CLINICAL CHEMISTRY LAB
LECTURE 4: AMINO ACIDS AND PROTEINS:
LABORATORY METHODS AND PROCESSING
Prof. Bea Angeli D. Laude
Octobr 25, 2021
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TOTAL PROTEIN METHODS
Total Protein Specimen:
● Serum (sample of choice)
● Need not be fasting
○ Sample can be taken at any time of the day
whether or not the patient had a meal
● Lipemia may interfere
○ Must be contraindicated because it could
interferences in the procedures
● Hemolysis
○ Elevate Total Protein because off the release of
RBC proteins in the serum
Total Protein Methods
● Kjeldahl Method
● Biuret Method
● Dye Binding
● Refractometry
Kjeldahl Method
● Classic method (determine nitrogen)
● Not used in Clinical Laboratory
○ Time-consuming
○ The procedure is way too tedious for it to be a
part of routine activity
○ Requires many reagents
● Average of 16% nitrogen as in proteins
○ In order to calculate the protein concentration
○ The actual nitrogen content of protein may
actually vary ranging from 15.1% - 16%
○ An error can be introduced if the protein
standard which was calibrated by the Kjeldahl
method that it may actually differ in protein
composition compared to the sample
● No proteins of significant concentration in the unknown
specimen are lost in the precipitation step which is the
first step of the method
● Start with the precipitation of proteins
● Proteins will be precipitated with the presence of tungstic
acid
● Pag naa na si tungstic acid, the non-protein nitrogens
will also be removed with the supernatant
● Proceed with digestion
1. Digestion
● Will occur if you mix the sample with sulfuric acid and
heat between 340°C - 360°C
● To speed the reaction, a catalyst will be added. Cupric
sulfate is utilized. In some cases, another reagent is
added that is potassium sulfate in order to increase the
boiling point to improve the efficiency of the digestion
● Sulfuric acid will oxidize the carbon, hydrogen, and sulfur
in the protein to become CO2, CO, H2O, SO2
● Nitrogen in the protein will be converted to become your
ammonium bisulfite which is then utilized for
neutralization and distillation
2. Neutralization and Distillation
● In neutralization, your ammonium bisulfite was highly
exposed to an acid which is sulfuric acid which needs to
be neutralized before proceeding to distillation
● The neutralizing agent is by the addition of an alkaline,
sodium hydroxide
● Once it is neutralized, proceed with distillation into a
standard boric acid solution
● From ammonium bisulfite, because of the presence of
your boric acid, it now becomes your ammonium borate
3. Titration
● Ammonium borate will be titrated with standard HCl so
we can be able to determine the number of proteins that
is present on the patient sample
Biuret Method
● Most widely used method
● Recommended by IFCC (International Federation of
Clinical Chemistry) for determination of Total Protein
● Cupric ions complex with the groups involved in peptide
bond
○ There will be a violet-colored soln if there are at
least two peptide bonds detected in the sample
● Biuret Reagent
○ Potassium hydroxide
■ Provides an alkali medium so the rxn
can take place
○ Sodium potassium tartrate
■ Complex cupric ions to prevent their
precipitation in the alkaline soln
○ Potassium iodide
■ Acts as antioxidant
● Absorbance is measured at 540 nm
● Also determines the size of the protein particles that is
present on the samples
● When small peptides react, the color of the chelate
produced has a different shade than that seen with
larger peptide
● Alkaline medium and at least 2 peptide bonds, a
violet-colored chelate formed
● Color varies from pink to a reddish violet
● Color formed is proportional to the number of peptide
bonds and reflects the Total Protein level
○ If pink = lower TP level
○ If reddish violet = higher TP level
● In (abnormally small proteins) multiple myeloma,
c-protein concentration is underestimated due to lighter
shade of color produced
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 ○ Smaller peptides could produce lighter shade
compared to samples with larger protein
● Lipemia in the sample is interferent
○ High lipid content must be rejected as it will
interfere to the procedure
Dye-Binding Method
● Ability of most proteins in serum to bind dyes
● Most common dye utilized: Coomassie brilliant blue 250
relies on the binding to protein
○ When it forms complexes with the protein in the
sample, it will be causing a shift of the
absorbance of the dye from 465-595 nm
● A shift absorbance maximum of the dye from 465-595
nm
● Absorbance at 595 nm is used to determine the protein
concentration
● Aside from coomassie brilliant blue, other dyes may be
used such as:
○ Bromothymol blue
○ Ponceau S
○ Amido black 10b
○ Lissamine green
● Although it can be a simple and fast method, caution
must still be applied in utilizing this method because
individual proteins tend to have different affinity to the
dyes to be utilized
Principle of Dye-binding Method in quantifying TP
● Coomassie Brilliant Blue - dye utilized
● With the presence of the protein in the sample, it is going
to form a dye-protein complex
● With this formation, we have a change in the maximum
absorbance of the dye which started at 465 - 490 nm to
shift to its maximum absorbance of 595nm
● Increased in absorbance at 595 nm, is utilized to
determine the protein concentration of the sample
Refractometry
● A quick alternative to chemical analysis of serum total
protein when a rapid estimate is required
● Measurement of RI due to solutes in serum
○ Refractive Index (RI) can be accurate in
measuring the serum protein concentrations,
but not in urine protein measurements because
of the excess amounts of solutes in relation to
the protein
SUMMARY: TOTAL PROTEIN METHODS
