Spectrophotometry and electrophoresis of haemoglobin

Question 1

Hb

Answer 1

in RBC 
transport O2 
structure senn by crystallography 
tetramer of 4 subunits 
2B 2a
a helical arrangements

Question 2

mutation in Hb

Answer 2

responsible for sickle cell and thalassemia

Question 3

what is electrophoresis

Answer 3

analysing molecules
on basis of charge
measure migration in electric field

Question 4

what is spectrophotometry

Answer 4

analysing molecules

spectral properties

Question 5

beginning electrophoresis

Answer 5

on cellulose acetate strips 
tris-glycerine buffer at pH 9.5 
Hb -ve charge 
migrate to anode
to avoid contamination wear gloves

Question 6

microapplicator

Answer 6

comes in 2 parts
lower tray - hold liquid samples
and upper applicator

Question 7

process of electrophoresis

Answer 7

pipette 20ul Hb samples into transparent sample trays of semi-micro applicator
remove 2 tabs from bridge which will secure the gel strips to the bridge
push flaps at end of bridge so they’re protruding
take gel strip from buffer - blot between filter paper
position strip on bridge- porous section uppermost - mitred edge at upper R or lower L corner
fold the flaps on the bridge - keep strip taut
return the pins
place multiple applicator in tray containing Hb
place index finger on orange button of applicator
push down 5/6times
teeth of applicator will pick up 1ul of Hb
position applicator on bridge
press gently on orange button
for 10sec
remove the applicator
place gel strips in tank
put lid on tank and switch on power - 200V 15mA for 1hr

Question 8

role of the bridge

Answer 8

securely holds acetate gel strip while loading and separating samples

Question 9

why do we blot the gel strip

Answer 9

remove excess buffer

Question 10

absorbance

Answer 10

fraction of incident light absorbed by the solution

Question 11

how do you measure absorbance

Answer 11

using a spectrophotometer
measures proportion of light absorbed compared to light form a blank
can be made at any wave length

Question 12

calculation for absorbance A

Answer 12

also called optical density

A=log10(light transmitted through blank soln/light transmitted through test soln)

Question 13

process of a spectrophotometer

Answer 13

sample in transparent plastic cuvette
into holder
lid closed
reading appears on LCD display
side of cuvette facing light is clear not frosted
machine adjusted for wl and set to absorbance
use blank - identical to solution, just w/o substance being measured
fill cuvette 3ml of soln
complete spectrophotometry at diff wl
determine wl with max absorbance - graphically/with measurements at closer intervals over these ranges

Question 14

what pipette is used to measure 3ml

Answer 14

P1000 micropipette

Question 15

what do you do after measureing the absorbance at diff wl

Answer 15

measure colour at each wl

see if same as Hb

Question 16

why is Hb visible in visible light

Answer 16

haem gp
unsaturated organic molecule complexed to Hb - stabalise Fe2+ion to form site where O2 can bind reversibly
change the electron resonance of haem - changing absorbance spectrum

Question 17

effect of removing O2 on absorbance

Answer 17

add reducing agent sodium dithionate 
invert cuvette to mix
strip O2 from Hb and change eq between Hb and O2 
deoxyhb is more purple 
for oxyhb peak ab at 540 and 577nm 
for deoxyhb - peak at 560nm

Question 18

why do you need to re-zero the machine with water at every wl

Answer 18

the machine does not give the same level of illumination at every wl

Question 19

using spectrometer to determine conc

Answer 19

absorbance is proportional to concentration and distance of light travelled

Question 20

beer lambert law

Answer 20

A = Ecl
E is extinction coeff - constant for substance being measured at that wl
c - conc mol/l
l - path length cm

Question 21

why is spectrophotometry useful to measure conc

Answer 21

many substances absorb light at some vol

if product/sub absorb light - can be used to determine the progression of a rn

Question 22

when do substances fail the beer lambert law

Answer 22

at high conc proteins may form dimers with diff E
linearity needs to be checked by creating a standard curve
this si exploited to find conc of unknown when no abs

Question 23

preparing a standard curve

Answer 23

make diff conc of Hb in test tubes

measure absorbance for each

Question 24

diagnostic use of spectrophotometry

Answer 24

follow changes in O2 binding by Hb - check resp status of newborns
spectrum change for diff Hb

Question 25

spectrometry for methaemoglobin

Answer 25

Fe2+ oxidised to Fe3+
impaired O2 binding
bluish chocolate colour

Question 26

methaemoglobinaemia

Answer 26

hereditary - deficiency in NADH methaemoglobin reductase which maintains Fe2+
mutations - Hb M = hb unamenable to reduction
acquired due to exposure- chemicals eg aniline dyes - P-chloroaniline, nitrates, local anesthetics - benzocaine

Question 27

carboxyhb

Answer 27

hb binds CO in place of O2

Question 28

colour at peak abs

Answer 28

light is green
colour of soln light that is transmitted
max absorption happens when light is opp colour to colour of soln
for red this is green

Question 29

determination of mystery Hb solns

Answer 29

done at wl with max absorbance - ie 540nm
beer lambert holds, ie line is linear, up to abs 1.0
best fit straight line drawn to experimental results

Question 30

analysis of electrophoresis

Answer 30

wear gloves
remove cellulose acetate strip
transfer to red staining soln for 3-5mins
blot off excess dye
sip strip into successive citric acid solns until background of strip white
blot to remove excess fluid

Question 31

interpretation of results from electrophoresis

Answer 31

normalk HbA travel further than HbS
therefore HbA more -ve than HbS
because of point mutation in B chain
hydrophilic -vely charged glutamate switched for valine, hyrdophibic and uncharged
hydrophobic - mean react with itself and not aq env = smaller = less able to carry O2 - ppte in soln and distort RBC
mixed contains bands from both HbA and HbS - were heterozygous