Protein

Quantification
 Lecture outline
• Assessing plasma proteins
• Protein quantification/quantitation
• Spectroscopy
• Colorimetric methods
• Biuret assay
• Lowry assay
• Bradford assay
• BCA assay
• Qualitative methods
• Electrophoresis
• Electrophoretic patterns
• Electrophoretic patterns in disease
• Paraproteinaemia
• Bence Jones proteins
 Plasma Proteins

 Concentration = g/L
 Or - Concentration defined as activity of
a defined function, e.g. clotting time for
prothrombin
 Note that abnormal forms of protein
can be present at normal concentration
but with impaired function
 Total Proteins

 Estimation is of limited clinical value

 Acute changes reflects ratio of protein to
fluid in vascular compartment
 Acute changes more likely due to loss or
increase of protein-free fluid rather than
protein
 Total Proteins
 Misleading total concentration possible ~
normal despite marked changes, e.g.:
 Plasma albumin ↓↓ = Immunoglobulin ↑↑
 Aside from albumin, most proteins
contribute little to total protein concentration
≡ change in concentration of one protein
may not cause detectable change in total
protein
 Total Proteins
 Raised plasma total protein
 Loss of protein-free fluid during venepuncture
 Major increase in one of more Immunoglobulins
 Low plasma total protein
 Dilution – if sample taken near site of
intravenous infusion
 Hypoalbuminaemia
 Profound immunoglobulin deficiency
Methods for quantification/quantitation
of protein

 Spectroscopy

 Electrophoresis
Methods for quantification/quantitation
of protein
 Note: protein assays only detect the presence of
certain parts of proteins
 The abundance and presence of these parts
(such as particular amino acids) is highly
variable from one protein to another and will
greatly influence the choice and methodology of
the assay
 Protein Quantification
 Spectroscopy
 Direct absorption amino acids: 280 nm
 Peptide bonds: 205 nm
 Fluorescence spectroscopy: contains
fluorescent amino acid tryptophan
 Fluorescence spectroscopy: reaction of
amino acids with o–phthaldialdehyde in
the presence of reducing reagents
 Protein Quantification
 Absorption at 280 nm (semi-quantitative)
 Absorption of aromatic acids, tryptophan,
tyrosine residues
 Advantages:
 Quick
 Useful for identification of proteins before
using a more accurate method
 Disadvantages:
 Disturbance by other substances with
absorption at 280 nm
 Absorption different for different proteins, due
to variation in tryptophan content
 Protein Quantification
 Absorption at 280 nm (semi-quantitative)
 Standard curve ⇒
Accurate determination
of protein concentration

A280

 Rule of thumb:
 A280 = 1 corresponds to 0.5 – 2 mg/mL
protein
 Protein Quantification
 Colorimetric methods
 Biuret assay: Complex of Cu2+ with peptide
bonds (-CO-NH-) under alkaline conditions →
Cu+-protein complex, absorbance at 570 nm
 Lowry assay: Transfer of electrons from bound
copper ions and from aromatic side chains to
Folin reagent
 Bradford assay: Adsorption of Coomassie
Brilliant Blue G250 to protein (mainly Arg, but
also His, Lys, Trp, Tyr and Phe sidechains)
 Bicinchoninic acid (BCA) assay: chelation of
reduced copper with BCA
 A standard is used in all colorimetric assays –
usually different amounts of bovine serum
albumin (BSA)
 Protein Quantification
 Biuret reaction: the oldest protein quantification
method
 Cu2+ → Cu1+
 Based on the formation of copper coordination
complexes (biuret complexes) when Cu2+SO4 is
added to alkaline protein solutions
 The biuret complex allows spectrophotometric
measurement at 550 nm
 This method probes the peptide bond - therefore
not dependent on the amino acid composition of
the protein
 Sensitivity is limited to mg/mL protein
concentrations
 Protein Quantification
 Biuret reaction