METHOD PRINCIPLE COMMENT
Reference method;
Digestion of protein;
assume average
Kjeldahl measurement of nitrogen
nitrogen content of
content
16%
Formulation of Routine method;
violet-colored chelate requires at least two
Biuret
between Cu2+ ions and peptide bonds and an
peptide bonds alkaline medium
Protein binds to dye and
Dye binding causes a spectral shift in Research use
the absorbance maximum
of the dye
Refractometry is a rapid, simple method, but is not commonly
used for total protein analysis.
Fractionation, Identification, and Quantitation of Specific
Proteins
● Albumin
● Globulin
● These 2 proteins can give us useful diagnostic
information to determine the presence of kidney or liver
disease, we refer to it as A/G ratio
● Get total protein, and determine the levels of either
albumin or globulin (most often its albumin)
● Then, subtract to the total protein and we can get the
levels of globulin
● If there’s a reversal or significant change in the ratio of
albumin and total globulin, it will aid us in determining if
the patient has problems in the kidney or liver
Methods for Fractionation, Identification and Quantification of
Specific Proteins
● Salt fractionation
● Dye-binding
● Serum protein electrophoresis
Salt Fractionation
● Fractionation of proteins is commonly done using the
precipitation technique:
● Globulins are separated from albumin by salting out,
using sodium salt to precipitate globulins
○ Globulin - precipitate
○ Albumin - supernatant fluid (measured)
■ Measured thru routine total protein
technique.
● Biuret test
● Dye binding method
○ Salting is no longer used bcoz we have
available methods that can directly react with ur
albumin in the given sample.
Dye-binding
● Dye-binding procedures
○ most widely used methods for determining
Albumin.
● pH of the solution is adjusted so that albumin is positively
charged.
○ If it is in complex with the albumin, maximum
absorbance is going to change
● Albumin is attracted to and binds to an ionic dye
● amount of albumin
○ (calculated by) absorbance of the albumin-dye
complex
■ Methyl orange
■ 2,4’-hydroxyazobenzene-benzoic acid
(HABA)
■ Bromocresol Green (BCG)
■ Bromocresol Purple (BCP)
Methyl Orange
● nonspecific for albumin
● Beta-lipoproteins, alpha-1 and alpha-2 globulins also
bind to dye
2,4’-hydroxyazobenzene-benzoic acid (HABA)
● low sensitivity but more specific for albumin salicylates,
penicillin, conjugated bilirubin, and sulfonamides
○ interfere albumin to HABA
Bromocresol Green (Bcg)
● Not affected by any interfering substances that can be
present in the sample such as bilirubin and salicylates.
● hemoglobin binds to BCG
○ for every 100 mg/dL of Hgb, albumin increased
by 0.1 g/dL
● has been reported to overestimate low albumin values
○ nephrotic syndrome or end-stage renal disease
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 ○ This pattern is going to be the basis as to what
Bromocresol Purple (Bcp) type of protein has caused the abnormality that
● alternate dye, binding specifically to albumin led to the px condition
● not subject to interferences, precise, exhibits excellent ● The scanning densitometer will compute the area under
correlation with immunodiffusion reference methods the absorbance curve for each band and the percentage
● Disadvantage: In renal insufficiency, BCP method of total dye that appears in each fraction.
underestimates serum albumin ○ This percentage will be multiplied to the total
● Bilirubin interferes with BCP binding to Albumin protein which is measured separately
● Concentration is calculated as a percentage of the total
Total Globins protein
● If in case that the methods for albumin determination is ● Computation also be made by cutting out the small
not available, we can still get the albumin levels by bands from the membrane and eluting the dye in 0.1
calculation mol/L NaOH
● Albumin can be calculated by subtraction of the globulin ● Absorbances are added to obtain total absorbance, and
from Total Protein. the percentage of the total absorbance found in each
● Total Globulin level- direct colorimetric method using fraction is calculated
glyoxylic acid.
● Glyoxylic acid, in the presence of Cu2+ and in an acid Reference values
medium (acetic acid and H2SO4), condenses with Fraction Percentage Concentration
tryptophan found in globulins to produce purple color.
Albumin 53% - 65% 3.5 - 5.0 g/dL
● Read spectrophotometrically, kukunin yung absorbance
and will be utilized to compute the total globulin levels. Alpha-1-globulin 2.5% - 5% 0.1 - 0.3 g/dL
● Determination of TG based on the presence of Alpha-2-globulin 7% - 13% 0.6 - 1.0 g/dL
tryptophan is not commonly used because mas easier Beta-globulin 8% - 14% 0.7 - 1.1 g/dL
gamitin ang dye binding method for albumin
Gamma-globulin 12% - 22% 0.8 - 1.6 g/dL
determination.
● Reference values for each fraction (Bishop, 7th ed.)
Electrophoresis ● Rank according to % in the total protein
● After determining the total albumin and globulin levels, if ○ 1st = Albumin
abnormalities are noticed then we can proceed to ○ 2nd = Gamma
electrophoresis. ○ 3rd = Beta
● separates proteins on the basis of their electric charge ○ 4th = Alpha
densities. ○ 5th = Alpha - 1
● protein will move according to their density determined
by pH of a surrounding buffer. SELECTED DENSITOMETRIC PATTERNS OF PROTEIN
● direction of movement depends on charge ELECTROPHORESIS
○ Cations → cathode terminal (-)
○ Anions → anode terminal (+) Reference Pattern
● the speed of migration can be estimated from the
difference between the pIof the protein and the pH of the
buffer.
● velocity depends on electric field strength, size, and
shape of molecule, temperature and characteristics of
buffer
● Cellulose acetate or agarose gel- support media

