ENZYMES
Key words: biocatalysis, enzyme activity, apoenzyme, holoenzyme, coenzyme, substrate and
reaction specificity of enzyme, pH optimum, sucrase (EC 3.2.1.26), -amylase (EC 3.2.1.1.),
activation and inhibition of enzyme, ion strength
Reagents:
1. Trypsin solution (from Pancreolan, mixture glycerol: water 1:1)
2. Casein solution 40g/L in sodium carbonate 0,001mol/L
3.
concentrated nitric acid !CAUSTIC!
4.
Yeast sucrase solution (50 g yeast at 100 ml of ethanol sol. 500 g/l)
5.
-amylase solution (fresh saliva 10 x diluted with water and filtered through gauze)
6. Starch solution 10 g/L
7. Cellulose suspension 2,5 g/L
8. Inulin solution 2,5 g/l
9. Sucrose solution 5g/L
10. Fehling s solution I (copper (II) sulphate 70 g/l)
11. Fehling s solution II (sodium hydroxide 125g/L and sodium potassium tartrate 175g/L)
12. Lugol solution (iodine sol. 20g/L in potassium iodide 40 g/L) 50x diluted
13.
ALP kit (BioSystems):
a) Buffer N-methyl-D-glukamin, pH 10.1
b) 4-nitrophenylphosphate substrate 92 mmol/l
c) inhibitor, EDTA 30 mmol/l sodium hydroxide solution 1 mol/l
14. serum
-amylase
-amylase ( -1,4- glucan-4-glucanohydrolase; EC 3.2.1.1) is one of the glycosidases.
Glycosidases are among the hydrolytic enzymes that are specific for the type of glycosidic
bonds ( -glycosidases or -glycosidases) and carbohydrates. Amylase cleaves -1,4
glycosidic bonds in starch giving a mixture of oligosaccharides and non-specifically cleaves
inulin.
There are two isoenzymes of -amylase in organism (both are encoded with genes
from locus 1p21). -amylase occurs in saliva (S-amylase) and duodenum (pancreatic, Pamylase). Increased -amylase activity is observed in acute pancreatitis, but also in most
cases of abdominal pain. Higher diagnostic potential for pancreatitis has determination of
pancreatic isoenzyme (P-amylase). -amylase is excreted to the urine due to its small
molecular weight and its increased level is detected in acute pancreatitis. S-amylase is
elevated in diseases of the salivary glands.
Yeast sucrase hydrolyses sucrose to glucose and fructose.
The objective of this task is to demonstrate the presence of degradation products of salivary
-amylase and sucrase. Products of hydrolytic degradation (with free hydroxyl group on C1)
react with Fehling´s reagent, while polysaccharides forms coloured product with Lugol´s
reagent.
1. Evidence of substrate specificity – sucrase and α-amylase
Procedure:
a. Prepare -amylase solution - use your fresh saliva 10 x diluted with water and filtered
through gauze.
b. Pipette reaction mixtures according to table 1:
Test tube N°
Starch (ml)
1
1
Cellulose (ml)
2
3
6
7
1
1
1
0,25
0,25
8
1
Sucrose (ml)
-amylase (ml)
Fehling’s test
Lugol solution
5
1
1
Inulin (ml)
Sucrase (ml)
4
0,25
1
0,25
0,25
0,25
0,25
0,25
c. Incubate the tubes for 30 minutes at 37 C. Put out the tubes from the water bath.
d. Transfer about a half of the volume of the test tubes into other tubes and perform Fehling s
test to know in which tube the substrate has been hydrolysed. (Recall that Fehling s reagent is
prepared by combining Fehling I with Fehling II in ratio 1:1. A sample is mixed with
Fehling s reagent and heated in the boiling water bath. Red colour of Cu2O demonstrates
presence of reducing compounds.)
e. Test the second half of the volume in the tubes for the presence of starch using Lugol
solution.
f. Do the same reactions with the native polysaccharide and compare the colour.
g. Write down the results (use marks + or -) in table 1.
Notes:
2. Determination of Michaelis constant (Km) of enzyme Alkaline
phosphatase
The Michaelis constant Km is the substrate concentration at which the reaction rate is
half of maximal velocity Vmax (Graph 1). Km does not depend on concentration of the
enzyme in reaction mixture and remains constant for a given couple enzyme-substrate.
