PROTEINS

PROTEINS pH > (CHONs are negatively charge)

 Macromolecules composed of polymers
of covalently linked amino acids
 50% C, 7% H, 23% O, 16% N, 0.3% S/P
Functions:
 Tissue Nutrition
 Maintenance of water distribution
 Buffers
 Transportation of metabolic substances
 Part of defense system (antibodies)
 Hormones and receptors
 Connective tissue structure
 Biocatalysts (enzymes)
 Hemostasis and Coagulation
pH > (CHONs are positively charge)
CHONs differ on pl:5:5-8
 Basis separation of CHONs
 Used in electrophoresis
4. Solubility
In blood required of pH of 7:35 – 7:45
Net charge at surface makes proteins
hydrophilic
No net charge: protein – protein interaction
Basis of salting out
5. Nitrogen content (16%)
Nitrogen: most important component of
protein o sets proteins apart from
carbohydrates and lipids

Amino Acids
 Albumin – major transport protein
 Building blocks of proteins
Classification of Proteins:  Small biomolecules containing at least one
amino group (-NH2) and one carboxyl group
1. Simple: amino acids (-COOH) bonded at the q-carbon
- Fibrous (collagen, troponin, keratin, elastin)
- Globular (albumin, hormones, enzymes) Essential Amino Acids
 Histidine (His or H)
2. Conjugated: protein + prosthetic group  Isoleucine (Iie or I)
(lipids, CHO, porphyrins, metals)  Leucine (Leu or L)
Metalloproteins  Methionine (Met or M)
Glycoproteins  Phenylalanine (Phe or F)
Mucoproteinds / proteoglycans  Theronine (Thr or T)
Necleoproteinds  Trptophan (Trp or W)
Lipoproteins  Valine (Val or V)
 Lysine (Lys or K
Characteristics of Protein:
1. Molecular size Non-Essential
Macromolecules  Alanine (Ala or A)
Size: 6,000 daltons to several million daltons  Aspartic Acid (Asp or D)
2. Structure  Cysteine (Cys or C)
Peptide bond (dipeptide, tripeptide,  Glutamic Acid (Glu or E)
tetrapeptide, polypeptide)
1* (sequence), 2* (a/B protein twisting), 3* Functions of Amino Acids
(protein folding), 4* 1. Synthesis
3. Charge and isoelectric point (Amphoteric) 2. Sysnthesis of non-protein containing
Based on the pH compounds (purines, pyrimidines,
Isoelectric point: pH at which a protein has no porphyrins, Creatine, Histamine,
net charge Thyroxine, Epinephrine, NAD)
3. Body Energy (12-20%)
 4. Metabolism Glucogenic Amino Acids - Retest in 7-10 days to catch earlier false
that generate precursors of glucose E.g. negatives
alanine to pyruvate, arginine to a- - No meat, dairy products, dry beans, nuts
ketoglutarate, aspartate to and eggs or cereals, fruits and vegetables in
oxaloacetate. moderation

Ketogenic Amino Acids that give rise to ketone
bodies. E.g Leucine and lysine to acetyl-CoA or
acetoacetyl-CoA
AMINO ACIDOPATHIES
 Inherited disorders of amino acids
metabolism
 Abnormalities in the activity of a
specific enzyme in the pathway
 Membrane Transport system of amino
acid
*RA 9288: Newbord Screening Act of 2004
Tests for PKU
Guthrie’s Test: bacterial inhibition assay o PHE
allows bacterial growth in a culture medium
with an inhibitor
Agar with B subtilis and B-2-thienylalanine B.
