1. Isolation &
Purification of
Phosphorylase b
from Rabbit
Skeletal Muscle
Presented by,
SOURAV MONDAL
Reg No. 14YUST4021
Under the guidance of,
Dr. C R SUBHASHINI
Director, Aristogene Biosciences Pvt Ltd.
& co-guidance of,
Dr. SOMA CHAKI
HOD, Dept. of Biotechnology,
ABBS college, Bangalore.
July,2016

2. • Phosphorylases are enzymes that catalyze the addition of a phosphate group from
an inorganic phosphate (phosphate+hydrogen) to an acceptor.
• Phosphorylases are also a common name used for enzyme glycogen phosphorylase.
• Glycogen phosphorylase catalyzes the rate-limiting step in glycogenolysis in
animals by releasing glucose-1-phosphate from the terminal alpha-1,4-glycosidic
bond.
• The glycogen phosphorylase monomer is a large protein, composed of 842 amino
acids with a mass of 97.434 kDa in muscle cells. While the enzyme can exist as an
inactive monomer or tetramer, it is biologically active as a dimer of two identical
subunits
16-11-2016Sourav Mondal/14YUST4021/ABBS 2

3. • Phosphorylase b is used to study the conversion mechanism of inactive
phosphorylase b to active phosphorylase in muscle.
• Phosphorylase b is used to study which factors influence the conversion of
phosphorylase b to phosphorylase a such as temperature, AMP, fluoride and
detergents.
• It is used to study phosphorylase b deficiency mutations.
• To be used as a marker in electrophoretic studies.
16-11-2016Sourav Mondal/14YUST4021/ABBS 3

4. • Although, there is no hard and fast rule for selecting tissue and/ or organism for the
isolation of an enzyme, it is always preferable to select a source enriched in that
particular enzyme.
• First, it is required to check from the literature whether the enzyme occurs
universally (in animals, plants as well as microbes) or confined to a particular
Kingdom.
• In 1943 rabbit skeletal muscle phosphorylase b was isolated in the crystalline form
by Green and Cori. The crystalline enzyme possessed 60 to 70 per cent of its
maximal activity in the absence of AMP.
• In 1957, direct isolation and crystallization of rabbit skeletal phosphorylase b was
carried through by Edmond H Fischer and Edwin G Krebs.
16-11-2016Sourav Mondal/14YUST4021/ABBS 4

5. 16-11-2016Sourav Mondal/14YUST4021/ABBS 5
50% pellet was found to have
more enzyme hence it was
taken further (5.5ml) to carry
out dialysis followed by
removal of contaminants.

6. • 0.03 M cysteine and 0.5 mM EDTA was
added to the dissolved 50% pellet.
• pH was increased to 8.6 by 1 M Tris-
HCl buffer with heat tretment.
• pH was brought down to 7.0 by 1 N
CH3COOH
• 0.1 M AMP was added along with 1 M
MgCl2 to get the precipitate formation
over a period of 24 hours.
• Formed Pb precipitate was analysed on
SDS PAGE and to the supernatant
salting out process was repeated.
16-11-2016Sourav Mondal/14YUST4021/ABBS 6
Lan
es
Sample loading Pattern
1 Marker 20µl
2 Precipitate 60µl
3 50% pellet (ppt) 60µl
4 70% pellet (ppt) 60µl

7. • Bed volume used = 10
ml
• Bound proteins was
eluted using salt gradient.
• pH was set to be 6.0
• KCl concentration
difference (from 50mM
to 500mM) was
employed.
• 23 Fractions were
collected containing 4 ml
each eluted samples.
• Absorbance data was
analysed by plotting a
graph and through
loading them on to SDS
PAGE
16-11-2016Sourav Mondal/14YUST4021/ABBS 7
0
0.5
1
1.5
2
2.5
3
ODat280nm
Fraction Number
Lanes Sample loading Pattern
1 Marker 20 µl
2 Load 25 µl
3 Breakthrough 30 µl
4 Wash 60 µl
5 Elution 3 60 µl
6 Elution 4 60 µl
7 Elution 5 60 µl
8 Elution 7 60 µl
9 Elution 9 60 µl
10 Elution 11 60 µl

8. 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
ODat280nm
Fraction number
Purification of Rabbit Pb by
DE52 cellulose chromatography
in Buffer I & II (20*4ml),pH 6
16-11-2016Sourav Mondal/14YUST4021/ABBS 8
Lan
es
Sample loading Pattern
1 Marker 20 µl
2 Load 20 µl
3 Breakthrough 60 µl
4 Wash 60 µl
5 Elution 2 50 µl
6 Elution 3 30 µl
7 Elution 4 50 µl
8 Elution 6 60 µl
9 Elution 8 60 µl
10 Elution 10 60 µl

9. • Used to preserve a liquid by
withdrawing the water
through sublimation under
vacuum.
• Follows mainly 3 stages
• 70 ml of eluted fractions was
kept for lyophilization for 48
hours.
• From 5 ml of lyophilized Pb,
protein concentration was
measured (Lowry’s method)
• Purified enzyme was loaded
on to SDS PAGE too to
observe the single band
appearance.
16-11-2016Sourav Mondal/14YUST4021/ABBS 9
Sl no Amou
nt of
standa
rd
BSA(
l)
Concentr
ation
(µg/tube)
Amoun
t of
distille
d
water(
l)
3ml of
compl
ex
formi
ng
reagen
t
Incub
ate at
RT for
10min
s
0.3ml
of
Folins
reagen
t
Incub
ate at
RT
for
30mi
ns
OD
660nm
1. 0 Blank 200 0.00
2. 10 10 190 0.023
3. 20 20 180 0.034
4. 40 40 160 0.256
5. 80 80 120 0.455
6. 120 120 80 0.615
7. 160 160 40 0.760
8. 200 200 0 0.936
T1 10 25.3 190 0.122
0
0.2
0.4
0.6
0.8
1
1.2
0 50 100 150 200 250
ODat660nm
Concentration of BSA in µg
Protein estimation by Lowry's method

10. 10µl of test sample was found to
have a concentration of 25.3 g
of phosphorylase b, hence 1ml
of test sample will have the
concentration of;
25.3  1000 / 10
= 25.3100 g/ml
= 2530 g/ml
Or
2.53 mg/ml
Hence from 5 ml of lyophilized
sample we were able to purify 5
 2.53 = 12.5mg of
phosphorylase b.
16-11-2016Sourav Mondal/14YUST4021/ABBS 10
Lane
s
Sample pattern
1 Marker 10µl
10 µl
sample
loading
buffer
to all.
2 Pb Lyophilized 01µl
3 Pb Lyophilized 02µl
4 Pb Lyophilized 04µl
5 Pb Lyophilized 10µl
References
• Journals
Isolation & crystallization of Phosphorylase b from rabbit muscle BY EDMOND H. FISCHER
AND EDWIN G. KREBS (1957)
Journal of biological chemistry, 231: 65-72
Proposals for the catalytic mechanism of glycogen phosphorylase b prompted by crystallographic
studies on glucose 1-phosphate binding by JOHNSON LN, JENKINS JA, WILSON KS (1980)
Journal of molecular biology, 140 : 565-580
• E references
http://nsdl.niscair.res.in/jspui/bitstream/123456789/735/1/EnzymePurification.pdf
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