]- principle

Answer 44

a dye binds to protein molecules in solution [acidic solution] wavelength shifts - absorbance @ 595nm - directly related to protein concentration - blue colour