Title: Experiment 3: Kinetic studies with alkaline phosphatase Objective(s): 1. To prepare prepare the the standard standard curve curve for for p – nitrophen nitrophenol ol 2. To study study the effect effect of of the addi addition tion of of Mg 2+ ions an alkaline phosphate Introduction: Phosphatases are enzymes that catalyze the hydrolysis of esters of phosphoric acid. They occur in the cells and extracellular fluids of a ide range of organisms. This large and complex group of enzymes falls into four general types !ased on the chemical nature of the su!strate or the type of hydrolytic reaction that is catalyzed .This large and complex group of enzymes falls into four general types !ased on the chemical nature of the su!strate or the type of hydrolytic reaction that is catalyzed.
"ne group# the phosphomonoesterases# hydrolyzes monoesters of phosphoric acid such as $% glycerophosphate glycerophosphate or glucose &%phosphate. &%phosphate. 'ome phosphomonoesterases phosphomonoesterases are highly su!strate% specific. (or example# in gluconeogenesis# fructose%1# &%!isphosphatase specifically converts fruc fructo tose se
1#&% 1#&%!i !isp spho hosp spha hate te
to
fruc fructo tose se
&%ph &%phos osph phat ate e
and and
inor inorga gani nic c
phos phosph phat ate. e.
"the "ther r
phosphomonoeste phosphomonoesterases rases react ith a !road range of su!strates# hich share common structural motifs. motifs. The phosphomon phosphomonoest oesteras erases es that that lack su!strate su!strate specificity specificity are classifie classified d as acid or alkaline phosphatases !ased on their p) optima. *cid phosphatases function !est at around p) ., and are inhi!ited !y fluoride ion !ut not !y divalent cation%chelating agents. The alkaline phosphatases have p) optima of a!out -., and are not generally sensitive to fluoride ion !ut are inhi!ited !y divalent cation%chelating agents like /T* 0ethylene diamine tetraacetic acid# disodium salt. o level level of alkaline alkaline phospha phosphatase tase can cause# cause# )ypopho )ypophospha sphatasi tasia. a. )ypopho )ypophospha sphatasi tasia a is a genetic meta!olic !one disease that is 3uite rare in occurrence !ut fatal to the sufferer. 'ome of the iden identif tifia! ia!le le sympto symptoms ms are skelet skeletal al hypo hypomin minera eraliz lizat ation ion## mild mild respi respirat ratory ory pro!l pro!lems ems##
progress progressive ive osteomal osteomalacia acia## etc. etc. The patient patients s of hypoph hypophosph osphata atasia sia have very lo alkaline alkaline phosphatase levels in their !lood serum. 'uch patients often lose their primary teeth much !efor !efore e the standard standard age. "ther "ther than than that that it can also cause cause aola aolasti stic c anemi anemia a or chron chronic ic Myelogenous eukemia. 4n this experiment# the alkaline phosphatase5s kinetic studies are carried out on the effect of p)# inhi!itors and divalent cation. *t the !eginning of the experiment the standard curve is dran against the amount of p%nitrophenol. The p%nitrophenol p%nitrophenol amount used in the assay is determined determined from the formula of6 0*mount of ,.7mM p%nitrophenol x 1,%7 8 0,.7 8 1,%7mol9 This graph is used as standard to determine the amount of p%nitrophenol p%nitrophenol have !een consumed in the p)# inhi!itor and divalent cations reaction. The alkaline phosphatases have !inding sites for :n2+ and Mg2+# on hich enzyme activity is dependent. 4n this experiment# the effects of addition addition of Mg2+ Mg2+ ions on alkalin alkaline e phospha phosphatase tase are carried carried out. out.
