Anemia ( #RBC or Hb) Primary Anemia Disorder that directly affects bone marrow production of RBCs Bone marrow

aplasia, drug poisoning , Gamma irradiation Maturation Failure, Deficiency of B12, Folic acid B12 re uired for D!" synthesis, essential for nuclear maturation and di#ision, lac$ inhibits RBC production Folic acid also re uired for D!" synthesis %ernicious "nemia Failure to absorb B12 &'sually no lac$ in diet( !ormally a lac$ of intrinsic factor %roduced by mucous cells in fundus of stomach Binds to B12, pre#ents digestion, assists in absorption B12 stored in the li#er released slowly )econdary "nemia Results from loss of RBCs, ie* Bleeding +emolysis of RBC, e-ample .rythroblastosis fetalis Drug %oisoning Genetic Disorders (heredity diseases) Sickle Cell Anemia Low o y!en tension ca"ses cells to de#orm (sickle) Block blood $essels% hemolysis

)pleen, /argest lymphoid organ, located on the left side of the abdominal ca#ity beneath the diaphragm 0t is ser#ed by the splenic artery and #ein, which enter and e-it at the hilus &"nctions' )ite of lymphocyte proliferation 0mmune sur#eillance and response Cleanses the blood )tores brea$down products of RBCs for later reuse )pleen macrophages sal#age and store iron for later use by bone marrow )ite of fetal erythrocyte production &normally ceases after birth( )tores blood platelets S(leen )) 1he spleen is an imm"nolo!ic #ilter o# the blood* 0t is made up of B cells, 1 cells, macrophages, dendritic cells, natural $iller cells and red blood cells* 0n addition to capturing foreign materials &antigens( from the blood that passes through

the spleen, migratory macrophages bloodstream*

and dendritic cells bring antigens to the spleen #ia the

"n immune response is initiated when the macrophage or dendritic cells present the antigen to the appropriate B or 1 cells* 1his organ can be thought of as an imm"nolo!ical con#erence center* +n the s(leen% B cells become acti$ated and (rod"ce lar!e amo"nts o# antibody * "lso, old red blood cells are destroyed in the s(leen* ,hym"s " bilobed organ that secretes hormones &thymosin and thymopoietin( that cause 1 lymphocytes to become immunocompetent )i2e of the thymus #aries with age, 0n infants, it is found in the inferior nec$ and e-tends into the mediastinum where it partially o#erlies the heart 0t increases in si2e and is most acti#e during childhood 0t stops growing during adolescence and then gradually atrophies &"nction' 1he thymus differs from other lymphoid organs in important ways +t #"nctions strictly in , lym(hocyte mat"ration 0t does not directly fight antigens ,hym"s )) 1he function of the thymus is to produce mature 1 cells* 0mmature thymocytes, also $nown as prothymocytes, lea#e the bone marrow and migrate into the thymus* 1hrough a maturation process sometimes referred to as thymic education, 1 cells that are beneficial to the immune system are spared, while those 1 cells that might e#o$e a detrimental autoimmune response are eliminated* 1he mature 1 cells are then released into the bloodstream* L"m(h -odes'

" lym(h node is a small ball3shaped organ of the immune system, distributed widely throughout the body including the armpit and stomach4gut and lin$ed by lymphatic #essels* /ymph nodes are garrisons of B, 1, and other immune cells* /ymph nodes act as filters or traps for foreign particles* 1hey are important in the proper functioning of the immune system* 1hey become inflamed or enlarged in #arious conditions, throat infection, to cancers* $s* Lym(hocytes' a type of white blood cell, will meet the antigens, or proteins, in the peripheral lymphoid organs, which includes lymph nodes* 1he antigens are displayed by speciali2ed cells in the lymph nodes* "fter the lymphocytes speciali2e they will e-it the lymph node through the efferent lymphatic #essel* 1he lymphocytes continuously recirculate the peripheral lymphoid organs and the

