PROTEINS Flashcards
Bonds between amino acids
Peptide bonds = covalent bonds
Ang chemical reaction from aa -> proteins muragg
Esterification
Naay water na end product
are Complex composed of macromolecules amino acids linked by_____ bonds in a head to tail fashion
Proteins
peptide
Proteins are synthesized in the
Liver
Proteins
Net charge and electrophoretic mobility are determined by the_______ amino acid monomers
acidic and basic
p1
6.8
↑H+
Protein becomes
increasingly +ve
↑OH-
Protein becomes
increasingly -ve
molecule that contains an equal number of positively and negatively charged functional groups.
Zwitterion
structure sequence of a chain of animo acids
Primary protein
structure hydrogen bonding of the peptide backbone causes the amino acids to fold into a repeating pattern
Secondary protein
structure three-dimensional folding pattern of a protein due to side chain interactions
Tertiary protein
structure protein consisting of more than one amino acid chain
Quaternary protein
Structure of Hemoglobin
Quaternary
TOTAL PROTEIN SPECIMEN
_______(sample of choice)
_______
_______may interfere
_______- ↑ Total Protein
SERUM
Need not be fasting
Lipemia
Hemolysis
If hemolysis_______then reject
≥ 200 mg/dl
Digestion of protein; measurement of nitrogen content
Kjeldahl
Reference method;
assume average nitrogen content of 16%
Kjeldahl
Formation of violet-colored chelate between Cu2+ ions and peptide bonds
Biuret
Routine method;
requires at least two peptide bonds and an alkaline medium
Biuret
Protein binds to dye and causes a spectral shift in the absorbance maximum of the dye
Dye binding
Research use
Dye binding
is a rapid, simple method, but is not commonly used for total protein analysis
Refractometry
TOTAL PROTEIN: METHODS
• Classic method (determines______)
• Not used in_____
• Average of_____ mass in protein = protein concentration
KJELDAHL METHOD
Nitrogen
clinical laboratory
16% nitrogen
3 steps in kjeldahl method
Digestion
Neutralization and Distillation
Titration
TOTAL PROTEIN: METHODS
KJELDAHL METHOD
Step 1: DIGESTION
• The serum proteins are precipitated with an organic acid such as _____ or _____
• The nonprotein nitrogen is removed with the supernatant.
Trichloroacetic acid (TCA) or tungstic acid.
TOTAL PROTEIN: METHODS
KJELDAHL METHOD
(2) Neutralization & Distillation
The_____ oxidizes the C, H, and S in protein to CO2, CO, H2O, and SO2
H2SO4
TOTAL PROTEIN: METHODS
KJELDAHL METHOD
The protein pellet is digested in:
•________ in _______
• Catalyst =_______ (to hasten the reaction)
•________= 1 boiling point; improve the efficiency of digestion.
Sulfuric acid (H2SO4) with heat (340°C to 360°C)
Cupric sulfate
Potassium sulfate
TOTAL PROTEIN: METHODS
KJELDAHL METHOD
(2) Neutralization & Distillation
• The nitrogen in the protein is converted to__________, which is then measured by adding alkali and distilling the ammonia into a standard _______ solution.
ammonium bisulfite (NH4HS04)
boric acid
TOTAL PROTEIN: METHODS
KJELDAHL METHOD
(3) Titration
• The ________ formed is then titrated with a standard solution of____ to determine the amount of nitrogen in the original protein solution.
ammonium borate (NH4H2BO3)
HCI
TOTAL PROTEIN: METHODS
Most widely used method
BIURET METHOD
TOTAL PROTEIN: METHODS
• Recommended by____ for determination of Total Protein
• Cupric ions complex with the groups involved in the_______.
• Alkaline medium and at least 2 peptide bonds = a_______ formed
BIURET METHOD
IFCC - The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC)
peptide bond
violet-colored chelate
BIURET method
Copper (II) - color???
Peptide-copper complex - color???
Blue
Deep purple
BIURET REAGENT
• _______- to provide an alkaline medium so that the reaction can take place
• Contains _______ to complex cupric ions
•_______ as antioxidant
Potassium hydroxide
sodium potassium tartrate
Potassium iodide
Biurets method
• Absorbance is measured at_____
• When small peptides react, the color of the chelate produced has a different shade than that seen with larger peptide
540 nm
TOTAL PROTEIN: METHODS
BIURET METHOD
• Color varies from____ to ____
• Color formed is proportional to the number of _____ and reflects the____
pink to a reddish violet
peptide bonds
Total Protein level
TOTAL PROTEIN: METHODS
BIURET METHOD
• In Multiple Myeloma,____\ conc. is underestimated due to lighter shade of color produced.
•_____ in the sample is an interferent.
C-protein
Lipemia
TOTAL PROTEIN: METHODS
• Based on the ability of most proteins in serum to bind dyes
DYE-BINDING
TOTAL PROTEIN: METHODS
DYE-BINDING
• Used to stain protein bands after electrophoresis:
• Bromophenol blue
• Ponceau S
• Amido black 10B
• Lissamine green
• Coomassie brilliant blue
TOTAL PROTEIN: METHODS
DYE-BINDING
•________ relies on the binding to protein
• A shift absorbance maximum of the dye from______
• Absorbance at_____ = protein concentration
Coomassie brilliant blue 250
465 to 595 nm
595 nm
TOTAL PROTEIN: METHODS
• a quick alternative to chemical analysis of serum total protein when a rapid estimate is required
• Measurement of____ due to solutes in serum.
REFRACTOMETRY
Refractive Index