;ext# ;ext# ith varia!le varia!le p)#
!eginning !eginning from p) <.,# p) <.# p) =.,# p) =. and p) -.,. >arying >arying p) levels may have a direct effect on the alkaline phosphatase phosphatase due to the presence of ioniza!le residues in the catalytic site of the enzyme.
aterials:
?ovine or calf calf intestinal alkaline alkaline phosphatase phosphatase 0'igma# 0'igma# diluted diluted 1@1,#,,, 1@1,#,,, ith , mM Tris%)Al# Tris%)Al# p) =., !uffer# containing 1 mg9ml !ovine serum al!umin# ater !ath# p%nitrophenol phosphate in !uffer !uffer 02.<, 02.<, mM# mM#
p%nitrop p%nitropheno henoll in !uffer !uffer 0,.7 mM# mM#
phenylalanine# , mM Tris%)Al# p) <.,# <.# =.,# =.# -.
!rocedure:
mono!a mono!asic sic sodium sodium phospha phosphate# te# %
") !reparation o# standard curve #or p$nitrophenol Tu!e ,.7nM p%nitrophenol 0 ml /istilled ater0ml
1
2
7
B
&
% 7.,
,., 2.-
,.1 2.-,
,.2 2.=,
,.B 2.&,
,.& 2.B,
1. (olloing tu!es ere prepared 2. *!sor!ance as read at B,, nm using tu!e 1 as a !lank 7. The amount in Cmol of p%nitrophenol for each tu!e B. * graph has !een plotted 0 a!sor!ance against amount of p%nitrophenol in umol %) E##ects o# divalent cation Tu!e 1 2.<, mM su!strate0 ml ,mm tris%)Al p) =., 1nM MgAl 2
1 ,.& 2.B %
2 ,.& 2.7 ,.1
7 ,.& 2.2 ,.2
B ,.& 2., ,.B
,.& 1.= ,.&
& ,.& 1.& ,.=
< ,.& 1.B 1.,
1. The folloing tu!es ere prepared 2. ach of the a!ove reaction mixture is added to a suita!le cuvette and the reaction as started !y adding 2, ul of an enzyme. 7. Das mixed !y invert B. The cuvette as placed in the spectrophotometer . *!sor!ance as read for every 7, s for 7 min at B,, nm &. The *9min as determined.*graph as plotted in 0umol9min against the MgAl 2 concentration
&) E##ect o# the p'
1. The tu!es ere prepared same as in ? ta!le !ut the !uffer as su!stituted ith !uffer ith different p) hich are <.,#<.#=.,#=.#-., 2. * graph of activity against p) as plotted.
esults: (") !reparation o# standard curve #or p$nitrophenol
Tu!e
1
2
7
B
&
,.7mM p% nitrophenol 0ml
%
,.,
,.1
,.2
,.B
,.&
/istilled ater 0ml
7
2.-
2.-
2.=
2.&
2.B
Ep% nitrophenolF 0CM
%
,.,1
,.,7,
,.,&,
,.12,
,.1=
*!sor!ance at B,,nm
,
,.,1=
,.,B1
,.,&
,.,<2
,.17&
mM convert to CM Tu!e 2@ ,.7m
mol L
× ,.,,,, G 1.
