Amino Acids and Proteins Report

8/8/2019 Amino Acids and Proteins Report

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AMINO ACIDS ANDPROTEINS

GROUP 1 - MT0831

Azarcon, Dyna Mae Guia, Alexa

Dedace, Jose Celso Lim, Jeannine De Rueda, Gerald Barry Ong, Tanya

Fran, John David



Overview of Amino Acids



Amino Acids

*Building blocks of proteins.

*Responsible for the growth, repair andmaintenance of the cells.

*It determines the biologic activity of the

protein.



Basic Structure



Basic Structure

*Contains atleast one of both amino and carboxylic acid functionalgroups.

General Structure of an amino acid

* Polypeptide Chain of amino acids* Large polypeptide constitutes a protein.

* In human serum, proteins average about 100-150 amino acids inthe polypeptide.



Met abolism



Metabolism

*About half of the 20 amino acids

needed by humans cannot be

synthesized at a rapid enough rate to

support growth; they must be supplied

in food.



Metabolism

Essential amino Acids

Arginine

Histidine

Isoleucine

Leucine

Lysine

Methionine

Phenylalanine

Threonine Tryptophan

Valine



Metabolism

*Tyrosine is produced from phenylalanine.

*Glucogenic amino acids generate precursors of glucose

Alanine pyruvate

Arginine ketoglutarate

Aspartate oxaloacetate

*Ketogenic amino acids Generate ketone bodies



Metabolism

Amino Acids that the body can produce.

Alanine

Asparagine

Aspartic acid

Cysteine

Glutamic acid

Glutamine

Glycine

Proline Serine

Tyrosine



Metabolism



Essential Amino Acids



Essential Amino Acid

Also called Indispensable amino acid

Is an amino acid that cannot be

synthesized de novo (from the

beginning) by the organism (usually

referring to humans), and therefore

must be supplied in the diet.



Arginine (Arg)

A complex amino acid that isoften found at the catalytic site inproteins and enzymes due to itsamine-containing side chain.

Immediate precursor of NO, urea,ornithine and agmantine.

Stimulate the secretion of growthhormone.

Used in growth hormonestimulation tests.



Arginine (Arg)

Plays an important role in:

Cell division

Healing of wounds

Stimulation of protein synthesis

Immune function

Release of hormones



Arginine (Arg)

Required for:

The generation of urea.

The synthesis of creatine.

Found in a wide variety of foods,including:

Animal sources

Vegetable sources



Histidine (His)

One of the basicamino acids due to itsimidazole side chain.

Essential amino acidfor infants.

Important source of carbon atoms in thesynthesis of purines.



Histidine (His)

Functions:

Grow and repair body tissues.

Manufacture red and white blood cells Protect the body from heavy metal

toxicity.

Maintain the myelin sheaths thatprotect the nerve cells.



Histidine (His)

Direct precursor for histamine

A deficiency in this enzyme is present

in the rare metabolic disorder

histidinemia.



Isoleucine (Ile)

Classified as a hydrophobic amino

acid.

Can be synthesized in a multistepprocedure starting from 2-

bromobutane and diethylmalonate.



Isoleucine (Ile)

Functions:

Helps maintain, heal, and repairmuscle tissue, skin, and bones.

Helps to regulate blood glucose levels

Maintain energy levels.



Leucine (Leu)

Second most common aminoacid found in protein besideglycine.

Major component of the subunits in ferritin, astacin andother 'buffer' proteins.

In conjunction with valine

and isoleucine, boosts thehealing of muscle, skin, andbones.



Leucine (Leu)

It is utilized in the liver, adipose tissue,

and muscle tissue.

Functions:

Aids in recovery from surgery.

Lowers blood glucose level. Necessary for optimal growth of infants

and nitrogen balance for adults.



Lysine (Lys)

Has a net-positive charge.

Needed for proper growth andbone development in children

Lysine is metabolised inmammals to give acetyl-CoA,via an initial transaminationwith -ketoglutarate. The

bacterial degradation of lysineyields cadaverine bydecarboxylation.



Lysine (Lys)

Plays an important role:

Formation of collagen.

Functions: Production of antibodies.

Lowers triglycerides level.

Maintain proper nitrogen balance in

adults. Helps in absorption and conservation of

calcium.



Methionine (Met)

Source of sulfur.

Reacts with adenosine triphosphate to

contribute to the synthesis of manysubstances.



Phenylalanine (Phe)

Classified as a non-polaramino acid.

Found naturally in the

breast milk of mammals. Direct precursor to the

neuromodulatorphenyethylamine.

Uses an active transportchannel

Part of the composition of aspartame.