state of the lymph nodes depends on infection* During an infection of fermintation, the lymph nodes can e-pand due to intense B3cell proliferation in the germinal centers, a condition commonly referred to as 5swollen glands5*
Lym(h -odes )) 1he lymph nodes function as an imm"nolo!ic #ilter #or the bodily #l"id known as lym(h* /ymph nodes are found throughout the body* Composed mostly of , cells% B cells% dendritic cells and macro(ha!es* Lym(h nodes drain #l"id #rom most o# o"r tiss"es* Anti!ens are #iltered o"t o# the lym(h in the lym(h node be#ore ret"rnin! the lym(h to the circ"lation* 0n a similar fashion as the spleen, the macrophages and dendritic cells that capture antigens present these foreign materials to 1 and B cells, conse uently initiating an immune response* 1hey may become enlarged due to a tumor or infection* /ymph nodes are enlarged when the body is infected due to enhanced production of some cells and di#ision of acti#ated 1 and B cells* 6hite blood cells are located within honeycomb structures of the lymph nodes* .ain #"nctions o# the lym(hatic system 5to collect and return interstitial fluid, including plasma protein to the blood, and thus help maintain fluid balance, to defend the body against disease by producing lymphocytes, to absorb lipids from the intestine and transport them to the blood*5

Lym(h or!ans' 0nclude the bone marrow, lymph nodes, spleen, and thymus* %recursor cells in the bone marrow produce lymphocytes* B3lymphocytes &B3cells( mature in the bone marrow* 13lymphocytes &13cells( mature in the thymus gland* &"nction, Besides pro#iding a home for lymphocytes &B3cells and 13cells(, the ducts of the lymphatic system pro#ide transportation for proteins, fats, and other substances in a medium called lymph* *

/idney'

Main Function : Excretion of nitrogenous waste and regulation of water balance Nitrogenous Wastes:
Nitrogenous waste dissolved in H20,

therefore bond up with water balance considerations

Filtration System:

he Nephron:

he Nephron and !ollecting duct: regional functions of the transport epitheliu":

he wo #olute Model:

Hor"onal $nfluences lin% water balance, %idne& function and blood pressure: '(H)*asopressin:

+enin,'ngiotensin,'ldosterone #&ste":

Hemolyzation: 1he production or occurrence of hemolysis*

Hemolysis &or haemolysis(7from the Gree$ 898 &aima, haema, hemo3( meaning 5blood5 and :;<=> &lusis, lysis, 3lysis( meaning a 5loosing5, 5setting free5 or 5releasing5?1@7is the rupturing of erythrocytes &red blood cells( and the release of their contents &hemoglobin( into surrounding fluid &e.g., blood plasma(* +emolysis may occur in vivo or in vitro &inside or outside the body(*

Genetics' &itness &itness' &often denoted w in population genetics models( is a central idea in e#olutionary theory* 0t can be defined either with respect to a genotype or to a phenotype*

0n either case, it describes the ability to both s"r$i$e and re(rod"ce, and is e0"al to the a$era!e contrib"tion to the !ene (ool o# the ne t !eneration that is made by an a#erage indi#idual of the specified genotype or phenotype* 0f differences between alleles at a gi#en gene affect fitness, then the fre uencies of the alleles will change o#er generationsA the alleles with hi!her #itness become more common* 1his process is called natural selection* Absol"te #itness and Relati$e &itness' Absolute fitness (wabs) of a genotype is defined as the ratio between the number of indi#iduals with that genotype after selection to those before selection* 0t is calculated for a single generation and may be calculated from absolute numbers or from fre uencies* 6hen the fitness is larger than 1*B, the genotype increases in fre uencyA a ratio smaller than 1*B indicates a decrease in fre uency*

Relative fitness is uantified as the a#erage number of sur#i#ing progeny of a particular genotype compared with a#erage number of sur#i#ing progeny of competing genotypes after a single generation, i*e* one genotype is normali2ed at w C 1 and the fitnesses of other genotypes are measured with respect to that genotype* Relati#e fitness can therefore ta$e any nonnegati#e #alue, including B* 6hile researchers can usually measure relati#e fitness, absolute fitness is more difficult* 0t is often difficult to determine how many indi#iduals of a genotype there were immediately after reproduction* 1he two concepts are related, and both of them are e ui#alent when they are di#ided by the mean fitness, which is weighted by genotype fre uencies*

