AMINO ACIDS AND PROTEINS

SUNIL SHAHBOND KING

THE B

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AMINO A

CIDS

BASIC STRUCTURES An amino acid contains:

a central carbon bonded to a carboxyl group (-COOH), an amino group (NH2), an R-group, and a hydrogen.

The R-group is what varies between the 20 amino acids and gives them unique characteristics.

The covalent bonds between amino acids are called peptide bonds. It is a bond between the carboxyl group of one amino

acid and the amino group of another amino acid.

Abbrev.

Full Name

Side chain type Remarks

A Ala Alanine hydrophobic

Very abundant and versatile. It behaves fairly neutrally and can be located in both hydrophilic regions on the outside of the protein and hydrophobic interior areas.

C Cys Cysteinehydrophobic (Nagano, 1999)

The sulfur atom binds readily to heavy metal ions. Under oxidizing conditions, two cysteines can be joined together by a disulfide bond to form the amino acid cystine. When cystines are components of a protein, they enforce tertiary structure and makes the protein more resistant to unfolding and denaturation; disulfide bridges are therefore common in proteins that have to function in harsh environments, digestive enzymes (e.g., pepsin and chymotrypsin), structural proteins (e.g., keratin), and proteins too small to hold their shape on their own (eg. insulin).

D Asp Aspartic acid

acidic

Behaves similarly to glutamic acid. Carries a hydrophilic acidic group with strong negative charge. Usually is located on the outer surface of the protein, making it water-soluble. Binds to positively-charged molecules and ions, often used in enzymes to fix the metal ion.

E Glu Glutamic acid

acidicBehaves similar to aspartic acid. Has longer, slightly more flexible side chain.

Abbrev.

Full Name

Side chain type

Remarks

*F Phe Phenylalanine

hydrophobic

Phenylalanine, tyrosine, and tryptophan contain large rigid aromatic group on the side chain. These are the biggest amino acids. Like isoleucine, leucine and valine, they are hydrophobic and tend to orient towards the interior of the folded protein molecule.

G Gly Glycinehydrophobic

Because of the two hydrogen atoms at the α carbon, glycine is not optically active. It is the smallest amino acid, rotates easily, and adds flexibility to the protein chain. It is able to fit into the tightest spaces (e.g., the triple helix of collagen).

*H His Histidine basic

In even slightly acidic conditions, protonation of the nitrogen occurs, changing the properties of histidine and the polypeptide as a whole. It is used by many proteins as a regulatory mechanism, changing the conformation and behavior of the polypeptide in acidic regions such as the late endosome or lysosome, enforcing conformation change in enzymes.

*I Ile Isoleucine

hydrophobic

Isoleucine, leucine and valine have large aliphatic hydrophobic side chains. Their molecules are rigid, and their mutual hydrophobic interactions are important for the correct folding of proteins, as these chains tend to be located inside of the protein molecule. 

Abbrev.

Full Name

Side chain type

Remarks

*K Lys Lysine basic

Behaves similarly to arginine. Contains a long flexible side-chain with a positively-charged end. The flexibility of the chain makes lysine and arginine suitable for binding to molecules with many negative charges on their surfaces. (e.g., DNA-binding proteins have their active regions rich with arginine and lysine.) The strong charge makes these two amino acids prone to be located on the outer hydrophilic surfaces of the proteins.

*L Leu Leucine hydrophobic Behaves similar to isoleucine and valine.

*M

Met Methionine

hydrophobic

Always the first amino acid to be incorporated into a protein; sometimes removed after translation. Like cysteine, it contains sulfur, but with a methyl group instead of hydrogen. This methyl group can be activated, and is used in many reactions where a new carbon atom is being added to another molecule.

N Asn Asparagine

hydrophilic Neutralized version of aspartic acid.

Abbrev.

Full Name

Side chain type

Remarks

P Pro Prolinehydrophobic

Contains an unusual ring to the N-end amine group, which forces the CO-NH amide sequence into a fixed conformation. Can disrupt protein folding structures like α helix or β sheet, forcing the desired kink in the protein chain. Common in collagen, where it undergoes a posttranslational modification to hydroxyproline. Uncommon elsewhere.

