Antibodies and Antigens Antibodies and Antigens Your body s - - PDF document

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Antibodies and Antigens Antibodies and Antigens

Your body Your body’ ’s natural defense s natural defense system system

Antibodies Antibodies

The formal chemical name for antibodies The formal chemical name for antibodies is is immunoglobulins immunoglobulins. . Immunoglobulins Immunoglobulins are protein molecules are protein molecules which defend your body from foreign which defend your body from foreign toxins known as toxins known as antigens antigens. . Immunoglobulin G, IgG, is the major Immunoglobulin G, IgG, is the major antibody in serum (blood) and will be what antibody in serum (blood) and will be what we use for example purposes. we use for example purposes.

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Immunoglobulin Structure Immunoglobulin Structure

IgG, is comprised of 3 polypeptide chains IgG, is comprised of 3 polypeptide chains

• 4

4 “ “light light” ” chains, L chains, L – – 25 25-

• kd chains (shown in yellow)

kd chains (shown in yellow)

• 2

2 “ “heavy heavy” ” chain, H chain, H – – 50 50-

• kd chain (shown in blue)

kd chain (shown in blue)

A disulfide bond links L to H. A disulfide bond links L to H. H chains link to each other by 1or more disulfide bonds. H chains link to each other by 1or more disulfide bonds.

S S

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.4700

Immunoglobulin Cleavage Immunoglobulin Cleavage

IgG is cleaved by an enzyme IgG is cleaved by an enzyme called called papain papain into 3 fragments, into 3 fragments, F. F.

• F

Fab

ab = antigen binding fragment

= antigen binding fragment

• F

Fc

c = constant fragment (does

= constant fragment (does not bind to antigens) not bind to antigens)

The disulfide bonds connecting The disulfide bonds connecting F Fab

ab and F

and Fc

c are flexible and

are flexible and called called “ “hinges hinges” ”

• This flexibility allows for one

This flexibility allows for one antibody to bind to antigens antibody to bind to antigens with multiple bonding sites. with multiple bonding sites.

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.4701 http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.4703

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Antigen Cross Antigen Cross-

• linking

linking

Since each IgG Since each IgG contains two F contains two Fab

ab

chains it can cross chains it can cross-

• link multiple antigens

link multiple antigens

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.4702

Other Immunoglobulins Other Immunoglobulins

IgA IgA – – external secretions: saliva, tears, mucus, etc. external secretions: saliva, tears, mucus, etc. IgM IgM – – appears first in serum upon introduction of antigen, 10 binding appears first in serum upon introduction of antigen, 10 binding sites sites allows for excellent binding to multiple receptor antigens allows for excellent binding to multiple receptor antigens IgD IgD – – role not known role not known IgE IgE – – protection from parasites, can also cause allergic reactions protection from parasites, can also cause allergic reactions

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.4704

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Variable Regions Variable Regions – – Mechanism of Mechanism of Antibody Action Antibody Action

The end terminus of the The end terminus of the F Fab

ab chains are HIGHLY

chains are HIGHLY

• variable. This is why our
• variable. This is why our

bodies are able to form bodies are able to form antibodies to nearly all antibodies to nearly all antigens. antigens.

• V

VL

L = variable light

= variable light

• V

VH

H = variable heavy

= variable heavy

• C

CL

L = constant light

= constant light

• C

CH

H = constant heavy

= constant heavy

Why can they vary so Why can they vary so widely? widely?

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.4707

V VL

L and V

and VH

H Chemistry

Chemistry – – the the REAL REAL BASICS! BASICS!

V VL

L and V

and VH

H are complex

are complex proteins which adopt a proteins which adopt a common structure called common structure called the the immunoglobulin fold. immunoglobulin fold. The The immunoglobulin immunoglobulin fold fold is constructed from is constructed from two two β β-

• sheets. The beta
• sheets. The beta

sheets contain anti sheets contain anti-

• parallel strands. These

parallel strands. These strands surround a strands surround a hydrophobic core. The hydrophobic core. The two sheets are connected two sheets are connected via disulfide bond. via disulfide bond.

Key aspect: Key aspect:

• Three loops at the end are

Three loops at the end are “ “hypervariable, hypervariable,” ” meaning that their meaning that their amino acid sequences can be varied amino acid sequences can be varied in many, many ways. in many, many ways.

• These loops are the variable regions

These loops are the variable regions we same in the previous slide. we same in the previous slide.

• This region is also known as the

This region is also known as the complementarity determining region complementarity determining region

• r
• r CDR.

CDR.

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.figgrp.4709

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Big Picture Big Picture

So if V So if VH

H can vary immensely and V

can vary immensely and VL

L can very

can very immensely then: immensely then:

(VL)i (VH)j

Σ

+ =

i,j=1 i,j

∞

Ok, so more like a really big number!

How How’ ’d he do that? d he do that?

So then you say So then you say “ “How can our body make How can our body make all these different antibodies? all these different antibodies?” ” I say : I say : “ “That is what B That is what B-

• cells are for!

cells are for!” ”

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B B-

• cells and T

cells and T-

• cells

cells

Both develop in the bone Both develop in the bone marrow marrow B B-

• cells mature in the

cells mature in the B Bone marrow as well

• ne marrow as well

whereas, T whereas, T-

• cells mature

cells mature in the in the T Thymus. hymus. B B-

• cells produce

cells produce antibodies with the help antibodies with the help

• f T
• f T-
• cells.

cells. Some T Some T-

• cells can act as

cells can act as antibodies themselves antibodies themselves (i.e., against viruses but (i.e., against viruses but we won we won’ ’t get into that) t get into that)

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/B/B_and_Tcells.html

Why do we care and how the heck Why do we care and how the heck does this relate to Chemistry? does this relate to Chemistry?

