Enzymes - Manfred Maitz

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Enzymes - Manfred Maitz

Methods of Biomaterials Testing

Lesson 3-5

Biochemical Methods

- Enzymatic Reactions -


Chemical Reactions

The Mass-Action Law (I)

CO 2 + H 2 O → H 2 CO 3- + H +

H 2 CO 3- + H + → CO 2 + H 2 O

CO 2

time

Substrate 1 + Substrate 2 +...+ Substrate n

Product 1 + Product 2 +...+ Product n

In the steady state situation:

[Product 1]x[Product 2]x...x[Product 3]

[Substrate 1]x[Substrate 2]x...x[Substrate n]

= K


The Mass Action Law (II)

Substrate 1 + Substrate 2 +...+ Substrate n

Product 1 + Product 2 +...+ Product n

[Product 1]x[Product 2]x...x[Product 3]

[Substrate 1]x[Substrate 2]x...x[Substrate n]

= K

Consequences

• The mass action law gives no information about the speed of a

reaction

• The increase of any substrate concentration will increase the

concentration of all products in the reaction

• Removal of any single product will increase the formation of the

other products

• Addition of one or more of the products can reverse the

reaction and lead to the formation of more substrates.


Enzymes I

Enzymes are biological catalysts: They facilitate a chemical reaction

• They do not change the mass action law

• They do not change the K

• They do not prefer one of the reaction directions

• They act by reducing the activation energy of the reaction or the energy of

an intermediate step

• Terminology: Name: Substrate + Type of reaction + ‘-ase’

‘Activity’ (measured in ‘Units’ U) instead of ‘Concentration’

Energy

Substrates

Products

Energy

Substrates

Products

Reaction Process

Reaction without Enzyme

Reaction Process

Reaction with Enzyme


Enzyme-Substrate Complex

+

Enzyme Substrate Enzyme-Substrate

Komplex

Reaction/ release

of the products

By random (Brownian) movement enzyme and substrate connect

to the enzyme substrate complex, where the reaction happens

Consequences:

• Very high substrate concentration → The enzyme is the limiting step.

Reaction speed is proportional to the enzyme activity

• Very high enzyme concentration → The substrate is the limiting step.

Reaction speed is proportional to the substrate concentration.

In this case: Michaelis-Menten Equation:

v =

V max

x [S]

K m

+ [S]

; K m : Michaelis constant


Enzymes III

Enzymes are proteins

Enzymes mostly have strict requirements to their environment

(temperature, pH, salt concentration...)

Enzymes can be ‘intoxicated’ by simulators of their substrate

– They cannot be processed at all

– They are processed much slower

– The simulator or the products react chemically with the

enzyme

→This can be used for selective testing of specific pathways

• Many enzymes are produced in inactive form, which get

activated on demand: pro-enzymes

Enzymes become degraded/ inactivated after some time

• According to their type and function enzymes can be located

inside the cell/ in special compartments of the cell/ on the cell

surface or freely outside the cell.


The Lambert-Beer‘s Law

Solution with

concentration c

Horizontal light beam

Lamp

d

Intensity I 0 Intensity I

I = I 0 e -κcd

Ln(I/I 0 ) = - κcd

• High precision by exact

pathlenght

• High volume required

(1-2ml)

• High handling time

Vertical light beam

• High throughput

• Reduced precision,

dependent from pipetting

E = log(I 0 /I) = εcd; E: Extinction

• Low volume required (50-

250µl)

• The extinction E (Absorbance A) is directly given from the photometer

• The extinction/absorbance is proportional to the concentration c

• The extinction is proportional to the pathlengh d

– Take care of pipetting errors at vertical set-ups (microplates)


Alkaline Phosphatase

• Low Substrate Specifity: R-OPO(OH) 2

• Reaction:

ALP

R-O-PO 3

2-

+ H 2 O R-OH + PO 4

3-

+ H +

• For Analysis frequently 4-Nitrophenly Phosphate as substrate:

The Product has yellow color: λ(E max )=405nm

Kinetic of ALP Catalyzed Hydroxylation of 4-NPP

5

1000

Reaction Velocity vs ALP Activity

4

10U/mL

5U/mL

2.5U/mL

Extinction [OD]

3

2

∆OD/∆t

∆OD/∆t

1.25U/mL

0.625U/mL

∆OD/∆t

1

0.313U/mL

0.156U/mL

0.078U/mL

0

0 5 10 15 20

Time [min]

∆E/∆t [mOD/min]

100

10

0.1 1 10

ALP Activity [U/mL]


Examples of Enzymes: LDH

• Lactate Dehydrogenase (LDH)

– Necessary for the energy metabolism

– Very constant activity in all cells

– Only inside the cell

– Presence outside of a cell indicates cell damage

(leakage)

• Reaction

LDH

Lacatate + NAD + Pyruvate + NADH + H +


LDH-Measurement Protocol

• Tris Buffer: pH 7.2

• Reagent solution:

– NADH

– Pyruvate

• Start reaction by addition of LDH or sample

• Measure absorption (kinetic) at λ=340nm

(this is an absorption maximum of NADH)

Lacatate + NAD + Pyruvate + NADH + H +

Obviously the test measures the

reverse reaction

Extinction [OD]

0.24

0.22

0.20

0.18

0.16

0.14

Kinetic for Different LDH Activities

0.12

12.5aU/L

0.10

50aU/L 25aU/L

0.08

0 5 10 15 20

time [min]

∆E/∆t

0.78aU/L

1.56aU/L

3.13aU/L

6.25aU/L

-Slope [OD/min]

10

1

0.1

neg. Slope vs. LDH Activity

1 10 100

LDH Activity [aU/L]

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