Q Gln Glutamine

hydrophilicNeutralized version of glutamic acid. Used in proteins and as a storage for ammonia.

*R Arg Arginine Basic Functionally similar to lysine.

S Ser Serine Hydrophilic

Serine and threonine have a short group ended with a hydroxyl group. Its hydrogen is easy to remove, so serine and threonine often act as hydrogen donors in enzymes. Both are very hydrophilic, therefore the outer regions of soluble proteins tend to be rich with them.

Abbrev.Full

NameSide chain

typeRemarks

*T Thr Threonine hydrophilic Behaves similarly to serine.

*V Val Valine hydrophobicBehaves similarly to isoleucine and leucine. See isoleucine.

*W Trp Tryptophan

hydrophobicBehaves similarly to phenylalanine and tyrosine (see phenylalanine). Precursor of serotonin.

Y Tyr Tyrosine hydrophobic

Behaves similarly to phenylalanine and tryptophan (see phenylalanine). Precursor of melanin, epinephrine, and thyroid hormones.

GENERALITIES

Classification

• Neutral

• Acidic

• Basic

Neutral• Aliphatic• Aromatic• Heterocyclic• S-containing AA

GENERALITIES - NEUTRALA. Aliphatic

1. Glycine (Gly) (G) – alpha– amino acetic acid

2. Alanine (Ala) (A) – alpha – amino propionic acid

3. Valine (Val) (V) – alpha– amino – iso – valeric acid

4. Leucine (Leu) (L) – alpha – amino – iso – caproic acid

5. Isoleucine (Ile) (l) – alpha – amino – B – methyl valeric acid

Above = Simple monoamino monocarboxylic acids

Hydroxy Amino Acids – they contain OH group in the side chains

6. Serine (Ser) (S) or alpha – amino β – hydroxy propionic acid

7. Threonine (Thr) (T) or alpha – amino β – hydroxybutyric acid

GENERALITIES B. Aromatic 8. Phenylalanine

(Phe) (F) or alpha – amino – β – phenyl propionic acid

9. Tyrosine (Tyr) (y) or parahydroxy phenylalanine or alpha – amino β – parahydroxy phenylpropionic acid

C. Heterocyclic AA 10. Tryptophan (Trp) (W) –

alpha – amino β – 3 – indole propionic

- often considered as aromatic AA since it has aromatic ring in its structure.

11. Histidine (His) (H) – alpha – amino – β - imidazole propionic acid

Histidine is basic in solution on account of the imidazole ring and often considered as Basic AA

GENERALITIES D. Imino Acids 12. Proline (Pro) (P) or

Pyrrolidone – 2 – carboxylic Acid

13. Hydroxyproline (Hyp) or 4 – Hydroxy pyrrolidone – 2 – carboxylic acid

Proline & Hydroxyproline do

not have a free _NH2 group but

only a basic pyrrolidone ring in which the Nitrogen of the Imino group is in ring but can still function in the formation of peptides.

Are called Imino Acids.

E. ‘S’ containing Amino Acids

- contains 2 sulfur containing AA

14. Cysteine (Cys) (C) or alpha – amino – β – mercaptopropionic acid

S – S linkage is called a Disulfide bridge

15. Methionine (Met) (M) or alpha – amino y – methylthio - - butyric acid

GENERALITIES - ACIDIC II. Acidic AA

- AA with 2 _COOH groups and 1 _NH2 group

- monoaminodicarboxylic Acids 16. Aspartic Acid (Asp) (P) or alpha – amino succinic

acid Asparagine (Asn) (N) or delta amide of alpha –

amino succinic acid 17. Glutamic Acid (Glu) (E) or alpha aminoglutaric

acid Glutamine (Gln) (Q) – amide of glutamic acid or 8

– amide of alpha – amino glutaric acid

GENERALITIES - BASIC III. Basic AA - AA with 1 – COOH group and 2 –

NH2 groups - Diamino monocarboxylic acids

18. Arginine (Arg) (R) or alpha – amino – 8 – guanidino - valeric acid

19. Lysine (Lys) (K) or alpha – E diamino 8 – hydroxy - - valeric acid

As already mentioned Histidine – is also classified as Basic AA

GENERALITIES

Classification of Amino Acids based on polarity

of the R group:• 4 groups

• Polarity reflects the functional role of AA in protein structure

GENERALITIES1. Non-polar AA

• hydrophobic (water hating)