1lectro(horesis and Amino Acids' 1he distribution of charged species in a sample can be shown e-perimentally by obser#ing the mo#ement of solute molecules in an electric field, using the techni ue of electro(horesis* an ionic buffer solution is incorporated in a solid matri- layer, composed of paper or a crosslin$ed gelatin3li$e substance* " small amount of the amino acid, peptide or protein sample is placed near the center of the matri- strip an electric potential is applied at the ends of the strip 1he solid structure of the matri- retards the diffusion of the solute molecules, which will remain where they are inserted, unless acted upon by the electrostatic potential* 0n the e-ample shown here, four different amino acids are e-amined simultaneously in a p+ D*BB buffered medium* !ote that the colors in the display are only a con#enient reference, since these

amino acids are colorless*

Res"lts' "t p+ D*BB alanine and isoleucine e-ist on a#erage as neutral 2witterionic molecules, and are not influenced by the electric field* "rginine is a basic amino acid* Both base functions e-ist as 5onium5 conEugate acids in the p+ D*BB matri-* 1he solute molecules of arginine therefore carry an e-cess positi#e charge, and they mo#e toward the cathode* 1he two carbo-yl functions in aspartic acid are both ioni2ed at p+ D*BB, and the negati#ely charged solute molecules mo#e toward the anode in the electric field* )tructures for all these species are shown to the right of the display* res"lt o# this e (eriment is critically de(endent on the (H o# the matri b"##er* 0f we were to repeat the electrophoresis of these compounds at a p+ of F*GB, the aspartic acid would remain at its point of origin, and the other amino acids would

mo#e toward the cathode* +!norin! di##erences in molec"lar si2e and sha(e% the ar!inine wo"ld mo$e twice as #ast as the alanine and isole"cine beca"se its sol"te molec"les on a$era!e wo"ld carry a do"ble (ositi$e char!e +soelectric Points and Side Chains o# Amino acids (/a 3al"es o# Poly#"nctional Amino Acids 4) C56H (/a7
2*1 2*1 1*J 2*2 1*G 2*2 2*2

Amino Acid
"rginine "spartic "cid Cysteine Glutamic "cid +istidine /ysine 1yrosine

4)-H 8 6 (/a H*B H*G 1B*K H*J H*2 H*B H*1 12*I F*H G*F K*F D*B 1B*I 1B*1

Side Chain (/ 8 a

(+

1B*G F*B I*B F*2 J*D H*G I*J

)ome amino acids ha#e additional acidic or basic functions in their side chains*

,he +soelectric (oint% (+,

is the (H o# an a0"eo"s sol"tion o# an amino acid (or (e(tide) at which the molec"les on a$era!e ha$e no net +n other words% the (ositi$ely char!ed !ro"(s are e actly balanced by the ne!ati$ely char!ed !ro"(s* For simple amino acids such as alanine, the p0 is an a#erage of the pL groups* 1hus, the p0 for alanine is calculated to be, &2*FK N H*DH(42 C D*B2, the e-perimentally determined #alue* +# additional acidic or basic !ro"(s are (resent as side)chain #"nctions, the (+ is the a$era!e o# the (/a9s o# the two most similar acids* 1o assist in determining similarity we define two classes of acids* 1he first consists of acids that are neutral in their protonated form &e*g* CO2+ P )+(* N(* 1he second includes acids that are positi#ely charged in their protonated state &e*g* 3!+F 0n the case of aspartic acid, the similar acids are the alpha3carbo-yl function &pL carbo-yl function &pLa C F*H(, so p0 C &2*1 N F*H(42 C F*B* For arginine, the similar acids are the guanidinium species on the side3chain &pLa C 12*I( and the alpha3ammonium function &pLa C H*B(, so the calculated p0 C &12*I N H*B(42 C 1B*JI* a C 2*1( and the side3chain aMs of the carbo-yl &2*FK( and ammonium &H*DH(

char!e*

+soelectric Point and 1lectro(horesis' +soelectric Point' 1he p+ at which a protein carries no net charge or protein has an e ual number of positi#e and negati#e charges Below the isoelectric (oint (roteins carry a net (ositi$e char!e% abo$e it a net ne!ati$e char!e* At a (H below the isoelectric (oint% (roteins carry a net (ositi$e char!e At a (H abo$e the isoelectric (oint (rotein has a net ne!ati$e char!e*

