TOPICS IN (NANO) BIOTECHNOLOGY

Lecture 5

19th April, 2006

PhD Course

An important set of proteins: Enzymes

• Thousands of biochemical reactions proceed at any given instant within living cells. These reactions are catalyzed by enzymes;

• Enzymes are mostly proteins. But two important enzymes are most certainly to be RNA (ribozymes). One is the ribosome (peptidyl transfer) and the other is the spliceosome (splicing of intron);

• Enzymes are the agents of metabolic function. Enzymes play key functions in controlling rate of reaction, coupling reactions, and sensing the momentary metabolic needs of the cell.

Enzymes

Enzymatic Catalysis Suited to Biological systems

• Higher reactions rates• Milder reaction conditions• Greater reaction specificity• Capacity for regulation

Enzymatic Catalysis Suited to Biological systems

http://www.chem.uwec.edu/Chem150_S06/Pages/Lecture-slides/C150_lect07_enzyme.html

• All chemical reactions require some amount of energy to get them started. This energy is called activation energy.

• The way enzymes operate is by effectively lowering the amount of activation energy required for a chemical reaction to start. 

• Sometimes this happens because enzymes might weaken a covalent bond within a substrate molecule. In other cases this lowering of activation energy seems to happen because the enzyme holds the substrate molecules in a particular position that increases the likely that the molecules are going to react.

Enzyme-substrate interactions-a prerequisite for catalysis

• Forces Important for substrate recognition

• Active Site Characteristics

Models for Enzyme Substrate Interactions

Example: Carboxypeptidase

N

N

N

H

2 O

OO

O

N

N

CH

H2

H H

H

P

O

O

O

P

O

O

O

P

O

O

O

-

- - -

ATP

Enzyme Cofactors

+

N

H

2 O

OO

O CH

H H

H

P

O

O-

N

N

N

H

2 O

OO

O

N

N

CH

H2

H H

H

P

O

O-

O

O

C-NH2

H

+N

H

2 O

OO

O CH

H H

H

P

O

O-

N

N

N

H

2 O

OO

O

N

N

CH

H2

H H

H

P

O

O-

O

O

C-NH2

H H

+ H + 2e+ -

-- H - 2e

..

NAD NADH+

HS-CH -CH -N-C-CH -CH -N-C-C-C-CH-

N

N

N

H

2 O

OO

ON

N

CH

H2

H H

H

P

O

O-

OP

O

O22

O

H

CoASH

O

-

2

O OH

H

CH3

CH3

2H

2

ATP

NAD(P)

CoASH

Enzyme Cofactors

CH3CH2OH + NAD+ ---> CH3CH=O + NADH + H+

The active site of ADH has two binding regions. The coenzyme binding site, where NAD+ binds, and the substrate binding site, where the alcohol binds. Most of the binding site for the NAD+ is hydrophobic as represented in green.

This is an oxidation reaction and results in the removal of two hydrogen ions and two electrons which are added to the NAD+, converting it to NADH and H+. This is the first reaction in the metabolism of alcohol.

Enzyme Classifications

23 3

ALCOHOL DEHYDROGENASE

CH -CH -OH + NAD CH -CH=O + NADH + H+ +

Enzyme Classifications

Oxido-reductases

Transferases

Enzyme Classifications

Hydrolases

PROTEASE

R-NH -CH-C-NH-CH-C-NH-R

OO

R R1 2

+ H O2

R-NH-CH-C-OH

R1

O

NH -CH-C-NH-R2

R2

O

+

Lyases

ENOLASE

O

O

O P-

-

CH -OH

O-C-H

C-O

O

- O

O

O P-

- C-O

O

2

-

CH

O-C + H O2

2

Enzyme Classifications

Isomerases

Ligases

CO H

C

H N

2

CH3H

CO H

CHN

2

CH3H

D-ALANINE

L-ALANINE

3

3

+

+

CO H

C

2

CH3

O + NH +

4

D-AMINO ACID OXIDASE

Enzymatic Reactions with Stereochemical Specificity

An important set of proteins: Antibodies

So, what is an antibody?

What is an antigen?

Any substance capable of producing a specific immune

response

So, what is an antibody?

http://www.cat.cc.md.us/courses/bio141/lecguide/unit3/viruses/opsonvir.html

http://www.cat.cc.md.us/courses/bio141/lecguide/unit3/viruses/adcc.html

http://www.learner.org/channel/courses/biology/archive/animations/hires/a_hiv1_h.html

B cells and T cellsThe two major classes of lymphocytes are B cells, which grow to maturity in the bone marrow, and T cells, which mature in the thymus, high in the chest behind the breastbone.