Phenylalanine (Phe)

Functions:

Promotes alertness and vitality, elevates mood,decreases mood pains, aids in memory andlearning.

Used in the manufacture of food and drinkproducts and sold as a nutritional supplement forits reputed analgesic and antidepressant effects.

Used to treat arthritis and depression.

Used by brain to produce norepinephrine.

Key Role:

Biosynthesis of other amino acids.



Threonine (Thr)

Alcohol-containing amino acidthat is an important componentin the formation of protein,collagen, elastin, and tooth

enamel. Important in the production of

neurotransmitters and health of the nervous system.

Functions: Helps maintain proper protein

balance in the body.

Aids liver function, metabolism,and assimilation.



Threonine (Thr)

Threonine is metabolized in two ways:

It is converted to pyruvate via threoninedehydrogenase. An intermediate in this

pathway can undergo thiolysis with CoAto produce acetyl-CoA and glycine.

In humans, it is converted to alpha-ketobutyrate in a less common pathway

via the enzyme serine dehydratase, andthereby enters the pathway leading tosuccinyl-CoA.



Tryptophan (Trp)

Formed from proteins during digestion by the action of proteolytic enzymes.

Natural relaxant.

Used in the treatment of migraine headaches.

Functions:

Helps alleviate insomnia by inducing sleep, soothes anxiety,and reduces depression

Aids in weight control by reducing appetite.

Helps control hyperactivity in children.



Valine (Val)

Another branched-chain aminoacid that is a constituent of fibrous protein in the body.

Needed for muscle metabolismand coordination, tissue repair,and maintenance of nitrogenbalance.

Used by muscle tissue as an

energy source.

Treatments for muscle, mentaland emotional problems,insomnia, anxiety, and liver andgallbladder disease.



Nonessential Amino Acid



Nonessential Amino Acids

Are amino acids that can be produced

in our body.

Their uses and functions in our bodyare equally as important as the limiting

amino acids.



Alanine (ala)

One of the simplest of the amino acids.

Involved in the energy-

producing breakdownof glucose.

Product of:

± breakdown of DNA or

the dipeptides(anserine andcarnosine)

± conversion of pyruvate



Plays a major role in the transfer of

nitrogen from peripheral tissue to the

liver.

Helps in reducing the buildup of toxic

substances.

Strengthens the immune system.

Alanine (ala)



-amide of aspartic acid synthesized

from aspartic acid and adenosine

triphosphate.

Converts one amino acid into another

via amination.

Required by nervous system and plays an

important role in the synthesis of

ammonia.

Aspargine (Asn)



Aspartic acid (Asp)

Alanine with one of the hydrogens replaced bya carboxylic acid group.

Plays a vital role in

metabolism duringconstruction of otheramino acids andmetabolites in citric acidcycle.

A metabolite in the ureacycle and participates ingluconeogenesis.



Cysteine (Cys)

May be essential forinfants, the elderly andindividuals with certainmetabolic diseases or

malabsorptionsyndromes.

An important structuraland functional

component of manyproteins and enzymes.

Named after cystine, itsoxidized dimer.



Potentially toxic and is catabolized in the

gastrointestinal tract and blood.

Absorbed during digestion as cystine.

Used as a constituent in the food,

pharmaceutical and personal care

industries.

Involved in production of flavors.

Cysteine (Cys)



Glutamic acid (Glu)

Synthesized from a number of aminoacids.

One of the two amino acids that have

a net negative charge (by pH).

Has been linked to epileptic seizures.

A neurotransmitter that is important in

the metabolism of sugars and fats.



Aids in transporting potassium into spinal

fluid.

Present in wide variety of foods and is

responsible for one of the five basic

tastes of the human sense of tastes.

Often used as a food additive and flavor

enhancer in the form of its sodium salt,

monosodium glutamate (MSG).

Glutamic acid (Glu)



Glutamine (Gln)

The most abundant amino acid in the body.

Involved in more metabolic processes than anyother amino acid.

Over 61% of skeletal muscle tissue is glutamine.

Converted to glucose. Assists in maintaining the proper acid/alkaline

balance in the body.

Provides fuel for a healthy digestive tract.



Glutamine (Gln)

The basis of the building blocks for

synthesis of RNA and DNA.

Useful in treatment of serious illnesses.

Marketed as a supplement used for

muscle growth in weightlifting and

bodybuilding.

Transports ammonia.



Glycine (Gly)

Glycine was discovered in1820, by Henri Braconnotwho boiled gelatin withsulfuric acid.

An organic compound withthe formula NH2CH2COOHwith only two hydrogenatoms as its 'side chain'.

The simplest amino acidsynthesized in the body.