Due to a preponderance of wea$ly acid residues in almost all proteins, they are nearly all negati#ely charged at neutral p+* 1he isoelectric (oint is of significance in (rotein ("ri#ication because it is the (H at which sol"bility is o#ten minimal mobility in an electro#oc"sin! system is 2ero &and therefore the point at which the (rotein will acc"m"late(* 1he property of isoelectric point has important biochemical implications in (rotein ("ri#ication and electro(horesis* 0n electrophoresis, if the (H o# the b"##er is hi!her than the isoelectric (oint of the protein, it will migrate towards the (ositi$e 6hile if the (h o# the b"##er is lower than the isoelectric (oint of the protein, then it will mi!rate towards ne!ati$e terminal* :hen the b"##er (H is e0"al to the (+ o# a (rotein it will not mi!rate at all* " protein can e-hibit different charges depending on the p+ of the medium* "t their electrostatic point, proteins e-hibit at least electrostatic repulsion, hence they ha#e the lowest solubility at this point and can easily precipitate* 1his property is useful in crystalli2ation of proteins* 0n general, positi#e and negati#e charges on the surface of protein are balanced around neutral p+* 1he electrostatic attraction pro#ides the compact shape and greater stability to the protein* On the other side, at e-tremely low p+, the carbo-yl groups are protonated and negati#e charges decrease* 0n this condition, proteins gain more electrostatic repulsion and become denatured* 6hen acidic proteins are denatured in an acidic condition, they aggregate easily* On the other hand, when basic proteins are denatured in acidic conditions, they do not aggregate much because the proteins ha#e greater number of positi#e charges in the acidic condition and the electrostatic repulsion is high* 6hen the p+ is brought bac$ to neutral, non3precipitated basic proteins may return to their nati#e structure* 1his is not generally true for precipitated acidic proteins, which often re uire treatment with strong denaturants, such as urea or guanidine hydrochloride* 1he e-amples of isoelectric point, terminal* and at which

1he isoelectric point of "lbumin in +uman serum is K*D Q I*F 1he isoelectric point of "mylase in +uman sali#a is D*2 Q D*I 1he isoelectric point of %rolactin in +uman pituitary is D*I 1he isoelectric point of 1hrombin in +uman plasma is J*1

Thin Layer Chromatography: 1hin layer chromatography can be used to,

Monitor the progress of a reaction 0dentify compounds present in a gi#en substance Determine the purity of a substance

General Strate!y o# Protein P"ri#ication)#ractionate based on di##erent characteristics %roteins are purified by fractionation procedures in a series of steps* 1he goal is not necessarily to preser#e all of the protein of interest, although it is important to preser#e as much as possible* One must, howe#er, eliminate all of the other proteins in the mi-ture, and this usually means that much of the protein of interest will be lost* One needs a criterion of purityA with en2ymes this is the specific acti#ity C amount of catalytic acti#ity per unit mass &mg(* )imilar criteria can be established for other proteins based upon their biological acti#ities* 7* Char!e

ion e-change chromatography electrophoresis


6* Polarity

isoelectric focusing

8* Si2e

adsorption chromatography paper chromatography re#erse3phase chromatography hydrophobic interaction chromatography

dialysis and ultrafiltration gel electrophoresis gel filtration chromatography ultracentrifugation

;* S(eci#icity o# bindin!)a##inity chromato!ra(hy

thin layer chromatography for example--and upon the physical principle--ion exchange chromatography, partition between phases of different polarity, size exclusion chromatography etc. All depend upon having a mobile phase, usually a liquid, and a stationary phase, usually a solic or a solid coated with a liquid. The sample is applied in the mobile phase which passes down the column (or paper or thin layer plate) and the different solutes move at different speeds depending upon their relative affinities for the mobile and stationary phases; we say that they partition between the mobile and stationary phases. A. Ion Exchange Chromatography--adsorption chromatography 1. Stationary Phase -- chemically bound charged groups with counter ions bound; these may be positively charged groups which bind anions (anion exchanger) or negatively charged groups which bind cations (cation exchanger) Table 5-2 2. Mobile Phase -- an aqueous buffer solution characterized by: pH and ionic strength

Covalent Structures of Proteins Primary structure (1 structure) is the amino acid sequence of its polypeptide chain(s). Secondary (2) structure is the local spatial arrangement of a polypeptide's backbone atoms without regard to the conformations of its side chains. Tertiary (3) structure refers to the three-dimensional structure of an entire polypeptide. The line between 2 and 3 structure is somewhat vague. Quaternary (4) structure is present in only some proteins, those which contain more than one polypeptide chain, referred to as subunits, held together by noncovalent bonds or, in some cases, disulfide bonds.

