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LECTURE NO:

1+---1 DONE BY: Ha. ' j a..A ~ - C " : 'l-- '-_ '

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Faculty OFMedicine

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Antibodies are formed of two light chaios and two heavy chains.Example:IgGThe oumber of amino acids in the light chain is about 214 amino acidsThe oumber of amino acids in the heavy chaio is about 446 amioo acid( which is twice as much as the light chain)

As the figure above shows :Heavy chaios are coooected together by disulfide boods and each lightchain is booded to the heavy chain by disulfide bood.

re.)1 .:-.)1 .1>1 eLecture #:4

Diversity: is the preseoce oflarge oumber (millioos) of differeot types ofantibodies.

Immunoglobulins (antibodies):We discussed previously the molecular basis of antibody specificity; howthe specificity of the antibody is determioed ? what part of the moleculedetermines the specificity and how the antibodies diversity is produced??

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The cleavage with papain (that produce partial digestion) produce halfof thetwo heavy chains as Fc fragment and two Fab fragments.

The heavy chains which are formed of 446 amino acids, also have onevariable region oflbe same length (108 a.a) that have a unique sequencewhich varies from one patient to the other. The rest from 109-446 aresimilar in many patients .

Within the variable region ( 1-108) , it was found that there are strechesofamino acids that shows prominent variability ( the sequence vary greatlyfrom one patient to anotheF::>hypervariability ) , where there are someregions that show less variahlility.

we conclude that there are two regions:I-variable region (because the amino acid sequence vary from one type ofantibody to another)2-constant region( halfthe molecule is variable and the other halfis constant)

whereas the amino acid sequence from 109- 214 is the same in many patient( not necessary exactly the same in all patient. we expect a norrnallittlevariation in amino acid sequence from one patient to another)

The amino acid sequence of the light chain like any amino acid sequencerequires purified protein 1 we cant study the amino acid sequence on amixture of antibodies, we need one type of antibody (multiple myloma).

Amino acid sequence of the light chains ofa multiple myloma patient showthat the amino acids from 1-108 vary (differ) from one patient to the other.i.e: for each patient there is a unique sequence of the amino acids from the Nterminal to almost half of the light chain.

Q One type of antibody can be obtained from a patient sufffering frommultiple myloma ; malignant growth of one single plasma cell. Thus all theantibodies produced in these cancer cells will have the same type andtherefore the same amino acid sequence!

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So both light and heavy chains have variable and constant regions!!Note that:the variable region of the light chain (VL) equals in length to the variableregion of the heavy chain (Vh)VL= VhThe constant region of the heavy chain is almost three times the constantregion of the light chain.Ch = 3 CLSo the variability is more in three segments of the variable region: these areknown as hypervariable segments.The function of these hypervariability segments is probably these are thesegments or a.a sequence where antigen binds.It means they form antigen binding sites and this was proved by a techniquecalled Xray crystallography.What is Xray crystallography???It is a technique that1- shows the three dimensions of the protein molecules.( in it we convert protein to protein crystals)2- determine the location of every a.a in the three dimension.It was found in Xray crystallography that the hypervariability regions are thebinding site for the antigen. Therefore they were named ComplementarityDetermining Regions ( CDR).Complementarity Determining Regions ( CDR):These are the regions which are complementary in shape to the antigen.Binding an antigen to the antibody is like the binding of a substarte to anenzyme. They fit together like key and lock ( they complement each other).

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Two b J Sh.oebiL>f a,..,hF- II ..1Sl-,..", ... J .

In the figure above note that in the light chain the length of the variableregion is almost the same as the length of the constant region.Where the constant region of the heavy chain is found in three domains andit is three times in length as the constant region of the light chain.

The three dimensional structure was studied for the variable and constantregIOns:

This shows us the shape of the constant domain and variable domain in thelight chain.This represents Bsheets as if you have two layers ofBsheets together andthere are loops that connect the streches of the Bsheets.The Complementarity Determining Region is the region that connects one Bstrand with the other.The variable region contain loops where the antibody binds the antigen.

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[fthe number of the anribodies is huge (millions) :How can the nucleotide sequence in the gene produces this large number ofpeptides (antibodies).Are there million of genes that produce these million ofantibodies??Actually NO, because the antibody specificity is dtermined by the lightchain sequence and the heavy chain sequence.

This is not the only way:The production of proteins from a gene occurs through this flow chart ofinfonnation:

Example:I fwe have 100 kinds of light chains and 100 kinds of heavy chain, whenthey combine in a random way knowing that we can combine every heavychain with every light chain, we will have100 100 = 10000 moleculeThis is one way of how we can get a large number of antibodies; every cellwill produce one type of light chain and one type of heavy chain and theycombine in different cells to produce different antibodies!

1- Double helix ofDNA( this is a gene of a protem)2- Synthesis ofmRNA by transcriptionmRNA contains the sequence of nucleotides that produce the polypeptidechain but in many mRNA types, its sequence may contain regions that arenot translated.