• No charge on the ‘R’ group

• Examples are:

Alanine MethionineLeucine

PhenylalanineIsoleucine

TryptophanValine Proline

GENERALITIES 2. Polar AA with (+) ‘R’ group

• carries (+) charge• Examples:

Histidine Arginine Lysine

3. Polar AA with (-) ‘R’ group• carries (-) charge• Examples:

Glutamic Acid Aspartic Acid

GENERALITIES 4. Polar AA with no charge on ‘R’ group

• no charge on the ‘R’ group

• possess groups hydroxylsulfhydrylamide

• participate in hydrogen bonding of • protein structure

• Examples:

Asparagine Glycine CysteineTyrosine Serine Threonine

Glutamine

GENERALITIES Zwitterion or dipolar ion:

Zwitter from German word –

means “hybrid”

Zwitter ion (or dipolar ion) a hybrid molecule

containing (+) and (-) ionic groups

GENERALITIESAA rarely exist in a neutral form with free carboxylic (-COOH) and free Amino (-NH2) groups

Strongly acidic pH (low pH) AA (+) charged

(cation)

Strongly alkaline pH (high pH) AA (-) charged (anion)

Each AA has a characteristic pH (e.g. Leucine, pH – 6.0), at which it carries both (+) and (-) charges and exist as zwitterion

GENERALITIES Isoelectric pH (symbol pI)

the pH at which a molecule exists as a Zwitterion or dipolar ion and

carries no net charge

Molecule is electrically neutral

GENERALITIES

P V T

T I M

H A L L

Tryptophan

Valine

Phenylalanine

Threonine

Isoleucine

Methionine

Histidine Arginine

Leucine

Lysine

Essential Amino Acids

    Essential Amino Acids (Body cannot make these amino acids, they must come from food or amino acid supplements.)

 Amino Acid

Sym

Abbr

Min.Dailymg

AcidBaseNeu.

 # of pathways - One Pathway

Generates

Works With Augments

1  Isoleucine* I Ile 10 n  1 - threonine -->

isoleucine - - muscles

2  Leucine* L Leu 14 n  1 - keto-isovalerate --> leucine - -

blood, muscle, hormone

3  Lysine K Lys 12 B  2 - asparate --> lysine - calciu

m

herpes, triglycerides

4  Methionine M Met 13 n  7 - homoserine -->

methionine cysteine selenium, zinc

hair, skin, chelator

5  Phenylalanine F Phe 14 n  3 - chorismate -->

phenylalanine tyrosine B6 depression

6  Threonine T Thr 7 N  3 - aspartate --> threonine

glycine, serine -

collagen, tooth enamel

7  Tryptophan W Trp 3.5 n  1 - chorismate -->

tryptophan

niacin, seratonin

- depression

8  Valine* V Val 10 n  1 - pyruvate --> valine - - muscles

          Food Sources: Fish - meat - poultry - cottage cheese - peanuts - lentils          A= acid, B= base, N= Neutral, n= non-polar          *= BCAA, Branched-Chain Amino Acids, all important in muscle recovery

  Non-Essential Amino Acids (The body can make these amino acids from the above essential amino acids.)

Amino Acid Sym. Abbr

Min.Dailymg.

AcidBaseNeu.

 # of pathways - One Pathway Generates

Works With Augments

1  Alanine A Ala - n  4 - valine pyruvate --> alanine - - -

2  Arginine* R Arg - B  4 - citrulline --> arginine polyamines, creatine

zinc (lysine)

immune, healing, muscles

3  Asparagine N Asn - N  3 - asparate and

ammonia - - CNS

4  Aspartic Acid D Asp - A  3 - glutamate -->

aspartate - - CNS, brain

5  Cysteine C Cys - N  4 - serine --> cysteine homocysteine, taurine B6, Vit.E skin, hair

6  Glutamic Acid E Glu - A  3 - ketoglutarate -->

glutamate glutamine, GABA B6 brain

7  Glutamine Q Gln - N  2 - glutamate --> glutamine - - brain

8  Glycine G Gly - N  2 - serine --> glycine glutathione GABA, taurine body protein

9  Histidine* H His - B  1 - histidinol --> histidine histamine - blood,

allergy, sex

10  Proline P Pro - n  4 - l-glutamine --> proline

hydroxyproline Vitamin C collagen,

elastin

11  Serine S Ser - N  1 - phosphoglycerate --> serine

cysteine, glycine choline blood sugar

12  Tyrosine Y Tyr - N  2 - phenylalanine --> tyrosine

thyroxin, melanin  B6 thyroid

              * = These are Essential for infants, since their bodies cannot produce them yet.