× 1,% H 1,,, Cmol
G ,.,1 Cmol Tu!e 7@ ,.7m
mol L
× ,.,,,1 G 7 H 1, % H 1,,, Cmol G ,.,7, Cmol
Tu!e B@ ,.7m
mol L
× ,.,,,2 G & H 1, % H 1,,, Cmol
G ,.,&, Cmol Tu!e@ ,.7m
mol L
×
,.,,,B G 1.2 H 1, %B H 1,,, Cmol G ,.12, Cmol
Tu!e&@ ,.7m
mol L
×
,.,,,& G 1.= H 1, %B H 1,,, Cmol G ,.1=, Cmol
Graph of absorbance vs p-nitrophenol concentration 0.16 0.14 0.12
f(x) = 0.74x R² = 0.97
0.1
absorbance
0.08 0.06 0.04 0.02 0 0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
concentration of p-nitrophenol
i*ure + ,raph o# absorbance a*aint amount o# p$nitrophenol
0.16
0.18
0.2
(%) E##ect o# divalent cations
Tu! e Time 0min , ,. 1., 1. 2., 2. 7.,
*!sor!ance at B,,nm B
1
2
7
,.,77 ,.,7 ,.,7,.,B1 ,.,B ,.,B= ,.,1
,.,7, ,.,7 ,.,7= ,.,B2 ,.,B& ,.,B,.,7
,.,7,.,B= ,.,2 ,.,& ,.,&1 ,.,& ,.,<,
,.,, ,.,B ,.,= ,.,&7 ,.,&= ,.,<7 ,.,<=
,.,72 ,.,7& ,.,B1 ,.,B< ,.,2 ,.,,.,&B
Table +: "bsorbance vs Time (min) Aalculation of p%nitrophenol
*!sor!ance G slope 0m H concentration I G mx# I is a!sor!ance# m is slope# x is concentration 'lope from standard curve from part *# so m G ,.<7<1 4n order to find concentration# use the a!sor!ance 9 slope xample@ Tu!e 1 ith Time G ,@ 0.033
Aoncentration G
0.7371
G ,.,B Cmol Time G ,. min 0.035
Aoncentration G
0.7371
G ,.,B< Cmol Time G 1 min 0.039
Aoncentration G
0.7371
G ,.,7 Cmol
&
<
,.,B ,.,, ,., ,.,&, ,.,&& ,.,<1 ,.,<<
,., ,.,,.,& ,.,<1 ,.,<& ,.,=2 ,.,==
Time G 1. min 0.041
Aoncentration G
0.7371
G ,.,& Cmol
e Time 0min , ,. 1., 1. 2., 2. 7.,
Tu! Ep%nitrophenolF 0Cmol 1 2 7
,.,B ,.,B< ,.,7 ,.,& ,.,&1 ,.,& ,.,&-
,.,B1 ,.,B< ,.,2 ,.,< ,.,&2 ,.,&& ,.,<2
,.,7 ,.,& ,.,<1 ,.,<& ,.,=7 ,.,== ,.,-
B
&
<
,.,&= ,.,<7 ,.,<,.,= ,.,-2 ,.,-,.11,
,.,B7 ,.,B,.,& ,.,&B ,.,<1 ,.,=, ,.,=<
,.,&1 ,.,&= ,.,< ,.,=1 ,.,-, ,.,-& ,.1,B
,.,< ,.,=, ,.,== ,.,-& ,.1,7 ,.111 ,.12,
Table -: . p$nitrophenol/ (0mol) vs Time (min)
Tube 1: [p-nitrophenol] vs Time 0.08 0.07 0.07
f(x) = 0.01x + 0.04 R² = 0.99
concentration of p-nitrophenol 0.06 0.06 0.05 0.05 0 0.5 1 1.5 2 2.5 3 3.5
time (min)
,raph +: Tube + .p$nitrophenol/ (0mol) vs Time (min)
Tube 2 : [p-nitrophenol] vs Time 0.08 0.07 0.07
f(x) = 0.01x + 0.04 R² = 1
0.06
concentration of p-nitrophenol
0.06 0.05 0.05 0.04 0 0.5 1 1.5 2 2.5 3 3.5
Time (min)
,raph -: Tube - .p$nitrophenol/ (0mol) vs Time (min)
Tube 3 : [p-nitrophenol] vs Time 0.1 0.09 0.08
concentration of p-nitrophenol
f(x) = 0.01x + 0.06 R² = 0.98
0.07 0.06 0.05 0 0.5 1 1.5 2 2.5 3 3.5
Time (min)
,raph 3: Tube 3.p$nitrophenol/ (0mol) vs Time (min)