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Gene is present in three segments VJC (comple gene) with differentcombinations.Example:V, to V100J,toJ,C,

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V2J4 are joined ,the rest are removed not by splicing but it is on DNA level(gene rearrangement)Only one gene ofV segment will join only one gene of J segment as wehave in the above example:We have40 V genes5 J genesso 40 5 200 possibilities of genes for the light chain.Then transcription ofmRNA followed by splicing that will remove thesequence between the VJ and the C - - . light chain.

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--+ The body produces initially IgM type then change it to IgG withoutchanging specificity (variable region stays the same, we only change theconstant region.) we change the class from IgM to IgG.Bioenergetics

Bioenergetics: is the study of energy In the living systems.Life reqiures both energy and matter.We eat or need nutr ients In order to susta in life. Cellularly weneed energy in order to sustain l ife.The energy Is very Important to the cell.No energy, no l ife.The cause o f death Is almost loss of energy.Example:Hemorrhage:Severe blood loss (bleedlng)that means no blood is reaching tothe brain so the brain cells cannot produce adequate energy.Myocardial Infarction will occur, arteries will be blocked, no moreO2 will reach the cells.Energy is one of the major purposes of nutrients (nutrients are forenergy).Why energy Is needed??1- mechanical work: includesa) muscular contractionb) beating o f f lagella and ciliac) movement of chromosomes during cell division.d) any motion requires energy.2- active transport:the cells cannot stay alive without act ive transport.Nerve cells work by active transport I constantly there ispumping of sodium outside the cell I and potassium to the Insideof it .

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Ex:

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3- biosynthesis:synthesizing la rg e m ole cu le s fr om s ma ll m o no m er s requiresenergy.Proteins a re p ro du ce d by j oi ni ng a m in o acids.

Flow of energy (unidirection):: .>"~ ; " , \ r - :J'-""- ~ Q L : . - - , " " " " ' , - - _ ~~ - .....,.

........ '" ......:---..u.

The function o f the cel l depends on this membrane pot ent ia l thaIs a chi eve d by active transport which requires energy. Also,absorption o f nutr ients (absorption o f glucose through intest inalcel ls) requires energy.

How come???

4- heat (not the least important):It Is produced as by-product in all o f the above cases.Active transport needs energy, energy conversion Is not 100% Isome o f the energy wil l be l iberated as heat .The l iberat ion o fheat Is useful , i t keeps the body warm even In cold climates.S o m e ti m es , h e a t production in newborns i s e x tr em e ly importanto keep the b ab y w a rm .Sun is t h e u lt im a t e source o f energy fo r all l iving oraganisms.Even our body depends on the sun's energy.

Glucose compared to CO2 and H 20 Is rich In energy.

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transformation In th e c ell from

Energy Qeneratlon Is a mojor purpose o f metabol ism.Other purposes:1- synthesis o f building blocks.2- synthesis o f macromolecule3- degredatlon o f blomoleculesMetabolism: is the sum of all chemical reactions in all l ivingorganisms.Can be divided Into 2 types:1) catabolism: lar ge molecules (energy y e ll di ng n u tr ie n ts ) a redegraded Into energy p oo r e nt p ro du ct s l ike: CO2 , H 20.2) anabolism: the small molecules l ike amino acids, sugars ,fatty a c i d s , nitrogenous b as es a re c on ve rte d i nt o la rg emolecules l ike proteins I polysaccharides, l ipids and nucl iecacids.Bioenergetics:deals with energyone form to another .We use chemical energy (n':ltrients) converted to:1) concentrat ion gradient: Is the presence o f molecules or Ions indifferent concentration across the membrane.Example:Na is more concentrated outside the cel l than inside (high energystate).2) el ectri ca l gradient3) mechanical gradient4) heat and even l ightBioenerget ics Involve chemical processes and quanti tativeaspects o f energy t ransformat ion. This energy transformationfol lows the s am e r ul es that govern the nonliving s y st ems.Prediction o f spontaneity or react ions:Spontaneous means possible ( possible doesn't mean i t is a fastreaction or i t wil l a c tu a ll y o c cu r In the cell.)Non Spontaneous (not possible): cannot occur unless energy isprovide.

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Which thermodynamic function can be used to determine theposibil ity of a rxn??

Thank you,Your colleague,Haya AI-Qudah

Loss of heat is a Spontaneous process.Heat can f low from the system to the surrounding or from thesurrounging to the system.

The thermodynamic funct ion that thought to be useful Inpredicting the reaction possib i li ty Is the enthalpy! heat content!heat transformation.

Note:Thermodynamics help to understand the spontaneity of thereaction.

Hot object can be a t es t t ube , a cel l ,an organism ,or an animal.The heat can f low easi ly f rom a hot ob ject to cold object .This hot object wil l lose i ts energy! enthalpy to the surrounding.