Other Amino Acid Factors

-  Carnitine - Ca

r - -  carnitine --> butyrobetaine -->

Lysine, methionine

Vitamin C

fat metabolism

-  Citrulline - Cit - -

 argenine --> citrulline --> ornithine

Ornithine zinc urea cycle

-  Hydroxyproline - Hy

p - -  proline --> hydroxyproline - Vitamin

C collagen

-  Ornithine - Or

n - -  argenine --> citrulline --> ornithine

- - urea cycle

-  Taurine - Tau - -  methionine -->

cysteine --> taurine GABA, glycine B6 heart, bile

              The above are precursors for, or important products of, the 20 "true" amino acids listed above.

AMINO ACID SYNTHESIS ALL ARE SYNTHESIZED FROM COMMON

METABOLIC INTERMEDIATES

NON-ESSENTIAL Transamination of -KETOACIDS that are

available as common intermediates All except tyrosine are derived from one of the

following common intermediates: pyruvate, oxalacetate, -KG, 3-phosphoglycerate

ESSENTIAL Their -KETOACIDS are not common

intermediates (Enzymes needed to form them are lacking)

Amino Acids Glucogenic Glucogenic and Ketogenic

Ketogenic

Non-Essential AA

AlanineAsparagineAspartateCysteine GlutamateGlutamineGlycineProlineSerine

Tyroxine  

Essential AA ArginineHistidineMethionineThreonineValine

IsoleucinePhenylalanineTryptophan

LeucineLysine

Glucogenic – amino acids that give rise to pyruvate and citric acid cycle intermediates that can be turned into glucoseKetogenic – amino acids that give rise to acetoacetate and acetyl-co-A, which do not yield glucose

Amino Acid

Metabolism

AMINOACIDOPATHIES•

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TYROSINEMIA

DEFECTIVE PROCESS: AMINO ACID TRANSPORT – EXCRETION OF TYROSINE AND TRYPTOPHAN CATABOLITES IN URINE

DEFECTIVE ENZYME: • TYROSINE AMINOTRANSFERASE (II); • 4-HYDROXY-PHENYLPYRUVATE OXIDASE (III); • FUMARYLACETOACETATE HYDROLASE (I)

ALKAPTONURIA DEFECTIVE PROCESS: TYROSINE

DEGRADATION DEFECTIVE ENZYME: HOMOGENTISATE-

1,2-DIOXYGENASE• Original “inborn error of metabolism• Darkening of urine at long standing• OCHRONOSIS – generalized

pigmentation of tissues and arthritislike degeneration

PHENYLKETONURIA

DEFECTIVE PROCESS: CONVERSION OF PHENYLALANINE TO TYROSINE

DEFECTIVE ENZYME: PHENYLALANINE HYDROXYLASE (Phenylalanine-4-mono-oxygenase)

An autosomal recessive trait – urine has a musty odor

Compounds seen in both urine and blood: Phenylpyruvic acid – primary catabolite Phenyllactic acid – product of deaminzation Phenylacetylglutamine – produced from oxidation

of phenylpyruvic acid

PHENYLKETONURIA NOT RESPONSIVE TO DIET: DEFICIENCY OF ENZYME S NEEDED

FOR THE REGENERATION AND SYNTHESIS OF TETRAHYDROBIOPTERIN (BH4)• High phenylalanine and deficiency of production of

neurotransmitters from tyrosine and tryptophan • Administration of L-dopa and 5-OH tryptophan