subtilis is inhibited by B-2-thienylalanine which
is inhibited by PHE
(+) result: bacterial growth
(-) result: no growth
O sensitivity: 180-240 umol/L (3-4 mg/dL) False
negative if infant is less than 24 hours old

Phenylketonuria (PKU) Microfuorometric Assay

 Absence of phenylalanine hydroxylase o Based on fluorescence of a complex formed
(PAH) of PHE-ninhydrin-copper in the presence of
 PHE up to 1200 umol/L a dipeptide
 Catabolites: phenylpyruvic acid, o Blood in filter is pre0treated with TCA
phenyllactic acid, phenylacetic acid, o Extract + ninhydrin, succinate and
phenylacetyglutamine leucylalanine = copper tartrate
 Urine: Musty Odor  Excitation wavelength: 360nm
 Types:  Emission wavelength: 530 nm
o Mild PKU (600-1200 umol/L)
o Non-PKU mild Alkaptonuria
Hyperphenylalaninenemia (180-600umol/L; no  Accumulation of Homogentisic acid
phenyketones) (HMA)
 1-5% of all cases: due to lack of  Blackening of urine after 24 hours
enzymes needed for the regeneration  Ochronosis and arthritis like
of tetrahydrobioperin (BH4) degeneration
 Effect: high PHE and deficient
neutransmitters from TYR and TRP
 Can cause:
o Mental retardations
o Convulsions
o Behavior Problems
o Skin rash
o Musty body odor
Tyrosinemia
 Type I: fumarylacetoacetate (FAA)
hydrolase
 Type II: tyrosine aminotransferase
 Type III: $-hydroxyphenylpyruvate
dioxygenase
 Diagnosed using Ms/MS
 Confirmed with elevated level of
succinylacetone
o Maple Syrup Urine Disease
 Reduced or absence of branched chain
a-ketoacid decarboxylase blocking the
metabolism of LEU, ILE and VAL
 Maple Syrup or burnt sugar odor of
urine, breath and skin
Test for MSUD
Modified Guthrie’s Test
o Bacteria: B sublitis
o Inhibitor: 4- azaleucine Microfluorometric
Assay
o Blood in filter paper treated with methanol
and acetone
o To denature Hb o Leucine dehydrogenase is
added
o Fluorescence of NADH at 450 nm and 360
nm
o MSUD > 4mg/dL leucine
Isovaleric Acidemia
 Autosomal recessive disorder
 Deficiency of isovaleryl-CoA
dehydrogenase
 Accumulation of isovaleryglycine
 Sweaty feet odor urine
Homocystinuria
 Lack of systathionine-B-synthase
 Elevated homocysteine and methionine
Test for Homocystinuria
Modified Guthrie’s Test
 Bacteria: B. subtilis
 Inhibitor: L-methionine sulfoximine
 Confirmatory Method: (-) HPLC
Citrullinemia
 Type I: (1:57,000 births)
o Lack of argininosuccinic acid
systhethase
o High citrulline and ammonia in
blood
 Type II (1:100,000 births)
o Mutation of gene producing
citrin
o Inhibits transport of molecules
needed in urea cycle and
production of proteins and
nucleotides
Argininosuccinic Aciduria
 Lack of argininosuccinic acid (ASA) lyase
 High argininosuccinic and citrulline
 Abnormal urea cycle: high ammonia
 Citrulline: diagnostics marker for these
disorders
 Ornithine and arginine also increases in
argininosuccinic aciduria
Cystinuria
 Autosomal recessive disorder
 Defect in the amino acid transport
system
 20-30 fold increase in the urinary
excretion of cysteine
 Cystine forms, urinary calculi (insoluble,
unless alkalinize)
PLASMA PROTEINS
Prealbumin (Transthyretin)
 Rich in TRP with 0.5% CHOTransports
T3 and T4, and binds with
retinolbinding protein to