#1 Everything relates to #1 Everything relates to CHEMISTRY CHEMISTRY!!! !!! #2 #2 – – You should care because this is how You should care because this is how your body protects itself. your body protects itself. #3 #3 – – One of the many ways this relates to One of the many ways this relates to Chemistry is the relationship between Chemistry is the relationship between antigens and antibodies both antigens and antibodies both kinetically kinetically and in terms of and in terms of equilibrium equilibrium. .

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Antibody/Antigen Affinity Antibody/Antigen Affinity

A very important aspect of the relationship A very important aspect of the relationship between antibodies and antigens is their between antibodies and antigens is their affinities for each other. Some affinities for each other. Some antibody/antigen pairs have a relatively antibody/antigen pairs have a relatively high affinity for each other and some have high affinity for each other and some have a somewhat lower affinity for each other. a somewhat lower affinity for each other.

Equilibrium Equilibrium

Think back to when we studied Think back to when we studied equilibrium. equilibrium. We showed that when species are We showed that when species are associated with each other we can associated with each other we can measure the degree of association with an measure the degree of association with an ???? ????

Equilibrium Constant, K Equilibrium Constant, K

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Example Example

We We’ ’ll call antigens G and antibodies A. ll call antigens G and antibodies A. K Kd

d stands for Equilibrium of dissociation

stands for Equilibrium of dissociation

[ ][ ] [ ]

GA A G K A G GA

d =

+ ⇔

Try these problems! Try these problems!

Suppose that the dissociation constant of an Suppose that the dissociation constant of an F Fab

ab-

• hapten complex is 3 x 10

hapten complex is 3 x 10-

• 7

7 M at 25

M at 25° ° C. C.

A) A) Immunologist often speak of affinity (K Immunologist often speak of affinity (Ka

a), the

), the reciprocal of the dissociation constant, in comparing reciprocal of the dissociation constant, in comparing

• antibodies. What is the affinity of this F
• antibodies. What is the affinity of this Fab

ab?

? B) B) What is the standard free energy of binding? What is the standard free energy of binding? C) C) The rate constant of release of hapten from the The rate constant of release of hapten from the complex is 120 s complex is 120 s-

• 1
• 1. What is the rate constant of

. What is the rate constant of association? association?

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.section.4770

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A. A.

1 6 7

10 3 . 3 10 3 1 1

− −

× = × = = M M K K K

a d a

B. B.

( )

kJ G J G M K K mol J G K be would this binding for K RT G

• f
• f
• f

a

• f

37 37214 ) 10 3 . 3 ln( 298 3145 . 8 ln

1 6

− = Δ − = Δ × ⋅ − = Δ − = Δ

−

10

C. C.

[ ] [ ][ ] [ ] [ ][ ] [ ] [ ][ ] ( )

1 1 8 1 6 1

10 96 . 3 10 3 . 3 120 : @

− − − −

× = × = = = = = = = s M k M s k K k A G GA k k A G k GA k n associatio rate release rate m equilibriu A G k n associatio rate GA k release rate

n associatio n associatio a release release n associatio n associatio release n associatio release

Here is another problem! Here is another problem!

The standard free energy of binding of The standard free energy of binding of F Fab

ab derived from an antiviral IgG is

derived from an antiviral IgG is -

• 7

7 kcal/mol at 25 kcal/mol at 25° °C. C.

A) A) Calculate the dissociation constant of this

Calculate the dissociation constant of this interaction. interaction.

B) B) Predict the dissociation constant of the IgG,

Predict the dissociation constant of the IgG, assuming that both combining sites of the assuming that both combining sites of the antibody can interact with viral epitopes and antibody can interact with viral epitopes and that the free that the free-

• energy cost of assuming a

energy cost of assuming a favorable hinge angle is +3 kcal/mol. favorable hinge angle is +3 kcal/mol.

http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=stryer.section.4770

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A. A.

( )( )

M M K M K e K e K RT G K K RT G mol J cal J kcal cal mol kcal

d a a RT G a

• fa

a a

• fa
• fa

6 1 5 1 5 298 315 . 8 29260

10 4 . 7 10 3 . 1 1 10 3 . 1 ln ln 29260 1 18 . 4 1 1000 7

− − − Δ −

× = × = × = = = Δ − = − = Δ − = ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ −

B. B.

( )( )

M M K M K e K e K RT G K K RT G mol J mol J sites because it double now mol J cal J kcal cal mol kcal

d a a RT G a

• fa

a a

• fa
• fa

5 6 1 6 298 315 . 8 33440

10 3 . 7 10 38 . 1 1 10 38 . 1 ln ln 33440 16720 2 : 2 16720 1 18 . 4 1 1000 4 × = × = × = = = Δ − = − = Δ = × = ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ −

− − − − Δ −

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References References

This presentation was prepared with the This presentation was prepared with the aid of the following texts: aid of the following texts:

• Garrett and Grisham, 2002

Garrett and Grisham, 2002

• Berg, Tymoczko, and Stryer, online @

Berg, Tymoczko, and Stryer, online @ http://ncbi.nlm.nih.gov/books/bv.fcgi?rid=strye http://ncbi.nlm.nih.gov/books/bv.fcgi?rid=strye r.section.5576 r.section.5576

The following website was also used: The following website was also used: http://users.rcn.com/jkimball.ma.ultranet/Bi http://users.rcn.com/jkimball.ma.ultranet/Bi

• logyPages/B/B_and_Tcells.html
• logyPages/B/B_and_Tcells.html