Tube 4 : [p-nitrophenol] vs Time 0.12 0.11 0.1
concentration of p-nitrophenol
f(x) = 0.01x + 0.07 R² = 0.99
0.09 0.08 0.07 0 0.5 1 1.5 2 2.5 3 3.5
Time (min)
,raph 1: Tube 1.p$nitrophenol/ (0mol) vs Time (min)
Tube 5 : [p-nitrophenol] vs Time 0.09 0.08
f(x) = 0.01x + 0.04 R² = 1
0.07
concentration of p-nitrophenol
0.06 0.05 0.04 0 0.5 1 1.5 2 2.5 3 3.5
Time (min)
,raph 2: Tube 2.p$nitrophenol/ (0mol) vs Time (min)
Tube : [p-nitrophenol] vs Time 0.11 0.1
f(x) = 0.01x + 0.06 R² = 1
0.09
concentration of p-nitrophenol
0.08 0.07 0.06 0 0.5 1 1.5 2 2.5 3 3.5
Time (min)
,raph : Tube .p$nitrophenol/ (0mol) vs Time (min)
Tube ! : [p-nitrophenol] vs Time 0.13 0.12
f(x) = 0.02x + 0.07 R² = 1
0.11
concentration of p-nitrophenol
0.1 0.09 0.08 0 0.5 1 1.5 2 2.5 3 3.5
Time (min)
,raph 4: Tube 4 .p$nitrophenol/ (0mol) vs Time (min)
Aalculation of MgAl2 concentration 0CM for the graph of activity 0Cmol9min vs MgAl 2 concentration 0CM
Tu!e 1 1 mM MgAl2 % 0m MgAl2 % concentration 0CM
2 ,.1
7 ,.2
B ,.B
,.&
& ,.=
< 1.,
1.
7.,
&.,
-.,
12.,
1.,
Table 35 &onversion o# *&l - concentration #rom m6 to 0 Aonversion of the concentration from mM as converted to CM !y@ Tu!e 2 1 m
mol %B L x 1x1, x 1,,, Cmol G 1. CM
Tu!e 7 1 m
mol %B L x 2x1, x 1,,, Cmol G 7., CM
Tu!e B 1 m
mol %B L x Bx1, x 1,,, Cmol G &., CM
Tu!e 1 m
mol %B L x &x1, x 1,,, Cmol G -., CM
Tu!e & 1 m
mol %B L x =x1, x 1,,, Cmol G 12., CM
Tu!e < 1 m
mol %7 L x 1x1, x 1,,, Cmol G 1., CM
MgAl2 concentration 0CM 1. 7 & 12 1
nzymatic 0Cmol9min ,.,1,1 ,.,171 ,.,17& ,.,1B,.,1B7 ,.,11
activity
Table 15*&l- concentration (0) vs En78matic "ctivit8 (0ol9min)5
Graph of "n#$matic %ctivit$ (&mol'min) vs of anesium *hlori+e concentration (&) 0.02 0.01 0.01
f(x) = 0x + 0.01 R² = 0.68
0.01
Enzymatic Activity (µmol/min)
0.01 0.01 0 0 0 0
2
4
6
8 10 12 14 16
Magnesium chloride concentration (µM)
,raph 5 En78matic "ctivit8 (0mol9min) vso# a*nesium &hloride concentration (0)
&) E##ect o# p' on the reaction
Tu!es ith *!sor!ance >alue Time 0minute , ,. 1., 1. 2., 2. 7.,
1 ,.
2 ,.12 ,.17 ,.1 ,.1& ,.1,.22 ,.2&
7 ,.&-7 1.,17 1.,2B 1.,2< 1.,7, 1.,7& 1.,B2
B ,.=-= ,.<-= ,.<-2 ,.<- ,.=,1 ,.=,,.=17
,.-,, ,.=-,.-1, ,.-22 ,.-7& ,.-, ,.-&B
Ta!le 1. *!sor!ance recorded in 7, seconds time interval ith different p) of Tris%)Al Aalculation of Tris%)Al concentration Aoncentration G
Absorbance slope
'lope# m G ,.<7<1 xample@ Tu!e 1# time G , minute 0.749
Aoncentration of Tris%)Al G
0.7371
G 1.,1& Cmol
Tu!e Time 0min
, ,. 1., 1.