Serum Protein Electrophoresis

● Serum are applied close to the cathode end of a support
medium (ph 8.6)
● All major serum proteins carry a net negative charge at
ph 8.6 and migrate toward the anode.
● 5 bands:
○ Albumin- travels farthest to the anode
○ alpha-1-globulins
○ alpha-2-globulins
○ beta-globulins
○ Gamma-globulins
● Width of the band = number of proteins present in that
fraction.
● After separation, protein fractions are fixed by immersing ● All protein fractions are in normal level
the support medium in an acid solution (acetic acid)
● Next step, proteins are stained.
○ Dyes
■ Ponceau S
■ Amido black
■ Coomassie blue
● Cleared transparent medium is placed in a scanning
densitometer for reading
● The pattern on the membrane moves past a slit through
which light is transmitted to a phototube to record the
absorbance of the dye that is bound to each protein
fraction
○ The absorbance is going to determine the
quantity of the protein that is present
● Absorbance is recorded on a strip-chart recorder to
obtain a pattern of the fraction
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Monoclonal Increase
● Sharp peak in the gamma globulin area
● Increased production of gamma globulins
Alpha-1-Antitrypsin Deficiency
● Deficiency in alpha-1
Nephrotic Syndrome
● Px tend to lose some of their serum albumin and low
molecular weight proteins including some globulin
gamma
● Increased alpha-2 macroglobulin, beta lipoprotein,
complement components and haptoglobin
Inflammation
● Decrease in albumin but increase in alpha-1, alpha-2,
and beta bcs of increased production of acute phase
reactants
● Alpha-1 = acid glycoprotein, alpha-1-antitrypsin
● Alpha-2 = ceruloplasmin & haptoglobin
● Beta = C reactive protein
● Commonly seen in trauma, burns, infarction, malignancy,
and liver disease
● Why acute phase reactants?
○ Bcs they are increased mainly in the serum
within a few days following trauma or exposure
to inflammatory reagents
○ Fibrinogen, haptoglobin, ceruloplasmin, serum
amyloid a = will increase several folds
○ C reactive protein & alpha-2 macroglobulin will
increase several hundred folds
○ Good indicators of the presence of
inflammation
● If gamma is increased = chronic infection
Cirrhosis
● Distinct bcs of the presence of beta gamma bridge
○ Caused by the presence of fast moving gamma
globulins in the px sample that prevents distinct
bands from appearing between the gamma and
beta globulin fractions therefore, forming a
bridge between two
○ Continuous pattern
■ Caused by fast moving
High Resolution Protein Electrophoresis
● Modifying electrophoretic parameters (12 bands)
● Uses higher voltage coupled with a cooling system and
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 more conc. buffer.
● Agarose gel
○ support medium
● semiquantitative estimates of protein
○ Densitometer
● useful in detecting small monoclonal bands and
differentiating unusual bands
*

