M1: Specific Protein I Flashcards
(79 cards)
0
Q
Make up 15% of the cell. Function as enzyme, transporter, structural support, motor function, storage, signaling(pathway receptors), receptors, gene regulation and other special functions.
A
Protein
1
Q
Specific Proteins
A
Globulin, Albumin & Creatinine “GAC”
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Q
Shape of proteins are from
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AA sequence
3
Q
Transporters
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Apolipoproteins
4
Q
All enzymes are protein but not all proteins are enzyme. T/F
A
False
5
Q
Proteins are made of _____ AAs linked by __________, polypeptide backbone. Consist of repeating sequence of AA. R group/side group/side chain not part of the backbone.
A
- Peptide bonds.
6
Q
Polar
A
Hydrophilic
7
Q
Nonpolar
A
Hydrophobic
8
Q
Determining factor as to chemical nature of proteins
A
Side chain
9
Q
Peptide bind allow for rotation around it
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Protein folding
10
Q
Protein can fold and orient the _______ in favorable positions linked by weak non covalent interactions.
A
R groups
11
Q
Weak non covalent interactions
A
Hydrogen bond, Ionic bond & Van der Waals attraction “HIV”
12
Q
Side chain/R groups help determine conformation in aqueous solution
A
Globular proteins
13
Q
Protein shape determined by
A
AA sequence
14
Q
Final shape. Has the lowest free energy possible
A
Conformation
15
Q
Process of unfolding the protein. Can be done with heat, PH& chemical compound.
A
Denaturation
16
Q
Folds back on itself as a ribbon (globular protein-muscle)
A
B-pleated sheet
17
Q
Type of B pleated: run in the same direction
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Parallel
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Type of B pleated: runs in the opposite direction of its neighbor
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Antiparallel
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Q
Protein turns like spiral. Fibrous proteins (hairs, nails & horns)
A
A-helix
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Q
Non covalent interaction. R groups within protein.
A
Tertiary structure
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Q
AA sequence of protein
A
Primary structure
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Q
Two polypeptides
A
Quaternary structure
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Q
Hydrogen bond in peptide chain backbone
A
Secondary structure
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Blood serum is placed into a gel or into liquid in a capillary tube, and exposed to am electric current to separate the serum protein components.
Electrophoresi
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In Electrophoresis, _____ serum protein fractions (at ____pH) were identified and quantified optically by change in _________. albumin, a, B & y.
Four. 7.6 Refractive index
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Separated proteins dissolved in an electrolyte solution by application of an electric current through a U-shaped quartz tube.
Tiselius
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Separation of the protein fractions into discrete bands or zones d/t the introduction of filter paper as an anticonvection support medium.
Zonal Electrophoresis
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In zonal electrophoresis, on solid support medium and at pH of _____, the a fraction further splits into two groups of proteins, a1 & a2.
8.6
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The buffer flow which carries the proteins with it by mechanical flow not by charge. Ex is Agar.
Endosmosis/Electro osmosis
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Electrophoresis Principle: are free to move under the electromotive force, and net result is flow of buffer toward the _________.
Positively charged ion. Cathode.
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Electrophoresis Principle: the electromotive force to which it is subjected tends to move it toward the _______.
Negatively charged ion. Anode.
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Determines the amount of current and movement of the proteins for a fixed voltage. If the electrodes are not properly aligned, the current may be denser on one side of the gel than the other; proteins will migrate farther on the side with more current.
Ionic Strength of the Buffer
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Ionic Strength of the Buffer: If ____, less current is carried by the proteins, which move a shorter distance.
High
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Ionic Strength of the Buffer: If _____, relatively more current is carried by the charged proteins.
Low
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Rate of migration factors: _______ on the particle.
Net charge
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Rate of migration factors: ______ & ______ of the particles.
Mass. Shape.
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Rate of migration factors: ____ & ____ of the medium
pH & Temperature
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Rate of migration factors: ______ of the electric field(buffer system)
Strength
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Rate of migration factors: _______ of supporting medium
Properties
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Support media
Polyacrylamide gel, Agarose gel, Cellulose acetate membrane & Starch gel "PACS"
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Have predominated in the clinical laboratory because of the ease of use, low cost and commercial availability.
Cellulose acetate & Agarose
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Have been devised to resolve albumin and the globulins into two or more fractions that can then be measured for protein content. Measure total proteins in the original serum & protein in the precipitate or the supernatant, values for albumin & globulin can be derived.
Precipitation
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Globulins can precipitate through the addition of
Methanol, Aluminum Sulfate, Sodium Sulfite & Sodium Sulfate "MASS"
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Precipitation: the ration of these values (______) has been used extensively because it accentuates abnormalities in serum protein composition, which generally involve depression of _______ and more elevation of _______ fractions.
A/G ration. Albumin. Globulin.