The only amino acid that isnot optically active becauseit has no isomers.



It has no stereoisomers and is the only amino

acid that is not optically active.

Glycine is a colourless, sweet-tasting crystalline

solid and can fit into hydrophilic or hydrophobicenvironments, due to its two hydrogen atom

side chain and serves as a buffering agent in

antacids, analgesics, antiperspirants, cosmetics,

and toiletries.

Liver is the one that helps in synthesis of bile

acids and detoxification of compounds.

Glycine (Gly)



The smallest of the 20 amino acids commonly

found in proteins.

Its codons are GGU, GGC, GGA, and GGG. An inhibitory neurotransmitter in the central

nervous system.

A metal complexing agent, retards muscle

degeneration.

Improves glycogen storage and promotes

healing.

Glycine (Gly)



Glycine (Gly)

Essential for the

synthesis of:

± nucleic acids

± bile acids

± proteins

± peptides

± purines

± ATP

± Porphyrins

± Hemoglobin

± Glutathione

± Creatine

± bile salts

± glucose

± glycogen



Proline (Pro)

An -amino acid and one

of the twenty DNA-

encoded amino acids.

It is not an essentialamino acid and its codons

are CCU, CCC, CCA, and

CCG.

It is unique among the 20protein-forming amino

acids in that the -amino

group is secondary.



Proline (Pro)

Precursor of hydroxyproline.

Involved in wound healing.

Plays important roles in molecular recognition

Important component in certain medicalwound dressings.

Helps in the healing of cartilage and the

strengthening joints, tendons and heart

muscle.

Works with vitamin C to promote healthy

connective tissues.



Serine (Ser)

Plays an important role in the bodys

synthetic pathways for:

± Pyrimidines

± Purines

± Creatine

± Porphyrins



Serine (Ser)

An organic compound with the formula HO2CCH

(NH2) CH2OH.

One of the naturally occurring proteinogenic

amino acids.

Its codons are UCU, UCC, UCA, UCG, AGU and AGC.

It is not essential to the human diet. (Non-Essential

Protein)

First obtained from silk protein thus deriving from

the Latin word, sericum for silk.

Serine's structure was established in 1902.



Serine (Ser)

Second amino acid that is also an alcoholbecause of its methyl side chain.

Needed for the proper metabolism of fats

and fatty acids. Highly concentrated in all cell membranes.

A component of the protective myelinsheathes surrounding nerve fibers.

Aids in the production of immunoglobulinsand antibodies for the maintenance of ahealthy immune system.



Tyrosine (Tyr)

One of the 20 amino acids that are used by

cells to synthesize proteins.

Its codons are UAC and UAU.

It is a non-essential amino acid with a polar

side group.

The word "tyrosine" is from the Greek ty ri ,

meaning cheese, as it was first discoveredin 1846 by German chemist Justus von

Liebig in the protein casein from cheese.



Tyrosine (Tyr)

Metabolically synthesizedfrom the important aminoacid phenylalanine tobecome the para-hydroxy

derivative of phenylalanine.

Important in overallmetabolism.

Aids in the functions of

the adrenal, thyroid andpituitary glands.

Acts as a mood elevator.

Suppresses the appetite.



Tyrosine (Tyr)

Tyrosine is found in many high proteinfood products such as soy products,chicken, turkey, fish, peanuts, almonds,

avocados, milk, cheese, yogurt, cottagecheese, lima beans, pumpkin seeds, andsesame seeds.

It can also be obtained throughsupplementation.



Tyrosine (Tyr)

Precursor of:

± adrenal hormones:

epinephrine

norepinephrine

dopamine

± thyroid hormones:

Thyroxine

Helps reduce body fat

making it useful in the

treatment of:

± chronic fatigue ± narcolepsy

± anxiety

± depression

± low sex drive

± allergies

± headaches



TwoN

ew Amino Acids



Selenocysteine (Sec)

Discovered to be coded

genetically

Recognized as the 21st amino

acid.

Not coded for directly in the

genetic code.

Encoded by a UGA codon.

Has a specialized transfer RNA

(tRNA). Names as an amino acid in

2002 and found to be the

selenium analogue of

cysteine.



Selenocysteine (Sec)

Present in several enzymes such as:

± Formate dehydrogenases

± Glycine reductases

± some hydrogenase

HIV infection have been shown to have a

lower-than-average blood plasma selenium

level. Proteins that contain one or more

selenocysteine residues are called

selenoproteins.



Pyrrolysine (Pyl)

22nd naturally occurring geneticallyencoded amino acid used by some archaeain enzymes that are part of their methane-producing metabolism.

Produced by a specific tRNA andaminoacyl tRNA synthetase.



