Download Proteins Structure Globular Protein Structure Globular Protein

Proteins Structure
n
Primary structure
Secondary structure
Tertiary structure
Quaternary structure
Fibrous proteins:
n
n
Globular Protein Structure
fibrinogen, troponin, collagen, and myosin
Globular proteins:
n
n
most proteins of clinical interest
hemoglobin, enzymes, and plasma
proteins (soluble)
Globular Protein Structure
Properties of proteins can be used for
their separation, identification, and assay.
2º
n
Molecular size.
n
分子內
分子間
n
Differential solubility.
n
Electrical charge.
n
3º
dialysis, ultrafiltration, gel filtration
4º
n
n
electrophoresis, isoelectric focusing
Adsorption on finely divided insert materials.
n
n
affected by the pH, ionic strength, temperature
silica, alumina, or hydroxyapatite
Specific binding to antibodies, coenzymes, or
hormone receptors.
n
high specificity
1
Biological function of proteins
Plasma Protein:
hundreds of different proteins
n
n
n
n
n
n
n
n
General and local clinical symptoms
of the acute phase reaction
General symptoms
Local symptoms
fever
Calor
熱
increased heart rate rubor
紅
hyperventilation
dolor
痛
tiredness
tumour 腫
Acute phase proteins
Carriers
Fibrinogen
Other coagulation factors
Complement components
Immunoglobulins
Enzymes inhibitors
Many others
Typical changes of CRP, fibrinogen,
ESR and albumin during an acute
phase reaction
early
loss of appetite ࢴ慾
2
C-reactive protein
Structure and function
Three properties can
explain many functions
of CRP:
1) Binding to
phosphorylcholine
2) Activation of
complement
3) Binding to cell receptors
(Fc receptors or specific
CRP receptors)
Synthesis and turnover
n
Strong
response in
Induction and synthesis of
CRP in hepatocytes
Minor
response in
New Indications for
C-Reactive Protein
• From Acute Phase
Protein to
Cardiovascular
Risk Marker
n
By Rolf D. Hinzman,
M.D., Ph.D.
3
Relative risk for future
cardiovascular events
Table 19-1 Properties of
Selective Plasma Proteins
n
Listed in the order of their
electrophoretic mobilities in agarose
gels at pH 8.6
P22
那一蛋白屬於那一電泳區?
--> a high relative risk for a future cardiovascular events
Table 19-2 Interim consensus
reference intervals for 14 Plasma
Proteins in human serum
INDIVIDUAL PLASMA
PROTEINS
n
Prealbumin
n
n
n
n
n
n
retinol-binding protein & transthyretin
Albumin
a1-Acid Glycoprotein, a1-Antitrypsin, a1Fetoprotein,
a2-Macroglobulin, Heptoglobin,
Ceruloplasmin
Transferrin, complement, b2-Microglobin
Immunoglobulins, C-Reactive Protein
4
Albumin:
3.5 - 5.2 g/dL
Albumin: 66.3 kD
n
n
n
The most abundant protein in human plasma
n
representing 40-60% of total protein
n
synthesized in liver.
n
n
A marker of both glomerular and bloodbrain barrier function
n
pI lies between 4.0 and 5.8
n
an anion at pH 7.4
n
> 200 negative charges per molecule
Hyperalbuminemia:
>5.2 g/dL
n
n
Acute dehydration
No clinical significance
n
n
Acute & chronic inflammation
Hepatic disease: cirrhosis(肝硬化)
Urinary loss
n
是否脫水
實驗步驟是否正確
to transport and store a wide variety of
ligands
to maintain the plasma oncotic pressure
to serve as a source of endogenous amino
acids.