SCREENING: GUTHRIE BACTERIAL INHIBITION ASSAY – B. subtilis + β2-thienylalanine• Semi-qualitative method: phenylalanine >2-4 mg/dL will

result to bacterial growth indicative of a POS (+) result• Microfluorometric assay – based on the fluorescence complex

formed of phenylalanine-ninhydrin copper in the presence of dipeptides (L-leucyl-L-alanine)• Quantitative method: filter paper is pretreated with

trichloroacetic acid added with ninhydrin, succinate and leucylalanine in the presence of copper tartrate and read at excitation and emission wavelengths of 360nm and 530nm

HPLC – reference method (1.2 -3.4 mg/dL)

MAPLE SYRUP DISEASE DEFECTIVE PROCESS: METABOLISM OF THREE ESSENTIAL

BRANCHED-CHAINAMINO ACIDS (LEUCINE, ISOLEUCINE AND VALINE)

DEFECTIVE ENZYME: BRANCHED CHAIN α-KETO ACID DECARBOXYLASE COMPLEX

• Burnt sugar odor of urine, breath and skin• Screening: Modified Guthrie test – metabolic inhibitor of B. subtilis

which is 4-azaleucine is impregnated in the medium• POS (+) for MSUD = bacterial growth

• Microfluorometric assay using leucine dehydrogenase• Filter paper specimen is treated with methanol and acetone to

denature hemoglobin• The NADH fluorescence produced is measured at 450nm;

excitation wavelength at 360nm• Confirmed diagnosis is based on finding increased plasma and

urinary levels of the three branched-chain amino acids and their ketoacids with LEUCINE (highest: above 4mg/dL)

• Prenatal diagnosis: measuring decarboxylase enzyme concentration in cells cultured from amniotic fluid

CYSTINURIA DEFECTIVE PROCESS: AMINO ACID TRANSPORT

SYSTEM RATHER THAN A METABOLIC ENZYME DEFICIENCY

• 20 – 30 fold increase in the urinary excretion of cystine as a result of genetic defect in the renal resorptive mechanism

• Other amino acids excreted: ornithine, lysine and arginine• Cystine being relatively insoluble and once

accumulated will tend to precipitate in the kidney tubules forming calculi

• Remedy: high fluid intake and alkalinizing the urine: penicillin

• Diagnosis: Cyanide-nitroprusside (red-purple color)

ARGININOSUCCINIC ACIDURIA AND CITRULLINEMIA DEFECTIVE PROCESS: UREA CYCLE DEFECTIVE ENZYME: ARGININOSUCCINIC

ACID LYASE• Decrease in activity of ASA synthetase

Citrullinemia • Citrulline is elevated in MS/MS

• Argininosuccinic aciduria – milder elevations

• Citrullinemia – dramatic elevations• * Ornithine and arginine are seen

increased in older infants

ISOVALERIC ACIDEMIADEFECTIVE PROCESS: DEGRATIVE PATHWAY OF LEUCINEDEFICIENCY ENZYME: ISOVALERYL-CoA DEHYDROGENASE• “Sweaty feet” odor urine

• Elevations of glycine conjugate: isovaleric acid and isovalerylglycine

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PROTE

INS

GENERAL CHARACTERISTICS

Structure of Proteins• PRIMARY • SECONDARY• TERTIARY• QUARTERNARY

FUNCTIONS OF PROTEINS 1. Enzymatic catalysis 2. Transport and storage 3. Coordinated motion

responsible for movements in the body (muscles)

4. Mechanical support support of body - bones and muscles

5. Generation and transmission of impulses

6. Control of growth and differentiation AA - simplest form of proteins 20 AA

CHARACTERISTICS OF PROTEINS 1. Have more members 2. Fundamental component of protoplasm 3. Elements in protein C, H, O, N (I & Fe) 4. Proteins are considered

macromolecules, composed of a number of AA joined together by peptide bond or linkage

5. Only foodstuff when absent in the diet will cause death

6. Utilize in the body for growth

PROTEIN METABOLISMGASTRIC• Digestion of protein begins in the

stomach where the enzyme pepsin is secreted by the chief cells as proenzyme or zymogen (inactive form)