transportretinolo
 Elevated: pxs taking steroids,
alcoholism, chronic renal failure
  Decreased: hepatic damage, tissue  Increased: spina bifida, neural tube
necrosis,a cute phase inflammatory defects, atresia of GI, fetal distress,
response ataxia telangiectasia, tyrosinosis, HDN
 Marker of poor nutritional status  Decreased: Down syndrome, Trisomy
18
Albumin
 Major plasma protein a1-acid glycoprotein (Orosomucoid)
 Synthesized in the liver hours  Negatively charged even in acidic
 Significance environment
o Liver function  Composed of 5 CHO attached to a
o Nutrition polypeptide
o Inadequate nitrogen intake  Elevated: stress, inflammation, cancer,
o Malabsorption pneumonia, RA cell proliferation, tissue
o Protein losing enteropathy or GI damage, AMI, and surgery
loss
o Kidney loss or skin loss a1-antichymotrypsin
o Hypothyroidism  Member of serine proteinase inhibitor
o Mutation (serpin) family
 Functions  Targets cathepsin G, pancreatic
o Regulates oncotic pressure elastase, mast cell chynase,
o Buffers pH chymotrypsin
o Transports thyroid hormones, iron  Elevated: inflammation
and Fas  Decreased (hereditary): asthma, liver
o Effective binder disease
 Mutated in patients with Parkinson’s
Globulin disease, COPD, and Alzheimer’s disease
 Only about 1/3 of the total protein
 a1, a2, B, and y fractions Inter-a-trypsin Inhibitor (IT)
 TP- albumin=globulin  Serine protease inhibitor family
 Between a1 and a2
a1-antitrypsin (AAT)  Composed of H1, H2, and L (bikukin)
 Inhibits the protease trypsin  Inhibits trypsin, plasmin, chymotrypsin
(neutrophilelastase)  Elevated in inflammation
 Low levels cause emphysema or
infantile hepatitis Gc-globulin
 Acute phase reactant  Group-specific Component; Vitamin D-
 Inflammatory reactions, pregnancy, and Binding Protein
contraceptive use  Between a1 and a2
 Major carrier protein of Vitamin D
a1-fetiprotein (AFP)  Also transports Fas and endotoxin
 Principal fetal protein  Elevated: Pregnancy (3rdtri), pxs taking
 No adult function oral estrogen
 Increase levels in adult indicate  Decreased: liver disease, CHON losing
hepatocellular tumor and gonadal syndrome
tumors
Haptoglobin  Elevated: pregnancy, DM, Duchenne
 Binds and transports free hemoglobin type (Muscular Dystrophy),
 Increased: inflammation, burns, malignancies like melanoma
nephrotic syndrome  Decreased: hemolytic disorders,
 Decreased: IV hemolysis, HDN, administration of diphenhydramine
mechanical breakdown of RC (Athletic  Free heme causes oxidative damage
trauma)
LIPOPROTEINS
Ceruloplasmin
Chylomicrons, VLDL, LDL, HDL
 Copper transport protein (90%)
 Decreased in Wilson’s disease HDL: migrates between albumin and a-1-
o Cornea: Kayser Fleischerrings globulin zone
o Deposited in skin, liver, brain VLDL: migrates at the beginning of B-globulin
 Also in malnutrition, malabsorption, (pre-B)
liver damage, nephrotic, syndrome, LDL: appear as a separate b and in the B-
menke’s syndrome (kinky hair disease) globulin region
 Increased in copper toxicity
B2-Microglobulin (B2M)
a2-macroglobulin (A2M or AMG)  Light chain component of the major
 One of the largest proteins in plasma histocompatibility complex (HLA)