Aoncentration of Tris%)Al at different p) 0Cmol 1 0p) < 1.,1& 1.,2B 1.,71 1.,7=
2 0p) <. ,.&- ,.&-& ,.&-,.<,,
7 0p) =
B 0p) =.
0p) -.,
,.-B, 1.7
1.21= 1.,=7 1.,
1.221 1.22, 1.27 1.21
2., 2. 7.,
1.,B1.,< 1.,&
,.<,B ,.<,= ,.<1B
1.7-< 1.B,& 1.B1B
1.,=< 1.,-= 1.1,7
1.2<, 1.2=1.7,=
Ta!le 2. Aoncentration of Tris%)Al at 7, seconds time interval ith different p)
[Tris-,*l] vs Time (inutes) at p, !. 1.07 1.06 1.05
f(x) = 0.02x + 1.02 R² = 1
1.04
[Tris-,*l] (&mol)
1.03 1.02 1.01 1 0.99 0
0.5
1
1.5
2
Timr (inutes)
2.5
3
3.5
[Tris-,*l] vs Time (inutes) at p, !5 0.72 0.72 0.71
f(x) = 0.01x + 0.69 R² = 0.94
0.71
Concentration of Tris-HCl (µmol)
0.7 0.7 0.69 0.69 0
0.5
1
1.5
2
2.5
3
3.5
Time (minute)
[Tris-,*l] vs Time (inutes) at p, /. 1.6 1.4 1.2
f(x) = 0.11x + 1.17 R² = 0.45
1
*oncentration of Tris-,*l (&mol)
0.8 0.6 0.4 0.2 0 0
0.5
1
1.5
2
Time (inute)
2.5
3
3.5
[Tris-,*l] vs Time (inutes) at p, /5 1.25 1.2 1.15
Concentration of Tris-HCl (µmol)
1.1
f(x) = - 0.02x + 1.14 R² = 0.21
1.05 1 0
0.5
1
1.5
2
2.5
3
3.5
Time (Minutes)
[Tris-,*l] vs Time (inutes) at p, 0. 1.32 1.3 1.28
f(x) = 0.03x + 1.21 R² = 0.96
1.26
Concentration of Tris-HCl (µmol)
1.24 1.22 1.2 1.18 1.16 0
0.5
1
1.5
2
Time (minutes)
Tris%)Al p) <., <. =., =.
nzyme activity 0Cmol9min ,.,1& ,.,,&1 ,.1,&< %,.,21&
2.5
3
3.5
-.,
,.,71,
Ta!le of enzyme activity vs p) of the !uffer
"n#$me activit$ (&mol'min) 0.12 0.1 0.08 0.06
Enzyme activity (µmol/min)
0.04 0.02 0 6.50 -0.02
7.00
7.50
8.00
8.50
9.00
9.50
-0.04
H
;iscussion
* standard curve is Just a plot of to different parameters and the curve reveals the relationship !eteen the to parameters. Knder alkaline conditions# the p%nitrophenolate anion a!sor!s light at B,,%B, nm. The amount of enzyme present is therefore determined !y measuring the amount of p%nitrophenolate anion produced in the reaction. )oever to make this estimation a standard curve must !e prepared so that the amount of yello%orange colour can !e translated into the amount of p%nitrophenol produced.