B cells produce antibodies that circulate in the blood and lymph streams and attach to foreign antigens to mark them for destruction by other immune cells.

B cells are part of what is known as antibody-mediated or humoral immunity, so called because the antibodies circulate in blood and lymph, which the ancient Greeks called, the body's "humors."

B cells and T cells

B cells become plasma cells, which produce antibodies when a foreign antigen triggers the immune response.

B cells and T cells

Certain T cells, which also patrol the blood and lymph for foreign invaders, can do more than mark the antigens; they attack and destroy diseased cells they recognize as foreign.

T lymphocytes are responsible for cell-mediated immunity (or cellular immunity).

T cells also orchestrate, regulate and coordinate the overall immune response.

T cells depend on unique cell surface molecules called the major histocompatibility complex (MHC) to help them recognize antigen fragments.

http://www.bio.davidson.edu/courses/Immunology/Bio307.html

Recognition of antigen by B and T-cells

• B-cells can recognise an epitope alone• T-cells can recognise antigen only when

it is associated with an MHC molecule• There are four cell membrane molecules

that are involved in recognition:– membrane bound antibody (B-cells)– T-cell receptor or TCR (T-cells)– MHC class I– MHC class II

What is an antibody?

• Antigen-specific products of B-cells• Present on the B-cell surface • Secreted by plasma cells• Effectors of the humoral immune response,

searching and neutralising/eliminate antigens• Two functions:

– to bind specifically to molecules from the pathogen

– to recruit other cells and molecules to destroy the pathogen once the antibody is bound to it

Structure of the antibody molecule

• The antigen-binding region of the antibody molecule is called the variable region or V region

• The region of the antibody molecule that engages the effector functions of the immune system is known as the constant region or C region.

• They are joined by a polypeptide chain that is known as the hinge region

Structure of the antibody molecule

• X-ray crystallography has revealed that the overall shape is roughly that of a Y

• Each arm of the Y is formed by the association of a light chain with a heavy chain

• The leg of the Y is formed by the pairing of the carboxyl-terminal halves of two heavy chains

Light Chain

• There are two types of light chain– kappa (k) chains– lambda (l) chains

• No functional difference has been found between antibodies having l or k light chains

• In humans 60% of the light chains are k, and 40% are l

Heavy chain

• There are five heavy chain classes or isotypes– IgM (m chain)– IgD (d chain)– IgG (g chain)– IgA (a chain)– IgE (e chain)

• These determine the functional activity of an antibody molecule

IgG

• IgG– most abundant

immunoglobulin in the blood

– provides the bulk of immunity to most blood-borne infections

IgD

• IgD– present in low

quantities in circulation

– primary function is that of antigen receptor on B-cells

IgE

• IgE– present in the serum

at very low levels– plays a role in acute

inflammation and infection by parasites

IgA

• IgA– present in external

secretions, such as tears, milk, saliva

– first line of defense against microbial invaders at mucosal surfaces

IgM

• IgM– first antibody produced

and expressed on the surface of B-cells, also secreted

– 10 combining binding sites per molecule make it very effective in removal of microbes

Enzyme Linked ImmunoSorbent

Assay (ELISA)

ELISA

• An analytical method based on the exploitation of the highly specific and selective nature of antibodies

• Radioimmunoassay developed in mid-sixties and the first report of enzyme immunoassay was in 1976 (Rubenstein et al.)

How do we produce polyclonal and monoclonal antibodies?

Polyclonal antibodies

- larger quantities may be produced at a time

- sometimes better selectivity and sensitivity due to recogintion of multiple epitopes

- no guarantee of batch to batch reproducibility

Monoclonal antibodies

- long and expensive process

- sometimes lower selectivity and sensitivity in comparison to Pabs observed

- once cell line established constant reproducible supply of antibodies …. forever

Enzyme Labels• Enzymes are protein catalysts present in all living cells.• They catalyse all essential reactions to supply the energy and/or

chemical chnages necessary for vital activities.• Enzymes bind their corresponding substrates with high specificity.

E + S ES E + P• Release of this product may be monitored by measuring, for

example, colour change.

• With the substrate in excess, the signal observed is proportional to the amount of enzyme present.

• Following enzymatic action, the products of the reaction are released and the enzyme is free to bind another substrate molecule.

• The speed with which this occurs is known as the turnover rate.

• Enzymes are conjugated to antibodies to provide a means of measuring the mount of antibody present.

• Enzymes commonly used are horse radish peroxidase, alkaline phosphatase, -galactosidase and glucose oxidase.