 Major component of alpha 2 band in  Found in the surface of the most
protein electrophoresis inhibits nucleated cells and high in lymphocytes
proteases such as trypsin, thrombin,  Elevated: impaired kidney clearance,
kallikrein, and plasmin overproduction, inflammatory diseases
 Elevated in nephrotic syndrome, DM, (RA, SLE), HIV
and liver disease, use of contraceptive,
pregnancy Complement
 One of the natural defense mechanisms
Transferrin (Siderophilin) against infections
 Major component of B- globulin o Elevated: inflammatory
transports iron (2:1) to storage sites, diseases
and to cells o Decreased: malnutrition and
 Increased in iron deficiency anemia hemolytic anemia
 Decreased in inflammation, liver C3: most abundant
disease, malnutrition, kidney loss, complement protein in human
infection, malignancy serum
 Decreased: autoimmune disease
Atransferrinemia: neonatal distress syndrome, bacteremia
 Autosomal recessive disorder , tissue injury, and chronic hepatitis
 Absence of transferrin  C4: second most abundant
 Microcytic anemia and hemosiderosis in o Decreased:
the heart and liver disseminated
 Hemopexin: Removes circulating heme, intravascular
ferriheme, porphyrin coagulation (DIC), acute
glomerular nephritis,
chronic hepatilis, SLE
Fibrinogen IgAa
 Responsible for fibrin clot 2 280 170,000 6
 Only protein found in plasma and not in 1. Secretory antibody
serum 2. Agglutination

Methods: IgDo
1. Parfentjev 2 3 158,000 3
Fibrinogen is precipitated with (NH4)2SO4 and 1. Antigen receptor
NaCI. Read spectrophotometrically
2. Howe’s Method IgEE
Fibrinogen is precipitated with CaCI2 and 1 0.03 196,00 2
assayed using Kjeldahl method 1. Anti-parasitic antibody
2. Allergy antibody
NV: 200-400 ml/dL or 2.0-4.0 g/L
Other important proteins:
Immunoglobulins Proteins-General description/function/clinical
 82-96% proteins and 4-18% CHO Significance
 Composed of two heavy chains and two
light chains Myoglobin
 Oxygen carrying protein found in
PHYSICAL AND BIOLOGICAL PROPERTIES OF striated skeletal and cardiac muscles
HUMAN IMMUNOGLOBULIN CLASSES  AMI: 2-3hours of onset and peaks at 8-
12 hours. Muscle trauma, renal failure.
CLASS HEAVY CHAIN DESIGNATION
 NUMBER OF SUBCLASSESMEAN Troponin (cTn)
 SERUM CONCENTRATION (mg/dl)  For calcium binding to trigger
 MOLECULAR WEIGHT (daltons) production of muscular force
 MEAN SURVIVAL (days)  ACS, myocardial damage
BIOLOGIC ACTIVITY
Brain Natriuretic Peptide (BNP)
IgGy Found in highest concentration in the left
4 1250 150,000 23 ventricular myocardium; also found in atrial
1. Opsonin tissue & right ventricle
2. Complement Congestive Heart Failure (CHF)
3. Crosses placenta
Fibronectin
IgMu Cell adhesion, tissue differentiation, growth,
1 120 890,000 5 and wound healing
1. Fix complement Nutritional MarkerFN: predict short term risk of
2. Agglutination premature delivery
3. Antigen receptor
Adiponectin
Inversely correlated with BMI
Low: heart disease, DM2, MetS, obesity
Methods for Protein Measurement
 Kjeldahl
 Bluret
 Refractometry
 UV absorption method
 Fractionation
o Salt precipitation, dye binding
Glyoxylic acid, Electrophoresis
Kjeldahl Method
 Involves acid digestion of protein wit
measurement of total Na.