http://www.uni-regensburg.de/Fakultaeten/nat_Fak_IV/Organische_Chem
 Protein Quantification
 Lowry method: more sensitive than biuret reaction
 Lowry method: 2-step reaction
 First - a Biuret reaction reducing Cu2+ to Cu+ ⇒
biuret complex
 Second - combination of biuret complexes with
Folin-Ciocalteau reagent = Folin-Ciocalteau
reduction by Cu+ → blue coloured product with
absorption range of 500 – 750 nm
 Formation of molybdenum or tungsten blue ⇒
presence of reducing amino acids (tyrosin,
tryptophan, cysteine)
 Protein Quantification
 Lowry method
 Advantages
 Sensitive over a wide range
 The most commonly referenced procedure for
protein determination
 10 – 20 times more sensitive than UV detection
 Disadvantages
 Many substances interfere with the assay
 Takes a considerable amount of time to perform
 Assay is photosensitive - illumination during the
assay must be kept consistent for all samples
 Amount of colour varies with different proteins
 Protein Quantification
 Bradford: absorbance shift in Coomassie Brilliant Blue G-
250 (CBBG) when bound to arginine and aromatic
residues
 The anionic (bound form) has absorbance maximum at
595 nm whereas the cationic form (unbound form) has
absorbance maximum at 470 nm

Chemical Structure of Coommassie Brilliant Blue G-250

 Protein Quantification
 Bradford: popular method
 Simple, rapid, inexpensive and sensitive
 Coomassie brilliant blue G-250 dye (CBBG) dye
specifically binds to proteins at arginine [8x more
compared to other residues], tryptophan, tyrosine,
histidine and phenylalanine residues
 CBBG binds to residues in anionic form, which has an
absorbance maximum at 595 nm (blue)
 The free dye in solution is in the cationic form, which
has an absorbance maximum at 470 nm (red)
 The assay is monitored at 595 nm in a
spectrophotometer ⇒ measures the CBBG complex
with the protein
 Protein Quantitation
 Bradford Assay
 Protein Quantification
 BCA test: Cu2+ and BCA-
containing solution added to
protein samples → purple
BCA-copper complexes with
an absorption of 562 nm
 Protein Quantification

 BCA test: detection limit is enhanced to the

μg/mL range
 First - biuret reaction = formation of Cu+
complexes from Cu2+
 Takes place in the presence of reducing
agents, such as ascorbic acid or
hydroxylamine
 Second step - formation of BCA complexes
[Cu(BCA)2]3-
 Protein Quantification
 BCA test
 Advantages
 Less susceptible to interference from common buffer
substances
 Very sensitive and rapid at elevated temperatures
 Very little variation in response between different
proteins
 Broad linear working range
 Disadvantages
 The reaction does not go to completion when
performed at room temperature or 37oC – a problem
when assaying a large number of proteins
 Dilution is often necessary for concentrated protein
samples
 Protein Quantification
Assay Sensitivity Accuracy Interference
Biuret 0 – 1 mg Very high, Amino groups
independent [e.g. (NH4)2SO4]
of amino acid
composition
Lowry 0 – 0.1 mg Partially Acids, chelators (EDTA),
dependent on reductants (DTT, phenol),
amino acid (NH4)2SO4
composition
Bradford 0 – 0.01 mg Dependent on Detergents
amino acid (SDS, Triton X100, soap)
composition
BCA 0 – 0.05 mg Almost Reducing agents
independent (2-mercaptoethanol, DTT),
of amino acid chelators (EDTA
composition
 Qualitative Methods

 Proteins have acidic and basic sites spread

throughout their structure
 Changing pH changes charge structure
 Depending on the pH, proteins can be separated
on the basis of charge using electrophoresis
 Electrophoresis

 Separates proteins according to their

different electrical charges
 Small amount of serum or urine applied
to cellulose/agarose strip
 Current passed through strip
 Strip is stained, e.g. Coomassie stain
 Electrophoretic patterns: protein bands
 Electrophoresis
 Five bands ⇒ five main groups of proteins
 Albumin
 Most obvious band. Single protein.
 α1- globulins
 Almost entirely α -antitrypsin
1
 α2-globulins
 Mainly α -macroglobulin and haptoglobin
2
 β-globulins
 β – transferrin
1
 β – C complement
2 3
 γ-globulins
 Immunoglobulin
 Note: some Ig found also in α and β regions
1
Electrophoresis
 Electrophoresis

Cathod
Anode

e
 Electrophoresis

 If plasma rather than

serum used, fibrinogen
appears as distinct
band in β-γ region
 ∴ always allow blood to
clot and use serum for
electrophoresis
 Electrophoresis