ENZYME SUBSTRATE (nm)

Horseradish peroxidase o-phenylenediamine dihydrochloride (OPD) 492*

tetramethylbenzidine (TMB) 450*

2,2’-azino-di-(3-ethyl)benzthiazoline 414* sulphonic acid (ABTS)

5-aminosalicyclic acid (ASA) 450*

[* H2O2 added and reaction stopped with sulphuric acid]

Alkaline phosphatase p-nitrophenyl phosphate 405

-D-galactosidase o-nitrophenyl -D-galactosidase 405

Glucose oxidase Glucose

(H2O2 produced and HRP and substrate used)

HRP

TMB/OPD/APTS

(no colour)

Oxidised product

ALP

p-nitrophenylphosphate

(no colour)

p-nitrophenol

-GAL

p-nitrophenylgalacto-pyronasidase

(no colour)

p-nitrophenol

Measurement principle

Note: Can also label antigen with enzyme!

Microtitre plates

96-well ELISA plate

Surface of polystyrene is activated with amine groups for enhanced binding of antibody

NUNC plates - best well to well reproducibility in binding (also very useful web site www.nunc.com)

With the exception of checkerboard titrations, avoid using column 1 and 12 and rows A and H, due to uneven heating effects

A

B

C

D

E

F

G

H

1 2 3 4 5 6 7 8 9 10 11 12

Sandwich assay

substrate

product

substrate

product

substrate

product

Concentration

Res

po

ns

e

Useful for large molecules

Robust assay - all reagents in excess

Use with Pabs or different MAbs

Competition assay

substrat

e

product

substrat

e

product

Concentration

Res

po

nse

Useful for small molecules

Reportedly less sensitive

Concentrations of reagents critical

Displacement assay

substrat

e

product

substrat

e

product

Concentration

Res

po

nse One step assay

In practise difficulties to achieve - effect of non specific displacement

Sub-optimum haptens met with some success

http://www.biology.arizona.edu/immunology/activities/elisa/technique.html?

Aptamers are isolated from combinatorial libraries of synthetic nucleic acid by exponential enrichment via an in vitro iterative process of adsorption, recovery and reamplification, known as SELEX (systematic evolution of ligands by exponential enrichment).

APTAMER DEFINITIONAPTAMER DEFINITION

Artificial nucleic acid ligands that can be generated against amino acids, drugs, proteins and even cells.

They bind their target with selectivity, specificity and affinity equal and often superior to those of antibodies.

SELEXSELEX

SELEX

can be selected against toxins/molecules that do not elicit good immune response selection is in vitro process - does not need animals kinetic parameters (kon/koff) can be controlled can be regenerated in minutes, stable for long term storage, can be transported at ambient temperature can be used in non-physiological conditions produced by chemical synthesis no ‘batch to batch’ variation negative selection against structures similar to target structure can improve specificity

BUT low stability = short life

Can be solved by chemical modification, spiegelmers, mixed LNA/DNA structures

APTAMERS VS. ANTIBODIES

Aptamers vs Antibodies

Examples of molecules for which aptamers have been selected in vitro:

ATPArginine

Dopamine Reverse transcriptase of HIV

ThrombineMembrane receptors

Whole viruses

Structure of aptamers

Structure

Modes of assay

Molecular beacons

• Molecular beacons essentially contain two structural components, a loop and a stem, with the loop serving as a probe and the annealing of two complementary arm sequences that are flanked by the probe forms the stem.

• A fluorophore and fluorescent quencher are linked covalently at each end of the arm. The stem of the beacon brings the fluorophore and quencher into close proximity, resulting in no fluorescent signal.

Molecular beacons

• When the molecular beacon encounters a target molecule it forms a probe target hybrid that is stronger and more stable than the stem in the hairpin, with the resulting conformational change forcing the arms apart, thus permitting the fluorophore to fluoresce.

Fluorescence Resonance Energy Transfer (FRET)

Fluorescence resonance energy transfer (FRET) is a distance-dependent interaction between the electronic excited states of two dye molecules. Excitation is transferred from a donor molecule to an acceptor molecule without emission of a photon.

FRET is dependent on the inverse sixth power of the intermolecular separation, making it useful over distances comparable with the dimensions of biological macromolecules.

FRET is an important technique for investigating a variety of biological phenomena that produce changes in molecular proximity.

Primary Conditions for FRETDonor and acceptor molecules must be in close proximity (typically 10–100 Å).

The absorption spectrum of the acceptor must overlap fluorescence emission spectrum of the donor (see figure).

Donor and acceptor transition dipole orientations must be approximately parallel.

Fluorescence Resonance Energy Transfer (FRET)