Hypoalbuminemia: <3.5 g/dL
n
n
The chief biological functions
n
n
n
n
Nephrosis
Renal disorders
Gastrointestinal loss
Protein energy malnutrition
Edema & Ascites腹水
5
Alpha 1
n
a1-Acid Glycoprotein (AAG, orosomucoid)
n
n
n
n
n
Serine proteinese inhibitor, an APR
Deficiency: liver disease
a1-Fetoprotein (AFP)
n
n
antenatal diagnosis of neural tube defects
hepatocellular carcinoma & germ cell tumor
Alpha 2
n
n
low pI (2.7 - 3.5)
An APR whose exact role is unknown
a1-Antitrypsin (AAT)
n
Alpha 2
Ceruloplasmin (Cp)
Contain ~95% serum copper; blue color
n A late-reacting ARP
n Wilson‘s disease
a2-Macroglobulin (AMG, MW~725 kD)
⁄: effects of estrogen
n ÿ: acute pancreatitis & prostate cancer
n
n
Heptoglobin (HAP, Hp)
An APR, binds free hemoglobin in serum
n ⁄: effects of corticosteroid & NSAIDs
n ÿ: hemolysis
n
‫ܧ‬類固醇抗免疫藥　普拿疼
Beta
n
n
n
Absent a intracellular ATPase to add Cu2+ to
Cp
n plasma Cp is typically reduced
n dialyzable copper concentration is increased
Transferrin (TRF, siderophilin)
n
Next slide
b2-Microglobin (BMG, MW 1.8 kD)
n
n
n
Pass through the glomerular membrane =>
99% reabsorbed
⁄: renal failure, inflammation, and
neoplasma
6
Transferrin (TRF, siderophilin)
n
n
n
n
Most of total iron binding
capacity (TIBC)
One molecule bind 2
ferric ion (Fe3+)
A negative APR
n Inflammation or
malignancy
⁄: pregnancy &
estogren administration
n
n
n
n
Fe2+
ferric ion
(Fe3+)
n
n
n
n
n
Overload proteinuria
n
n
Mainly low-MW plasma proteins
Defective tubular reabsorption
n
n
Protein in amniotic fluid, saliva, and
feces
Specific quantitative assays of particular
proteins by immunochemical methods
n
n
n
From plasma to urine of an abnormal low-MW protein
Postrenal proteinuria
Albumin, TRF, TTR
Analysis of Proteins
Most common & serious; mainly albumin
Increased glomerular permeability
Tubular proteinuria
Only a small amount of protein
Most is albumin
Protein in cerebrospinal fluid (CSF)
n
Glomerular proteinuria
n
Urinary proteins
n
Proteinuria
n
Proteins in other body fluids
n
nephelometry, turbidimetry, RID, RIA, EIA
Detection and identification of proteins
by electrophoresis
Quantitative measurements of total
protein in serum, urine, and CSF
Analysis by mass spectrometry
Production of protein by the urinary tract
7
Serum Protein Electrophoresis-on cellulose acetate or agarose gel
• pH 8.6 barbital buffer
• Stains:
• Amido black &
Ponceau S in the past
• CBB is more sensitive
and more widely used.
• Densitometry
Serum Protein Electrophoresis-Polyclonal Gammopathy
Polyclonal
gammopathy usually
occurs secondary to
many chronic diseases
avoids the complication of a fibrinogen band
Serum Protein Electrophoresis-Nephrotic Pattern
The long term loss
of lower molecular
weight proteins
(albumin, IgG) and
retention of higher
molecular weight
proteins (alpha-2macroglobulin.)
Serum Protein Electrophoresis-Cirrhotic Pattern
The "beta-gamma
bridge" pattern:
distinction between
beta and gamma
globulin is blurred
8
Serum Protein Electrophoresis-Acute inflammatory pattern
The intensity of the
alpha-2 fraction is
greater that the gamma
globulin fraction
Serum Protein Electrophoresis-Monoclonal (M) Protein Present
A large increase in
serum IgG, but
decreased IgA and IgM.
Serum Protein Electrophoresis-Alpha 1 anti-trypsin deficiency
Alpha-1-anti-trypsin deficiency can be
congenital or acquired, typically
secondary to liver or pulmonary
diseases.
Serum Protein Electrophoresis-Monoclonal (M) Protein Present
IFE on this patient's
serum showed the M
protein was IgG kappa.
A diagnosis of multiple
myeloma was made.