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May be depressed owing to decrease synthesis (malnutrition, malabsorption, liver failure, diversion of synthesis to other proteins) and increased loss (proteinuria, accumulation of ascites fluid & enteropathy)
Albumin
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Precipitation methods are not as accurate as zonal electrophoresis, because some _________ may fail to precipitate, thus leading to an overestimate of the albumin fraction.
Alpha globulins
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Simplest & mildest of all chromatography (column separation) Separates molecules based in difference in size.
Gel Filtration
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In Gel Filtration, Particles the size of dissolved salt penetrate farthest into the anterior to the GF beads and come out after all the proteins have emerged in amount of applied buffer called ________.
Salt volume
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Gel Filtration: _______ doesn't enter gel pores and elute rapidly from the column in the void volume.
Large proteins
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Gel Filtration: _________ repeatedly enter and leave pores of the gel thus remain longer in the column.
Smaller proteins
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Gel Filtration: All protein species continuously move through a gel filtration column all at the same ______ but at different ______.
Time. Rates.
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Gel Filtration: A sample of proteins passing through a gel filtration column will separate based on _________. The big ones will elute ____ and smallest one will elute _____. And middle sized in the middle.
Molecular size. First. Last.
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Based on the reversible interaction between a charged protein and an oppositely charged chromatography medium. Retains analyte molecules on the column based on coulombic(ionic) interactions. Advantage is that biomolecules with even small differences in net surface charge can be separated.
Ion Exchange Chromatography
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Ion Exchange Chromatography: displays ionic functional groups interact with analyte ions of opposite charge. Further subdivided into cation exchange and anion exchange chromatography.
Stationary phase surface
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Keep positively charged cations and shows negatively charged
Cation Exchange Chromatography
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Proteins are usually applied at a basic pH(8.6) Either negatively charged. Neutral proteins pass through an anion exchange column. Anionic proteins stick to the (+) charged column matrix.
Anion Exchange Chromatography
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Anion Exchange Chromatography: are considered as anions
Albumin, A1, A2 & B globulins
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Anion Exchange Chromatography: No net charge
Y globulins
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Samples are applied at high salt; eluted with low salt. The support medium interacts with proteins with a hydrophobic nature.
Hydrophobic Chromatography
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Based on specific binding between a protein of interest and another protein that has been covalently linked to the solid support medium of a column.
Affinity Chromatography
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The most common use affinity chromatography is for the
Purification of recombinant proteins
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Based on a flow through a capillary tube. Can be tailored to resolution of different molecules based on size, hydrophobicity or stereospecificity. Applicable to large & small molecules. Similar to HPLC. Employs column with similar agarose. A detector at the effluent and detects & quantities protein bands. Equipment costs relatively high, reagent labor low costs, fast and very quantitative.
Capillary Electrophoresis
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Solvent is pumped through a column that retains/passes solutes according to chemical interactions
High Performance Liquid Chromatography (HPLC)
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Very basic. Ultimate reference method for determining concentration of proteins. Has the basic principle.
Nitrogen Analysis
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Acid digestion release of ammonium ions. Forms a nitrogen containing compound. Highly accurate but not commonly used in the lab.
Kjeldahl Technique
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Wherein double iodides (potassium & mercuric) forms a colored complex with ammonia in an alkali medium
Nesslerization
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Kjeldahl Technique: ammonium is quantitated by conversion into _________ and titration as a base or by nesslerization.
Ammonia gas
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Used to estimate specific gravity and by inference protein as well. Pipette drops of serum/blood into a graded series of this.
Copper sulfate solution
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Protein concentration is estimated from the __________ of the copper sulfate solution in which the drop remains stationary.
Specific gravity
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Forms to prevent dissolution as a drop falls to the bottom and remains stationary or rises to the top
Copper sulfate capsule
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Protein in a solution absorbs ultraviolet light at 280nm
Absorbance
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Measurement of turbidity. Protein forms a precipitate on the addition of ______________. Not specific for protein. Could be used to measure nucleic acids as well.
Turbidimetric Methods. Trichloroacetic/Sulfosalycilic Acids.
73
Highly specific for proteins and peptide. Biuret method. Copper salts in alkaline solution forms a purple complex with substances containing 2 or more peptide bonds. Extensively used among automated laboratories. Ex is chemistry analyzer.
Colorimetric Method
74
Used the Biuret method plus phenol reagent equals greatly enhanced color formation. Used for consistently accurate determination of protein concentration.
Lowry Assay
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Reacts with the Biuret complexes involving all peptide bonds
Phenol
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Standard dyes for Electrophoresis
Ponceau S, Amido black & Coomasie Brilliant Blue "PAC"
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Detects turbidity produced by precipitation of a reagent antibody with its target protein in serum sample. Method used for measuring major serum proteins in automated immunochemical analyzers.
Nephelometry
78
Immunologic method that detects proteins in lower concentration
Radioimmunoassay(RAI) & Enzyme-linked Immunosorbent Assay(ELISA)