Pyrrolysine (Pyl)

It is similar to lysine, but with an added pyrrolinering linked to the end of the lysine side chain.

Lysine derivative that is encoded by the UAGcodon.

It forms part of an unusual genetic code in theseorganisms

It is encoded in mRNA by the UAG codon, whichin most organisms is the 'amber' stop codon. Thisrequires only the presence of the pylT gene,

which encodes an unusual transfer RNA (tRNA)with a CUA anticodon, and the pylS gene, whichencodes a class II aminoacyl-tRNA synthetasethat charges the pylT -derived tRNA withpyrrolysine.



Amino Acid Analysis



Amino Acid Analysis

Methodology used to determine the

amino acid composition or content of

proteins, peptides, and otherpharmaceutical preparations.



Amino Acid Analysis

o For blood sample:

It must be drawn after at least 6-8 hour fast.

Blood plasma must be collected in a heparin tube.

Platelet and white cell layer must not be aspiratedto prevent contamination.

WBC levels of aspartic acid and glutamic acid areabout 100 times higher than those in plasma.

Hemolysis is unacceptable.

Deproteinization should be performed within 30minutes of sample collection.

Analysis should be performed immediately or frozeit at -20C to -40C.



Amino Acid Analysis

Urinary amino acid analysis can be

performed on a random specimen for

screening purposes. ± For quantitation, 24 hour urine sample

preserved with thymol or organic solvents

is required.

Amniotic fluid also may be analyzed.



Amino Acid Analysis

For screening test:

± Thin layer chromatography

Method of choice

Examples: One-dimensional separation (chromatography)

± Used for searching for a particular category of aminoacid

» Examples: Branched-chain amino acidsSingle amino acid

Two-dimensional separation (chromatography)

± Used for more general screening



Amino Acid Analysis

Amino acids migrate along one solvent frontand then chromatogram is rotated 90degrees and second solvent migration

occurs.

Kinds of solvent:

Butanol

Acetic acid

Water and ethanol

Water and ammonia



Amino Acid Analysis

Chromatogram

Is the visual output of the chromatograph.

Is viewed by staining with ninhydrin, which givesmost amino acids a blue color.

Techniques to separate and quantitate amino acids:

1. Ion-exchange chromatography

2. HPLC reversed phase system w/ fluorescence

detection3. Capillary electrophoresis

4. MS/MS Highly specific and sensitive method for measurement of

amino acid.



Protein in Other Body Fluids



Urinary Proteins

Kidney

Urinary tract

Vagina

Prostate

Sources of urinary

proteins



Urinary Proteins

Plasma proteins appear in the urinebecause they have passed through therenal glomerulus and have not been

reabsorbed by the renal tubules. Reagent Test Strip commonly used

qualitative test for proteinuria. It is basedthe change of an indicator dye in the

presence of protein, known as proteinerror indicators (ability of protein to alterthe color of some acid-base indicatorswithout altering the pH).



Urinary Proteins

In acid pH, the indicator that is yellow in

the absence of protein progresses

through various shades of green andfinally to blue as the concentration of

protein increases.

A protein concentration of 6 mg/dL orgreater produces a color change.

U i S i



Urinary Specimen

Collection

Most quantitative assays are

performed on urine specimens of 12

24 hours. The 24-hour timing allows for circadian

rhythmic changes in excretion at

certain times of day.

U i S i



Urinary Specimen

Collection

The patient should void, completely

emptying the bladder, and discard this

urine. At the end of the 24 hour period,

the bladder is completely emptied andthat urine included in the sample.

The results are reported generally in

terms of weight of protein per 24 hoursby calculating the amount of protein

present in the total volume of urine

collected during that time.



URINE PROTEIN METHODS

METHOD PRINCIPLE COMMENT

Turbidimetric methods

Sulfosalicylic acid

Trichloroacetic acid

Benzethonium chloride

Proteins are precipitated as fine

particles, turbidity is measuredspectrophotochemically

Rapid, easy to use;

unequal sensitivity forindividual proteins

Biuret Proteins are concentrated by

precipitation, redissolved in

alkali with Cu2+; Cu2+ formscolored comlplex with peptide

bonds

Accurate

Follin-Lowry Initial biuret reaction; oxidation

of tyrosine, tryptophan, and

histidine residues by Folin

phenol reagent; measurementof resultant blue color

Very sensitive

Dye binding

Coomassie blue

Ponceau S

Protein bind to dye, causes shift

in absorption maximum

Limited linearity;

unequal sensitivity for

individual proteins



Microalbumin Methods

Immunochemical assays

Immunoturbidimetry

Immunofluorescence ELISA (Enzyme Link Immunosorbent

Assay)

Zone electrophoresis



Methods for Quantifying

Urine Alb

umin:

Immunochemical

Chromatographic;

Liquid Chromatography-MassSpectrometry

Even a Fluorescence Resonance EnergyTransfer Assay for point-of-care testing of urinary albumin has been developed.