Typical method for Km determination is analysis of series enzyme assays with varying
substrate concentrations (at least 5 different concentrations) but with the same conditions (pH,
temperature, amount of enzyme).
Michaelis constant is actually affinity of the enzyme to the substrate. The lower value
of Km means higher affinity between enzyme-substrate. Alcoholdehydrogenase has lower Km
for ethanol than for methanol and its preferentially cleaves ethanol. This effect is used for
therapy of methanol intoxication. Different isoenzymes have different Km for substrate (e.g.
glucokinase vs. hexokinase and phosphorylation of glucose in livers and whole body –
regulation of glucose metabolism).
Graphical methods:
I.
Direct linear plot (Eisenthal, Cornish-Bowden)
Plot v (A420) against substrate concentration S as in graph 1 and fit a rectangular
hyperbola. Then plot S value onto the x-axis (horizontal) and the corresponding v (A420) onto
the y-axis (vertical). Connect these two points by a straight line. The process repeats for each
pair of points. Hence n lines are obtained for n pairs of points. If data fit equation of
rectangular hyperbola exactly, all the lines intersect in one point, its distance from x-axis is
Vmax and that one from y-axis is -Km.
Graph 1. Direct linear plot
II.
Double reciprocal plot (Lineweaver-Burke)
For better visualization of Km value is double reciprocal plot. When 1/v is plotted
against 1/S, a linear function is obtained (see graph 2). The intercept of the function with xaxis represents -1/Km, while intercept with y-axis gives 1/Vmax.
1
v
Km
Vmax
1
S
1
Vmax
Plot 1/v (1/A420) against1/S as in graph II, fit a straight line and obtain Km and Vmax
from the intercepts.
Graph 2. Double reciprocal plot
Alkaline phosphatase (ALP)
Alkaline phosphatase (ALP) cleaves phosphate groups (phosphoric acid residues)
especially from proteins and nucleics and maximal activity is in alkaline pH.
ALP occurs in most tissues of the body as an isoenzyme such as the liver, kidney, bone,
placenta and intestine.
Four genes encode ALP isoenzymes: ALP-L (L, B, K), ALP-I, ALP-P, ALP –PL, (GALP). The
same gene can produce different isoforms (different degree of glycation, saccharide structure).
1. Tissue non-specific alkaline phosphatase (TNAP), ALP-L/B/K or liver includes
three differently glycated isoforms: liver L (L1,2), bone (B), kidney (K)
L (liver) – partly inactivated (30%) at 56°C/10min
B (bone) – fully inactivated at 65°C/10min
2. Intestinal alkaline phosphatase, ALP-I (I1-3), occurs in three isoforms, produced in
the intestine. Non-glycosylated, inhibited by L-phenylalanine.
3. Placental alkaline phosphatase occurs in two isoforms:
a) ALP-P (P1) produced in placenta, is glycosylated and thermostabile. Increased
production at the end of the third trimester.
b) Pseudoplacental alkaline phosphatase (Placental Like Alkaline Phosphatase
– PLAP, ALP-PL, ALP-P2) is glycosylated dimmer isoform of ALP-P,
produced especially in the germinal cells – GALP (Germinal Alkaline
Phosphatase) and embryonic tissue. Occurs also in testis, thymus and some
cancers derived from germinal cells (e.g. seminoma, embryonic carcinoma).
In the Table 3 are basic characteristic of different ALP isoenzymes and isoforms.
Isoenzyme AP
Temperature
Specific inhibitors
neuraminidase, sodium
Stable at 70°C
deoxycholate,
PLAP/GCAP
L-phenylalanine
Stabile at 56°C
L-phenylalanine. L-tryptophan
IAP
Unstable at 55°C
Bone isoform TNAP
L-homarginin.
At 55°C more stable
neuraminidase, sodium
Hepatic isoform TNAP
than bone isoform
deoxycholate
Unstable at 45°C
Kidney isoform TNAP
Isoenzymes and isoforms of alkaline phosphatase are shown as below:
ALP-L (hepatic form) – participates in the membrane transport processes. ALP-L is localized
in the cytoplasmic membrane nearside to bile ducts of hepatocytes.
ALP-B (bone form) is part of the osteoblast membrane. Takes part in bone architecture, is
involved in Ca2+ incorporation.