• Pesinogen – activated by HCL secreted by the parietal cells

• By autoactivation – pepsin itself stimulates its own activation

PROTEIN METABOLISM PANCREATIC• Once protein reaches the duedenum,

exocrine pancreatic secretion: trypsin,chymotrypsin, elastase and carboxypeptidase in their inactive forms are release

• Endopeptidase – cleave protein in the internal sites

• Exopeptidase – cleave one amino acid from the carboxyl terminus of the polypeptide

Secretin stimulates the pancreas to produce a protein-free electrolyte solution rich in bicarbonates

Pancreozymin and cholecystokinin – mediates the secretion of enzyme rich pancreatic juice

PROTEIN METABOLISM

INTESTINAL• This mediated by peptidase produced by the

mucosal cells• Amino peptidases and dipeptidases

hydrolyze the residual peptides• The end products of protein digestion are

amino acids and some short peptides

AMINO ACID POOL

Primarily for the synthesis of body proteins:

• Plasma• Intracellular

proteins• Structural proteins

Also for the synthesis of nonprotein nitrogen-containing compounds

• Purines• Pyrimidines• Creatine• Porphyrins • Histamines

PROTEIN MEASUREMENT

PLASMA TOTAL PROTEIN:

• 60 – 70 g/L (SI units)

• 6.0 – 7.0 g/dL (Conventional unit)

ALBUMIN:• 32 – 45 g/L • 3.2 – 4.5 g/dL

GLOBULIN:• 23 – 35 g/L• 2.3 – 3.5 g/dL

FIBRINOGEN:• 2 – 4 g/L• 0.2 – 0.4 g/dL

DETERMINATION OF PROTEIN NITROGEN KJELDAHL• Reference (standard) method for the

determination of protein concentration• Liberation the nitrogen content from proteins

is measured by oxidation with heat at 350oC and strong sulfuric acid

• Catalysts used during the digestion: copper sulfate, mercuric sulfate or selenium oxychloride

• Nitrogen then is converted to ammonium ion

• Separated from the digestant by steam distillation

• Ammonium ion is liberated and are measured by titration with an alkali, Nesslerization or Berthelot reaction

NESSLERIZATION• Liberation of ammonium ions with Nessler’s reagent

– double iodide of potassium and mercury dimercuric ammonium iodide (yellow to orange brown product

• Colloidal stabilizer: gum ghatti

BERTHELOT • Idophenol reaction• Ammonia is allowed to react with alkaline

hypochlorite to form indophenol blue solution• Catalyst: sodium nitroprusside

BIURET REACTION Based on the ability of the peptide bonds to react

with copper ions to form purple “biuret” complex Biuret reagent contains: • Copper sulfate – biuret formation• Sodium hydroxide • Rochelle salt (potassium sodium tartrate) –

stabilizes the copper sulfate• Potassium iodide – keeps copper ions in cupric

state• The purple complex is measure at 540 – 560 nm Total proteins – measure by allowing to react with

the biuret reagent “Salting-out process” – precipitation of globulins

with 22-26% sodium sulfate to get the albumin content

FOLIN-CIOCALTEAU METHOD

based on the ability of phosphotungstomolybdic acid (phenol reagent or Folin-Ciocalteau’s reagent) to oxidize the phenolic structures of the amino acids tyrosine, phenylalanine, tryptophan and histidine.

LOWRY PROTEIN ASSAY• Uses biuret method followed by the

Folin-Ciocalteau’s method• Color obtained is enhance and

provide a more sensitive method• Consistently obtained accurate

protein determination

COOMASIE BRILLIANT BLUE DYE

• Free from interferences• It can detect proteins as low as 1 ug in

concentration

NINHYDRIN REACTION• Used to detect peptides and amino

acids after paper chromatography• Violet color formation upon reaction

to ninhydrin (tri-keto-hydrindine hydrate and amines

• Results are comparable to Coomasie dye method

REFRACTOMETRY

• Measuring refractive index of serum • Accurate levels are obtained at protein

concentrations greater than 2.5 g/dL

SPECIFIC GRAVITY

• Copper sulfate standards with known specific gravity

• Measurement of hemoglobin

TURBIDIMETRY

• Measures total proteins and globulins in urine and CSF

• Proteins are precipitated by sulfosalicylic acids, trichloroacetic acid, acetic acid-potassium ferricyanide solution