Figure (a): Normal electrophoresis pattern of blood serum

 Electrophoresis

Figure (b): Abnormal pattern, with elevated γ-globulin, indicating

the possibility of liver disease, collagen disorder, or infection.
Electrophoresis
Electrophoretic patterns in disease
Serum Protein Electrophoresis

Normal Pattern

Note the relative sizes and

shapes of the globulin fractions.
Electrophoretic patterns in
disease
Serum Protein Electrophoresis
Polyclonal Gammopathy

Polyclonal gammopathy usually

occurs secondary to many chronic
diseases.

This patient was a 39-year-old male

with sarcoidosis. The sequential
increase of the globulin fractions
illustrated "sarcoid stepping."

Sarcoidosis – granulomatous disease of

unknown aetiology; histological
resemblance to tuberculosis
 Electrophoretic patterns

Abnormal serum protein electrophoresis pattern in a patient with

multiple myeloma. Note the large spike in the gamma region.
Electrophoretic patterns
 Electrophoretic patterns

 Polyclonal response: B-cell response to antigenic stimulation

(e.g. infection) →→ synthesis of a range of different
immunoglobulin groups, i.e. clones
 Diffuse hypergammaglobulinaemia electrophoretic pattern
 If cells proliferate to form clones of like cells ⇒ excess production
of a single protein: monoclonal proliferation
 Electrophoretic patterns
Bands mistaken for monoclonal γ band

Band Condition

Alpha-2-Macroglobulin Nephrotic syndrome

Hemoglobin-haptoglobin Hemolysis
Beta-1-Lipoprotein Hyperlipidemia
Fibrinogen Inadequate clot
C-Reactive Protein Acute inflammation
Immune complex pattern Inflammation
Electrophoretic patterns in
disease
Serum Protein Electrophoresis
Nephrotic Pattern

The nephrotic pattern illustrates the long

term loss of lower molecular weight
proteins (e.g. albumin, IgG) and
retention of higher molecular weight
proteins (e.g. α2-macroglobulin)

This patient was a 39-year-old female

with SLE. Her daily total urinary protein
loss exceeded 3700 mg. (Normal loss is
less that 150 mg/day.)

SLE – systemic lupus erythromatosus

Electrophoretic patterns in
disease
Nephrotic Syndrome
Decreased albumin
Electrophoretic patterns in
disease
Serum Protein Electrophoresis
Cirrhotic Pattern

Patient was a 46-year-old male with

end stage liver disease secondary to
chronic alcohol abuse. He died 5
months after this sample was
obtained.

In the cirrhotic pattern, the distinction

between β and γ globulin is blurred
and is sometimes referred to as the
"β-γ bridge" pattern.
Electrophoretic patterns in
disease

Hepatic cirrhosis
Decreased albumin (synthesis)
Electrophoretic patterns in
disease
Serum Protein Electrophoresis
Acute inflammatory pattern

This patient was a 42-year-old female

who presented with a temperature of
40°C and was diagnosed with both
pneumonia and pyelonephritis.

In the acute inflammatory pattern

albumin and γ globulin are decreased
and α2-globulin becomes very
prominent.

Note that the intensity of the α2

fraction is greater that the γ globulin
fraction.
Electrophoretic patterns in
disease
Electrophoretic patterns in
disease
Electrophoretic patterns in
disease
Serum Protein Electrophoresis α
1 anti-trypsin deficiency

α1 anti-trypsin deficiency can be

congenital or acquired, typically
secondary to liver or pulmonary
diseases.

Congenital α1 anti-trypsin
deficiency is most commonly
associated with early onset
emphysema, pancreatic insufficiency,
or cirrhosis.
 Immunofixation
Electrophoresis
 Electrophoresis technique to identify individual
immunoglobulins
 IgG, IgM, IgA, lambda λ light chain, and kappa κ
light chain
 Identifies proteins in serum and urine
 Abnormal results indicate immune system
disorders including multiple myeloma and
Waldenstrom's macroglobulinemia.
 Immunofixation
Electrophoresis

Typing an M band by immunofixation. In this example, the M band

found on electrophoresis is identified as an IgG (type κ)
 Immunofixation
Electrophoresis (IFE)

http://www.immunologyclinic.com/defaul
t.asp
Electrophoretic patterns in disease
Serum Protein Electrophoresis
Monoclonal (M) Protein Present

The patient was a 72-year-old male

who presented with lower back pain.