PROTEINS IN OTHER BODY FLUIDS

Urinary Protein
● Can originate from the kidneys and the urinary tract, lso
vagina and the prostate
● Proteinuria
○ resulting from glomerular or tubular dysfunction
○ Useful indicator for problems of glomerular or
tubular portions of the kidney
● Qualitative test: Reagent test strip
○ Dip the strip in the urine
○ Observe for color
○ Protein error of indicators
● Quantitative test: 12-24 hour urine specimen
○ Results are generally reported in terms of
weight of protein for 24hrs by calculating the
amount of protein that is present in the total
volume of the urine collected during that time
● Precipitation methods: Sulfosalicylic Acid (SSA),
Trichloroacetic acid (TCA), Benzethonium chloride
● Chemical Methods: Biuret Method, Folin-Lowry Method
○ Biuret agent is mainly with the principle of the
complex formed by the cupric ions of the biuret
reagent with the peptide bonds which gives off
a pink to reddish-violet color
○ Folin-Lowry Method, it is going to use
Folin-Ciocalteu′s reagent which is a
phosphotungstomolybdic acid solution or
frequently called as phenol reagent because it
is able to oxidize phenolic compounds
■ The reagent will change in color from
Prealbumin - fastest to migrate yellow to blue during the reaction with
the following amino acids mainly
Other Methods: tyrosine, tryptophan, and histidine that
● Capillary Electrophoresis is present in the urine sample
○ separation of molecules takes place in silica ● Dye-binding methods: Coomasie blue, Ponceau S
capillaries
○ capillaries are typically 30-50 cm long Urine Protein Methods
○ Amount of sample is nm
● Isoelectric Focusing
Method Principle Comment
○ zone electrophoresis that separates proteins on
the basis of pI
○ When a protein is electrophoresed in the gel, it Turbidimetric Proteins are Rapid, easy to
migrates to a place in the gel where the pH is methods precipitated as fine use; (problem)
the same as its isoelectric point (sulfosalicylic acid, particles, turbidity is unequal
● Immunochemical Methods trichloroacetic acid, measured sensitivity for
○ specific proteins may be identified by or benzethonium spectrophotometrically individual person
immunochemical assays in which the reaction chloride)
of protein (antigen) and antibody is measured
○ radial immunodiffusion, immunoelectrophoresis, Biuret Proteins are Accurate
immunofixation electrophoresis, electro concentrated by
immunodiffusion, immunoturbidimetry, precipitation,
immunonephelometry redissolved in alkali,
○ Protein serves as the antigen (same with then reacted with Cu2+,
racket) Cu2+ forms colored
complex with peptide
bonds

Folin-Lowry Initial biuret reaction; Very sensitive

oxidation of tyrosine,

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 ● CSF is one of the most carefully handled
tryptophan, and samples in the lab because of the difficulty of its
histidine residues by extraction
Folin phenol reagent ● Means of collecting: lumbar of spinal tap
(mixture of ● The doctor will do the collection by inserting a
phosphotungstic and syringe between lumbar 4 and 5 vertebra
phosphomolybdic 1. Lie on table in fetal position
acids); measurement of 2. Doctor injects anesthetic to numb lower back
resultant blue color 3. Needle inserted between lower back bones
4. Small amount of cerebrospinal fluid drawn
Dye-binding Protein binds to dye, Limited linearity;
(Coomassie blue, causes shift in unequal
Ponceau S) absorption maximum sensitivity for
individual
proteins

CSF Protein
● The presence of proteins in the CSF is indicative of
infection, inflammation, or any other types of nervous
system disorder
○ Also, it can indicate a problem in the
capillary-endothelial barrier through which
ultrafiltration occurs
● Reference interval (10-40 years old)
○ 15-45 mg/dL
● Abnormal increased of total CSF Proteins
○ Conditions will include bacterial ,viral and
fungal meningitis, traumatic tap during CSF
collection, multiple sclerosis, obstruction in the ● The CSF must be separated into three tubes
neoplasm, and cerebral infarction ○ Tube 1: chemistry / serology
● Degree of permeability can be evaluated by measuring ○ Tube 2: microbiology
the CSF albumin comparing it with the serum albumin ○ Tue 3: hematology
○ Albumin is a very good indicator of nervous ○ The excess CSF must be kept in another tube
system problems because it is not produced in just in case there are additional tests to be
the CSF , but in the liver performed in the sample
○ In the event that albumin is higher in CSF than ● The accepted sample must appear clear
serum albumin, that is indicating increased ● If there is presence of blood, hemolysis, xanthochromia
permeability of capillaries or a problem in the because of the presence of bilirubin - it must be rejected
blood-brain barrier ● But if after centrifugation, you have a CSF with clear
supernatant and formation of red cell button in the bottom
- can still be accepted for processing

What to expect during a Spinal Tap