CSF Albumin Serum



CSF Albumin-Serum

Albumin Ratio

Albumin used as the reference protein forpermeability because it is not synthesized to anydegree in the CNS

Reference value:

< 2.7-7.3 = Normal Ratio

High Value Low Value

Damage in Blood-Brain

Barrier

Hyperthyroidism

Fluid leak on the CNS

Total CSF protein



Tot al CSF protein

determination

Sensitive chemical methods or

Spectrophotometric methods

Most commonly used:

Tur-bidimetric using TCA, sulfosalicylic

acid with sodium sulfate, or

benzethonium chloride

Total CSF protein



Tot al CSF protein

determination

Other methods:

Dye-Binding methods (e.g., Coomassie brilliant blue)

Definition: ± a kinetic biuret reaction, and the Lowry method using a

Folin phenol reagent.

Although total protein levels in the CSF are informative,diagnosis of specific disorders often requiresmeasurement of individual protein fractions.

The pattern of types of proteins present can be seen byelectrophoresis of CSF that has been concentrated.

Performed on: Cellulose acetate

Agarose gel

Total CSF protein



Tot al CSF protein

determination

The normal CSF pattern:

Prealbumin a prominent albumin band

1-globulin composed predominantly of 1-antitrypsin

2band consisting primarily of haptoglobin andceruloplasmin

1

band composed principally of transferrin

CSF specific transferrin that is deficient in carbohydrate,

referred to as protein, in the 2 zone

The globulin present in the band is typi-cally IgG with asmall amountof IgA.



B A ND

Electrophoretic patterns of CSF from patientswho have multiple sclerosis have multiple, distinct, oligo-clonal bands in the -zone.

The identifica-tion of discrete bands in the region that are present in theCSF but not in theserum isconsistent withproduc-tionof IgGintheCSF.

Note: This cannot be seen in routine celluloseacetate electrophoresis but require a high-resolution technique in which agarose isusually used.



B A ND

Oligoclonalbands can also be seen in

inflammatory conditions and infectious

neurologic diseases:

± Guillain-Barre syndrome

± bacterial meningitis

± viral encephalitis ± subacutescleros-ingpanencephalitis (SSPE)

± neurosyphilus.



IgG Index

To distinguish raised CSF IgG due to local CNSproduction from leakage ofplasma into the CSF, thelaboratory can compare CSF and serum IgG levelswith reference to albumin.

If:

CSF IgG:Albumin Ratio > serums (raised IgG Index)

Means a local CNS production of IgG

If:

Serum IgG:Albumin Ratio > CSFs (low IgG index)Suggest HYPERGAMMAGLOBULONEMIA or low serum albumin

Reference range for IgG index: 0.26-0.70



IgG Index

To identify the Source of the elevated CSF IgG levels, the IgG-albumin index can be calculated as follows:

CSF IgG Index:

= CSF IgG (mg/dL) x serum albumin (g/dL)

Serum IgG (g/dL) x CSF albumin (mg/dL)

Another index to aid in discriminating the source of the IgG inthe CSF:

IgG synthesis rate cal-culation using the formulaofTourtellotte.

The reference interval for the synthesis rate is -9.9 to +3.3mg/day.

In the investigation of



In the investigation of

multiple sclerosis:

Myelin basic proteins present in the CSF are also assayedbecause these proteins can provide an index of activedemyelination.

These are constituents of myelin, the sheath that surroundsmany of the CNS axons.

In very active demyelination:

concentration of myelin basic proteins of 17-100 ng/mL is

found on RIA.

In slowdemyelination:

values of 6-16 ng/mL occur and, in remission, the values areless than 4ng/mL.



Other conditions that induce

CNS demyelination: (elev ated

levels)

Meningoencephalitis

SLE of CNS

Diabetes Milletus

Chronic Renal Failure



Thank you

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Negative no turbidity (- 5 mg/dL or less)

Trace perceptible turbidity (- 20 mg/dL)

1+ Distinct turbidity, but no discrete granulation (- 50 mg/dL)

2+ Turbidity with granulation, but no f locculation (- 200 mg/dL)

3+ Turbidity with granulation and f locculation (- 500 mg/dL)

4+ Clumps of precipitated protein, or solid precipitate (- 1.0 g/dL or more).

Amino Acids and Proteins Report

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