ALP-K (kidney form) is localized in the kidney tubular cell’s membrane.
ALP-I (intestine) participates on the fatty acids transport and Ca2+ absorption.
Physiological ALP values in blood (serum, plasma):
ALP-L (L1,2)
– 30 - 50 %
ALP-B
– 60 - 70 %
ALP-K
– usually not detect in serum
ALP-I (I1-3)
–
<20 %
ALP-P (P1,2)
– appear during pregnancy
The ALP activity in serum increases as a result of many pathological conditions.
Determination of increased isoenzyme form is necessary for correct diagnosis.
Increased hepatic ALP is characteristic of biliary obstruction. The reason for this
obstruction is the most common gallstones or malignant tumour blocking the free flow of bile.
Bone ALP activity increases due to some bone disease (osteomalacia, Paget's disease) and is
physiologically higher in children.
Pathological changes ALP in blood (serum, plasma):
ALP-L – cholestasis, bile duct inflammation (cholecystitis and cholangoitis),
hepatopathy, cancer – especially hepatoma
ALP-B – osteosarcoma (osteoblastic), osteopathy, systemic diseases, some cancer,
therapy, osteoporosis
ALP-I – diabetes mellitus, intestine cancer, liver cirrhosis, renal insufficiency
ALP-P – epithelial carcinoma of ovaries, stomach, pancreas and sarcomas
In this experiment, the effect of substrate concentration on reaction velocity will be
demonstrated on alkaline phosphatase (ALP). ALP splits 4-nitrophenylphosphate into 4nitrophenol and phosphate. The enzyme activity is measured as the amount of the liberated 4nitrophenol spectrophotometrically at 420 nm.
Procedure:
- According to table 3. prepare a set of 6 marked “Eppendorf” tubes for dilution of the
stock substrate solution.
- Pipette 0,6ml of stock substrate into the tube n°1.
- Pipette 0,3 ml of distilled water to tubes 2-6.
- Add 0,3 ml of substrate solution from tube N°1 to tube N°2 and mix.
- From the tube N°2 do the same for the other tubes according the table 1:
Table 3.
Tube N°
1
2
3
4
5
6
4-nitrophenyl
phosphate
Stock substrate
From tube 1
From tube 2
From tube 3
From tube 4
From tube 5
substrate
ml
0,6
0,3
0,3
0,3
0,3
0,3
Distilled water
ml
–
0,3
0,3
0,3
0,3
0,3
c mmol/L
92
46
23
11,5
5,75
2,88
- Prepare a set of 7 marked glass tubes. Numbers of test tubes correspond to the numbers of
working concentrations of substrate in the “Eppendorf” tubes.
- Pipette 1 ml of buffer solution into tube N°1-6 and add 0,02 ml of sera.
- Pipette 1 ml of buffer solution into a glass tube N° 7 (blank).
- Incubate in water bath at 37°C for 5 min.
- Add 0,2 ml of diluted substrate from “Eppendorf” tubes to corresponding glass tubes. Keep
30 sec intervals between the tubes preparation. Do not put out the glass tubes from water bath.
- Add 0,2 ml of stock substrate solution (92 mmol / l) to tube N° 7.
- Mix and cover the tubes with aluminium foil and incubate in water bath at 37°C for 15 min.
- Put out the test tubes from water bath and add 0,5 ml of inhibitor. Keep 30 sec intervals
between the tubes.
- Add 0,02 ml of sera to tube N° 7 (blank).
- Read the absorbance of samples against blank at 420 nm and write down the measured
values into the table 4.
- Calculate the reciprocal values 1/ [S] and 1/A.
Table 4.
Tube N°:
[S] (mmol/l)
1
15,00
2
7,50
3
3,77
4
1,88
5
0,94
6
0,47
A
1/[S]
1/A
[Sr] = the real concentration of substrate in reaction mixtures [i.e.concentration of stock
solution multiply dilution 0,164 (0,2/1,22)].
From the obtained values [S], A, 1/ [S] and 1/A construct two graphs and determine the Km
value as a 4-nitrophenol molar substrate concentration.
Plot A against [S] (exp.):
Plot 1/A420 against 1/[S] (exp.):
Write down the results in Table 5.
graph
Km (mmol/L)
Notes:
1
2