 Kjeldahlization: conversion of nitrogen
to NH3
 NH3 measurement
o Nessler’s
o Berthelot’s
 Serum proteins are precipitated with
TCA or tungstic acid, supernatant is
removed
 Heating 340-360 C, sample digestion
with H2SO4 with heat and catalyst
NH4HSO4 neutralize with NaOH
distillation with boric acid
NH4H2BO3Tiration with HCL
Biuret Method
 Formation of violet colored chelate
between cupric ions and peptide bond
Biuret reagent:
 Cupric ions: breaks the peptide bonds
 Tartrate salt: keeps copper in solution
Potassium iodide: stablilizes cupric ion
(anti-oxidant)
Limitation: 2 or more peptide bonds
Refractometry
 Measurement of refractive index which
reflects the concentration of proteins
Dye Binding
 Bromphenol blue, Ponceau S, amido
black 10B, listamine green, Coomasie
brilliant blue 250
 Shift in absorbance maximum of the
dye from 465 to 595 nm
Refraction by Dye Binding
 Albumin dye binding technique pH
adjusted to make albumin positively
charged
 Dyes: methyl orange, hydroxyl-
azobenzene-benzoic acid (HABA),
bromcresol green (BCG), bomcresol
purple (BCP)
UV Absorption Method
 CHONs absorb UV at 200-280 nm (due
to TRP, TYR, PHE)
 Use beer-Lambert’s Law
A=ebcoC=Aux CsoAs
 Molar Absorptivity is necessary to
obtain the concentration (differ in
molecular structure)
Fractionation by Salt Precipitation
 Globulins are separated from albumins
by salting out
 Sodium salt will precipitate globulins
o Sodium sulfate
o Sodium sulfite
o Ammonium Sulfate
 Albumin in the solution is measured
using any of routine protein
measurement methods
Fractionation for Globulin
 Separates proteins on the basis of
electrical charge
 Uses barbital (veronal) with pH of 8.6
Stains: Ponceau S, bromphenol blue,
Coomassie Brilliant blue, Listamine
green, Amido black
 Quantitated using densitometer
Bands of Proteins
1. Albumin: fastest; most electronegative
2. a1-globulin (a1-antitrypsin, HDL)
3. a2-globulin (ceruplasmin, haptoglobulin, a2-
macroglobulin, VLDL)
4. B-globulin (transferri, C3,C4, plasminogen,
LDL)
5. y-globulin (immunoglobulins)
Pattern of Protein
Western Blot
 Detect a single protein within a mixture
of proteins from a biological sample
 Estimate the size of protein
 Confirm the presence of post-
translational modifications
 Compare quantitatively protein levels
between samples
PROTEINS IN URINE AND CSF
 Proteins in urine
 Mainly from the blood
 Causes
o Glomerular
 Diabetes, amyloidosis,
dysglobulinemia, collagen
disorders Mercury, heroine
(toxins)
o Tubular: fail to reabsorb
 Overflow proteinuaria:
overabundance of CHONs,
tubules are overwhelmed
Bence Jones Proteins
 An abnormal protein in the urine of
patients with multiple myeloma
 Demonstrated by Heat and acetic acid
method
o Precipitates when solution is
heated at 40-60C
o Soluble when heated at 100C
o Re-precipitates when the
solution cools at 40-60C
Tamm Horsfall Protein
 Found only in urine and not in serum
 Mucoprotein produced in renal tubules
 Component of urinary casts
 40mg/dl
Urine Protein Detection
 Strip: CHON error indicator (Y-G-B)
o Protein alters color of some acid-
base indicators without altering the
pH
o >6m/dl protein: color change
 Turbidimetric: SSA, TCA, benzethonium
chloride
 Doesn’t react equally with each CHON
fraction
Biuret
 Dye binding: coomasie brilliant blue and
pnceau S
 Folin Lowry: uses folin ciocalteau
reagent (phosphotungstomolybdic
acid)/phenol reagent
o 10x more sensitive
o Yellow to blue: TYR, TRP, and
HIS
o Modified: and biuret as initial
step (100x more sensitive)
Proteins in CSF
 From choroid plexuses
 Accepted CSF protein level:15-45 mg/dl.
Increased capillary endothelial barrier
permeability:
 Meningitis, traumatic tap,
multiple sclerosis, obstruction,
neoplasm, disk herniation,
cerebral inarction
CSF albumin: serum albumin
 Reference value: <2.7-7.3
 Increased: damaged BBB
 Decreased: fluid is leaking from CNS
CSF-IgG Index
CSF-IgG index=
CSF-IgG (mg/dl) x serum albumin (g/L)/
Serum IgG(g/dl) x CSF albumin (mg/dl)
 Normal: 0.26-0.70
 Elevated: local CNS production
o Multiple schlerosis, bacterial
infections, CNS inflammatory
disease
 Decreased: leakage from plasma
o BBB is destroyed (meningitis,
tumors)

Methods
 Turbidimetric
 Dye Binding
 Biuret
 Folin Lowry
 Electrophoresis
o Pre-albumin, albumin, a1, a2,
B1, B2 (Tprotein), and ybands