UV ABSORPTION METHOD

• Proteins absorb UV light at 280 nm• Mostly due to the presence of

tryptophan, phenylalanine and tyrosine• Quantifies protein in the range of 0.5 to

1.5 mg/dL

ELECTROPHORESISSodium dodecyl sulfate polyacrilamide gel electrophoresis

• Separates protein according to molecular weight and isoelectric focusing which separates proteins on the basis of isoelectric poins

• After separation, proteins are quantified by densitometry (preferred method and by elution and spectrophotometry

Qualitative characterization of proteins may be stained with appropriate dyes:1. Coomasie brilliant blue2. Ponceau S3. Amido black4. Silver staining - most sensitive and measures

proteins in nanogram concentration

PRECIPITATION

Precipitants:• Sodium sulfate• Ammonium sulfate• Methanol

PLASMA PROTEIN

PRE-ALBUMIN• Migrates faster than albumin toward

the anode• Has the ability to bind with thyroxine

(TBPA) and complex with vitamin A• Very rich with tryptophan • Marker of nutritional status• Crosses the plancenta

ALBUMIN• Single most abundant protein in normal

plasma • About 2/3 of the total plasma proteins

• Regulates intravascular oncotic pressure• Loss of albumin is seen in ascites, protein

losing nephropathy and protein losing enteropathy• Seen in peripheral edema

Functions of albumin:1. Regulation of oncotic pressure2. Amino acid reservoir3. Transport of small molecules

ALBUMIN• Bounded by thyroxine, bilirubin,

penicillin, cortisol, estrogen and free fatty acids

• Calcium and magnesium• Analbuminemia – absence of

albumin in the blood• Bisalbuminemia – two separated

albumin bands after electrophoresis

ALPHA1-ANTITRYPSIN• Major component of the alpha1-globulin

fraction• Acts as protease inhibitor

• Combines with and inactivates trypsin and elastase

• In lungs, elastase is released by the neutrophils during inflammatory conditions to combat the invading microorganisms• Deficiency will result to emphysema

• Also an intrinsic factor in homeostatic mechanism modulating endogenous proteolysis with the body

ALPHA2-MACROGLOBULIN• one of the largest non-

immunoglobulin proteins in the plasma

• Rises tenfold or more in nephrotic syndrome when other low molecular weight proteins are lost

• In nephrotic syndrome, the levels of alpha2-macroglobulin may be equal to or greater than that of albumin (2-3 g/dL)

HAPTOGLOBIN• Haptoglobin migrates in the alpha2-region• Combines with hemoglobin released by lysis

of red blood cells in order to preserve body iron and protein stores

• Hemoglobin-haptoglobin complex is then taken by the RES where the hemoglobin fraction is broken into iron and bilirubin

Hemoglobinuria vs myoglobinuria:• Peroxidase acitivity using dipstick or

chemstrips – urine specimen• Increased or normal free haptoglobinuria will

indicate myoglobinuria or rhabdomyolysis

BETA-LIPOPROTEIN

Low-density lipoprotein which has a characteristic sharp leading edge and a feathery trailing edge

TRANSFERRIN• Also known as siderophilin• Major beta-globulin protein• Normally at 200 to 400 mg/dL• Transport ferric ions from iron stores

of the intracellular or mucosal ferritin to bone marrow where the red blood cells precursors and other cells have transferrin receptor on their surfaces

• Measured in terms of iron-binding capacity – seen elevated in anemia

COMPLEMENT

• Travels with beta-globulins during the electrophoresis

• C3 is decreased in autoimmune diseases

FIBRINOGEN• Most abundant of the coagulation

factors• Elevation seen in pregnancies and

the use of birth control pills• Decreased during the activation of

coagulation factors• Migrates between beta and gamma

fractions• Measured by Parfentjev method

which uses ammonium sulfate and sodium chloride

MINOR PROTEINS• Ceruloplasmin – Wilson’s disease

(hepatolenticular degeneration)• Gc-globulin – binds with vit D• Hemopexin – prevent excretion of

heme and iron• Alpha1-acid glycoprotein – increased

during pregnancy• C-reactive protein – seen in tissue

necrosis; most sensitive acute phase reactant located at chromosome 1