Quantitative immunoglobulin
measurements showed a large
increase in serum IgG, but decreased
IgA and IgM.

Bone marrow exam revealed a large

increase in plasma cells that were
frequently aggregated.

IFE on this patient's serum showed

the M protein was IgG-κ. A diagnosis
of multiple myeloma was made.
Electrophoretic patterns in disease
Serum Protein Electrophoresis
Biclonal Gammopathy

This sample is from a 62-year-old

male who presented with weight loss
and fatigue.

He was found to have multiple

myeloma. In this disease, biclonal
gammopathies are rare, occurring in
about 1.7 % of patients.

IFE showed the 2 M proteins to be

IgG-κ and IgA-λ
Electrophoretic patterns in
disease

http://erl.pathology.iupui.edu/INDEX.HTM

http://erl.pathology.iupui.edu/ LABMED/GENER27.HTM

http://www.columbia.edu/itc/hs/medical/selective/AdvClinicalPa
thology/2005/lecture/ProteinBW_Pesce.pdf
Electrophoretic patterns in disease

 Paraproteinaemia
 Paraprotein: appearance of
abnormal, narrow, dense
band on electrophoresis strip
= the M band

Commonly found in γ region,
but may be anywhere in the α
2 – γ region
Electrophoretic patterns in disease

 Paraprotein: a monoclonal Ig or Ig light chain

in the blood or urine resulting from a clonal
proliferation of plasma cells or B-lymphocytes
 M band, monoclonal band, monoclonal
spike, monoclonal protein
 May consist of a whole Ig, or only the light
chain
 Ig light chain ↑↑ : most or all of the
paraprotein is in the urine because of the low
molecular weight
Electrophoretic patterns in disease
 Presence of a paraprotein in serum or urine can be
used as a diagnostic criterion in some B cell
malignancies
 In B cell malignancy, the paraprotein is a sensitive
tumour marker
 Immnoglobulin fragments (particularly Bence Jones
protein) are rarely found in non-malignant conditions
 Serum and/or urine gives an 'overview' of the B cells
products while B cell disease may be patchily
distributed
 Some symptoms may be directly related to the
presence of a paraprotein e.g. hyperviscosity with high
concentrations of IgM paraproteins (Waldenstrom’s
macroglobulinaemia)
Electrophoretic patterns in disease
 Paraproteinaemia
 Presence of a paraprotein is suggestive but not
diagnostic of malignancy
 Seen in:
 Myelomatosis (multiple meyloma)
 Macroglobulinaemia (Waldenstrom’s
macroglobulinaemia)
 B-cell lymphomas, including chronic lymphatic
leukaemia
 Heavy-chain disease (H chain: α, γ or µ)
Electrophoretic patterns in disease

 Paraproteinaemia
 Coexistent features very suggestive of B-cell
malignancy:
 Immune paresis
 Synthesis of Ig from other clones of cells
suppressed by the proliferation of a single
clone
 Narrow paraprotein band, otherwise, band
is reduced or absent
 Very low concentration of other Ig
Electrophoretic patterns in disease

 Paraproteinaemia
 Bence-Jones protein (BJP) – found in urine of
patients with B-cell malignancy
 Consists of monoclonal light chains
 Low molecular weight: 20,000 – 40,000 mwt
 ∴ filtered at glomeruli → urine
 BJP deposition in tubular cells ⇒ renal
glomerular dysfunction
 Precursor to amyloid deposition in tissues ⇒
amyloidosis
Electrophoretic patterns in disease

Note:
 Immunisations within previous six months can
increase Ig
 Increased Ig also seen with drugs such as phenytoin
(Dilantin), procainamide, oral contraceptives,
methadone, and therapeutic gamma globulin
 Aspirin, bicarbonates, chlorpromazine (Thorazine),
corticosteroids, and neomycin can affect protein
electrophoresis results
E.g. of evaluation procedure in the
event of an abnormal serum protein
electrophoresis (SPEP) result; in this
case, a monoclonal gammopathy.