Separation, Purification, and Characterization of Polypeptide Chains 1. Denature polypeptides by changing pH, [salt], or adding chemicals such as urea or guanidinium which disrupt structure. 2. Purify proteins using techniques described above: chromatography, electrophoresis, etc. 3. Mass spectrometry Specific Peptide Cleavage Reactions&endash;can sequence polypeptides 40-80 amino acid residues long ==> longer polypeptides and proteins must be cut into smaller pieces at specific locations 1. Endopeptidases: see Table 5-5 for specificities of endopeptidases


1/C

Trpsin cleaves peptide bonds whose carboxyl group is provided by positively charged residues (e.g. Lys or Arg)

Chymotrypsin cleaves after bulky hydrophobic residues (Phe, Trp, Tyr)

Endopeptidase V8 from Staph aureus cleaves after Glu

Chromatography is used to separate mi-tures of substances into their components* "ll forms of chromatography wor$ on the same principle* 1hey all ha#e a stationary phase &a solid, or a li uid supported on a solid( and a mobile phase &a li uid or a gas(* 1he mobile phase flows through the stationary phase and carries the components of the mi-ture with it* Different components tra#el at different rates*

The stationaryphase- silica gel Silica gel is a form of silicon dioxide (silica). The silicon atoms are joined via oxygen atoms in a giant covalent structure. However, at the surface of the silica gel, the silicon atoms are attached to -OH groups. So, at the surface of the silica gel you have Si-O-H bonds instead of Si-O-Si bonds. The diagram shows a small part of the silica surface.

1he surface of the silica gel is #ery polar and, because of the 3O+ groups, can form hydrogen bonds with suitable compounds around it as well as #an der 6aals dispersion forces and dipole3dipole attractions* 1he other commonly used stationary phase is alumina 3 aluminium o-ide* 1he aluminium atoms on the surface of this also ha#e 3O+ groups attached* "nything we say about silica gel therefore applies e ually to alumina* :hat se(arates the com(o"nds as a chromato!ram de$elo(s< "s the sol#ent begins to soa$ up the plate, it first dissol#es the compounds in the spot that you ha#e put on the base line* 1he compounds present will then tend to get carried up the chromatography plate as the sol#ent continues to mo#e upwards* How #ast the com(o"nds !et carried "( the (late de(ends on two thin!s'

How sol"ble the com(o"nd is in the sol$ent* ,his will de(end on how m"ch attraction there is between the molec"les o# the com(o"nd and those o# the sol$ent* How m"ch the com(o"nd sticks to the stationary (hase ) the silica !et% #or e am(le* ,his will de(end on how m"ch attraction there is between the molec"les o# the com(o"nd and the silica !el*

)uppose the original spot contained two compounds 3 one of which can form hydrogen bonds, and one of which can only ta$e part in wea$er #an der 6aals interactions* 1he one which can hydrogen bond will stic$ to the surface of the silica gel more firmly than the other one* 6e say that one is adsorbed more strongly than the other* "dsorption is the name gi#en to one substance forming some sort of bonds to the surface of another one* "dsorption isnMt permanent 3 there is a constant mo#ement of a molecule between being adsorbed onto the silica gel surface and going bac$ into solution in the sol#ent* Ob#iously the compound can only tra#el up the plate during the time that it is dissol#ed in the sol#ent* 6hile it is adsorbed on the silica gel, it is temporarily stopped 3 the sol#ent is mo#ing on without it* 1hat means that the more strongly a compound is adsorbed, the less distance it can tra#el up the plate*

0n the e-ample we started with, the compound which can hydrogen bond will adsorb more strongly than the one dependent on #an der 6aals interactions, and so wonMt tra#el so far up the plate* 6hat if both components of the mi-ture can hydrogen bondR 0t is #ery unli$ely that both will hydrogen bond to e-actly the same e-tent, and be soluble in the sol#ent to e-actly the same e-tent* 0t isnMt Eust the attraction of the compound for the silica gel which matters* "ttractions between the compound and the sol#ent are also important 3 they will affect how easily the compound is pulled bac$ into solution away from the surface of the silica* +owe#er, it may be that the compounds donMt separate out #ery well when you ma$e the chromatogram* 0n that case, changing the sol#ent may well help 3 including perhaps changing the p+ of the sol#ent*