AMINO ACIDS AND PROTEINS Flashcards by Unknown Unknown

Classic method (determine nitrogen)

Kjeldahl Method

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The procedure is way too tedious for it to be a
part of routine activity

Kjeldahl Method

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Requires many reagents

Kjeldahl Method

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Time-consuming

Kjeldahl Method

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Average of ______ nitrogen as in proteins

Kjeldahl Method - 16%

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No proteins of significant concentration in the unknown
specimen are lost in the precipitation step which is the
first step of the method

Kjeldahl Method

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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

Kjeldahl Method

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_____________ is a rapid, simple method, but is not commonly used for total protein analysis.

Refractometry

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Digestion of protein; measurement of
nitrogen content

kjeldahl method

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Reference method; assume average nitrogen
content of 16%

Kjeldahl method

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Formation of violet-colored chelate between
Cu2+ ions and peptide bonds

Biuret

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Routine method; requires at least two peptide
bonds and an alkaline medium

Biuret method

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Protein binds to dye and causes a spectral
shift in the absorbance maximum of the dye

Dye binding

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Research use

Dye binding

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Research use

Dye binding

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Will occur if you mix the sample with sulfuric acid and
heat between 340°C - 360°C

Digestion

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In neutralization, your ammonium bisulfite was highly
exposed to an acid which is sulfuric acid which needs to
be neutralized before proceeding to distillation

Neutralization and Distillation

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Sulfuric acid will oxidize the __________
in the protein to become CO2, CO, H2O, SO2

Carbon, hydrogen, sulfur

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Nitrogen in the protein will be converted to become your
___________ which is then utilized for
neutralization and distillation

ammonium bisulfite

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The neutralizing agent is by the addition of an _________

alkaline,
sodium hydroxide - Neutralization and distillation

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Once it is neutralized, proceed with distillation into a
___________

standard boric acid solution

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From ammonium bisulfite, because of the presence of
your boric acid, it now becomes your ________

Ammonium borate - neutralization and distillation

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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

titration

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Most widely used method

Biuret Method

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Recommended by IFCC for determination of Total Protein

Biuret method

There will be a violet-colored soln if there are at least two peptide bonds detected in the sample

Biuret method

Provides an alkali medium so the rxn can take place

Potassium hydroxide

Complex cupric ions to prevent their precipitation in the alkaline soln

Sodium potassium tartrate

Acts as antioxidant

Potassium iodide

Kjeldahl color

Red-green-pink

biuret method

Violet

Dye binding

Blue

Absorbance is measured at 540 nm

biuret method

Also determines the size of the protein particles that is present on the samples

Biuret method

When small peptides react, the color of the chelate produced has a different shade than that seen with larger peptide

Biuret method

Alkaline medium and at least 2 peptide bonds, a violet-colored chelate formed

Biuret method

Color varies from pink to a reddish violet

Biuret method

Color formed is proportional to the number of peptide

Biuret method

Color pink in Biuret method inidcates

Lower tp level

Color reddish violet in biuret method inidcates

Higher tp level

In (abnormally small proteins) multiple myeloma, c-protein concentration is underestimated due to lighter shade of color produced

Biuret method

Smaller peptides could produce _______ compared to samples with larger protein

lighter shade - biuret method

________in the sample is interferent

Lipemia - Biuret method

__________ content must be rejected as it will interfere to the procedure

High Lipid - Biuret method

Ability of most proteins in serum to bind dyes

Dye binding

Most common dye utilized: _________ relies on the binding to protein

Coomassie brilliant blue 250

A shift absorbance maximum of the dye from _____ nm

465-595 nm dye binding

Absorbance at ______ nm is used to determine the protein concentration

595 nm dye binding

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

Refractive Index (RI)

Methods for Fractionation, Identification and Quantification of Specific Proteins

● Salt fractionation ● Dye-binding ● Serum protein electrophoresis

Fractionation of proteins is commonly done using the precipitation technique

Salt Fractionation

- precipitate

Globulin

supernatant fluid (measured)

Albumin

Measured thru routine total protein technique.

Biuret test Dye binding Method

most widely used methods for determining Albumin.

Dye binding procedures

__________ is attracted to and binds to an ionic dye

Albumin

(calculated by) absorbance of the albumin-dye complex

M2BB ■ Methyl orange ■ 2,4’-hydroxyazobenzene-benzoic acid (HABA) ■ Bromocresol Green (BCG) ■ Bromocresol Purple (BCP)

nonspecific for albumin ● Beta-lipoproteins, alpha-1 and alpha-2 globulins also bind to dye

Methyl Orange

low sensitivity but more specific for albumin salicylates, penicillin, conjugated bilirubin, and sulfonamides ○ interfere albumin to HABA

2,4’-hydroxyazobenzene-benzoic acid (HABA)

Not affected by any interfering substances that can be present in the sample such as bilirubin and salicylates.

Bromocresol Green (Bcg)

alternate dye, binding specifically to albumin

Bromocresol Purple (Bcp)

not subject to interferences, precise, exhibits excellent correlation with immunodiffusion reference methods

Bromocresol Purple (Bcp)

not subject to interferences, precise, exhibits excellent correlation with immunodiffusion reference methods

Bromocresol Purple (Bcp)

hemoglobin binds to BCG ○ for every ______ mg/dL of Hgb, albumin increased by ______ g/dL

100 mg/dl - 0.1 g/dl

has been reported to overestimate low albumin values

nephrotic syndrome or end-stage renal disease - BCG

Disadvantage: In renal insufficiency, BCP method underestimates serum albumin

Bromocresol Purple (Bcp)

Cations →

cathode terminal (-)

Anions →

anode terminal (+)

the speed of migration can be estimated from the difference between the pIof the protein and the pH of the buffer.

Electrophoresis

Cellulose acetate or agarose gel-

support media - Electrophoresis

AMINO ACIDS AND PROTEINS Flashcards

(70 cards)