The e3uation for this reaction is p%nitrophenyl phosphate + )2"
p%nitrophenol L + )7P"B
*s shon !y the graph in result section * # it indicates that the rate of a!sor!ances is linearly proportional to against amount of p%nitrophenol in mol. *nd the e3uation for this graph is I G ,.<7218.
nzymes are proteins that act as !iological catalysts that are either essential for a reaction to occur or may increase the speed of the reaction. 'ome enzyme re3uire the presence of either a cofactor or a coenzyme to catalyse the reaction. 0?rennan# n.d. The specific function of the cofactor may vary according to the enzyme. ach enzyme has it5s respective se3uence of reaction steps hich the cofactor play a role in 0?rennan# n.d.. Aofactors are re3uired !y the enzymes to facilitate the electrons transfer needed in the formation and !reaking of !onds in the reaction mechanism. 0N*OK?"D'O4# 2,1B /ivalent cations are cations that have a charge of +2. Magnesium ion# Mg 2+ is a divalent cation. 4t is a cofactor to alkaline phosphathase. * cofactor is a non%protein molecule or ion re3uired !y the enzyme hen underdoing it5s enzyme activity. Throughout the experiment the concentration of enzyme# hich is alkaline phosphotase# and of the concentration of the cofactor in the form of magnesium chloride# MgAl 2. Tu!e 1 acted as a control to o!serve the enzymatic activity of phosphotase ithout the cofactor. ?ased on raph 2# the loest among all the tu!es. This indicates that the phosphotase is capa!le of hydrolyzing p%nitrophenyl phosphate to p%nitrophenol ithout the presence of Mg 2+. )oever# the presence of the cofactor does yield a much higher enzyme activity.
The concentration of MgAl 2 increases from Tu!e 2 – <. This ill help in identifying the optimal concentration from maximum product formation and also the effect of the concentration on enzyme activity hen too high# if any. ?ased on the nzymatic activity 0Cmol9min vs Magnesium Ahloride concentration 0CM graph it can !e clearly seen that hen the concentration of MgAl 2 increases# the enzyme activity increases. 4t can also !e deduced that the optimum concentration of MgAl 2 for maximum enzyme activity is higher than 1 Cmol# hich is the highest concentration tested ith 0 Tu!e < .
?inding of the su!strate to the active site of an enzyme involves interaction ith reactive groups provided !y the side%chains of amino acids at the !inding site. The p) of the incu!ation medium may affect the ionisation of !oth the su!strate and the amino acid side%chains and ill therefore this ill affect !inding. 4t may also affect the ionisation of reactive groups that catalyse the reaction# although in the micro%environment of the catalytic site# hen it is occupied !y the su!strate# this is less likely.
xtreme values of p) may also disrupt the tertiary structure of the enzyme# and so distort the active site# or even denature the enzyme protein. *s its name indicates# alkaline phosphatase is highly p)%sensitive. 4n the effective !uffering range for Tris%)Al# the initial velocity of the hydrolysis of su!strate !y alkaline phosphatases hould increases more than &%fold from p) <., to p) -.,.
4n ta!le 2# it shos that the concentration of Tris%)Al increases ith the p) value. )oever there is drop in concentration value at ,.min and 1., min on p) of =. and at ,.min on p) of -. ven the graphs sho concentration of Tris%)Al increases linearly except for graph 0p) of =. shos inverse linearly proportional graph.The values of the Om and >
to !e affected !y
max
the difference in p) value.
e#erences 1. N*OK?"D'O4# 02,1B. Chapter 7C - Cofactors and Electron Pushing . EonlineF mployees.cs!sJu.edu.
*vaila!le
[email protected]!sJu.edu9hJaku!oski9classes9ch7719catalysis9olelectronpush.htm E*ccessed 22 Nun. 2,1BF.
at@
2. ?rennan# N. 0n.d.. How Would the Lack of a Cofactor for an Enzyme Affect the Enzymes !unction" # $he Classroom # %ynonym. EonlineF 'ynonym. *vaila!le at@
[email protected]ould%lack%cofactor%enzyme%affect%enzymes%function% <,2.html E*ccessed 22 Nun. 2,1BF