9
Quantitative Measurements of
Total Protein in Body Fluids
n
Specific methods
n
n
n
Bovine or human albumin is used routinely
Cu2+
Reference intervals
n
n
n
An adult at rest:
Ambulatory:
Neonates:
6.0-7.8 g/dL
6.3-8.3 g/dL
4.6-7.0 g/dL
Biuret Method
n
Biuret reaction
Calibration of total protein methods
n
n
Biuret, direct photometric, dye-binding, Lowry,
Kjeldahl, refractometry, turbidimetric
An analogous reaction occurs between
cupric ion and the organic compound
n
n
Amino acids & dipeptides: not react
tri-, oligo-,& polypeptides:
n give pink to reddish-violet products
Biuret treated with
Cu2+ ions in a
moderately alkaline
medium, a colored
chelate is formed
Direct photometric methods
n
UV 280 nm absorption
n
n
n
aromatic amino acids
tyrosine, tryptophan, phenylalanine
UV 220 nm
n
n
研究用
peptide bond
Specific absorption at 220 nm is 10 to
30 times greater than at 280 nm
10
Dye-binding methods
n
Amido black 10B &
Coomassie brilliant
blue (CBB)
n Simple, fast, and
linear up to 150
mg/dL
Folin-Ciocalteu (Lowry)
method
n
Folin-Ciocalteu reagent
n
n
研究用
phosphotungstic-phosphomolybdic acid
Tyrosine and tryptophan
n
produce a blue color
ß More useful for assay
n
Urine: pyrogallol red,
Ponceau S and CBB
Kjeldahl’s method:
reference method
n
of a pure prorein
The Kjeldahl procedure used
to determine protein content.
Acid digestion
n
convert nitrogen in protein to ammonium ion
The concentration of ammonia nitrogen
was then evaluated by titration or
nesslerization
[Protein] =
n
Ammonia nitrogen value x 6.25 (100%/16%)
n
Well defined and reproducible; but timeconsuming and inconvenient
11
Auto Kjeldahl System B-339:
Refractormetry:
a fully automatic unit
n
A speedy and problem-free
nitrogen determination
n
n
The overall time for a
complete determination is
only 3-4 min.
Formation of a fine precipitate of uniform,
insoluble protein particles
n
n
n
n
n
performing distillation,
digestion, titration,
calculation and print-out of
the results
Turbidimetric or nephelometric
methods
n
Refractormeter
Sulfosalicylic acid alone
Sulfosalicylic acid + sodium sulfate
Sulfosalicylic acid + trichloroacetic acid (TCA)
TCA alone
If a solute is added to water, the refractive
index of the solution increases
n
n
proportion to concentration of solute
quick
Total Protein Methods
Method
Principle
Comment
Kjeldahl
Digestion of
protein;
measurement of
nitrogen content
Reference method;
assume average
nitrogen content of 16%
Measurement of
refractive index
Refractometry due to solutes in
serum
Rapid & simple;
assume nonprotein
solids are present in
same concentration as
in the calibrating serum
12
Total Protein Methods
Method
Biuret
Dye-binding
Principle
Comment
Formation of violetcolored chelate
between Cu2+ ions
& peptide bonds
Protein binds to dye
& causes a spectral
shift in the
absorbance
maximum of the
dye
Routine method;
requires at least two
peptide bonds & an
alkaline medium
Research use
Total Protein Abnormalities:
Hypoproteinemia
n
Excessive loss
n
n
n
n
n
Excretion in urine & renal disease
Loss of blood
Decreased intake: malnutrition
Decreased synthesis: liver disease
Accelerated catabolism
n
Total Protein Methods
Burns, trauma, other injuries
Total Protein Abnormalities:
Hyperproteinemia
n
n
Is not seen as commonly as
hypoproteinemia
Dehydration
n
n
Vomiting, diarrhea, excessive sweating,
diabetic acidosis, & hypoaldosteronism
Excessive production primarily of gglobulins
13
Determination of Albumin in
Plasma
n
Most: automated dye-binding methods
n
n
Albumin Methods
Bromcresol green (BCG) or bromcresol
purple (BCP)
Serum is reommended
BCG test is more sensitive dye
Determination of Total Protein
in Urine
14
Questions
1. What are five principal functions of plasma
proteins?
2. What plasma protein is present in the
highest concentration, and what are its
functions?
3. Separation of serum proteins by
electrophoresis depends on what
principles?
4. In what circumstances is the concentration
of urine protein elevated?
Questions
5. What are the characteristic serum protein
electrophoresis patterns in patients with (a)
nephrotic syndrome, (b) acute infectious
hepatitis, (c) multiple myeloma, & (d)
hypogammaglobulinemia?
6. What principles are involved in the
determination of total protein by the biuret
reaction? Of albumin by the bromcresol
gree method?
Questions
7. What are the functions of (a)
prealbumin & (b) acute phase reactants,
& how may their concentrations in
serum be measured?
8. What is the origin of cerebrospinal fluid
proteins, & what is the rationale for
measuring same?
15