M1: Specific Protein I Flashcards
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.
Protein
Specific Proteins
Globulin, Albumin & Creatinine “GAC”
Shape of proteins are from
AA sequence
Transporters
Apolipoproteins
All enzymes are protein but not all proteins are enzyme. T/F
False
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.
- Peptide bonds.
Polar
Hydrophilic
Nonpolar
Hydrophobic
Determining factor as to chemical nature of proteins
Side chain
Peptide bind allow for rotation around it
Protein folding
Protein can fold and orient the _______ in favorable positions linked by weak non covalent interactions.
R groups
Weak non covalent interactions
Hydrogen bond, Ionic bond & Van der Waals attraction “HIV”
Side chain/R groups help determine conformation in aqueous solution
Globular proteins
Protein shape determined by
AA sequence
Final shape. Has the lowest free energy possible
Conformation
Process of unfolding the protein. Can be done with heat, PH& chemical compound.
Denaturation
Folds back on itself as a ribbon (globular protein-muscle)
B-pleated sheet
Type of B pleated: run in the same direction
Parallel
Type of B pleated: runs in the opposite direction of its neighbor
Antiparallel
Protein turns like spiral. Fibrous proteins (hairs, nails & horns)
A-helix
Non covalent interaction. R groups within protein.
Tertiary structure
AA sequence of protein
Primary structure
Two polypeptides
Quaternary structure
Hydrogen bond in peptide chain backbone
Secondary structure
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
In Electrophoresis, _____ serum protein fractions (at ____pH) were identified and quantified optically by change in _________. albumin, a, B & y.
Four. 7.6 Refractive index
Separated proteins dissolved in an electrolyte solution by application of an electric current through a U-shaped quartz tube.
Tiselius
Separation of the protein fractions into discrete bands or zones d/t the introduction of filter paper as an anticonvection support medium.
Zonal Electrophoresis
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
The buffer flow which carries the proteins with it by mechanical flow not by charge. Ex is Agar.
Endosmosis/Electro osmosis
Electrophoresis Principle: are free to move under the electromotive force, and net result is flow of buffer toward the _________.
Positively charged ion. Cathode.
Electrophoresis Principle: the electromotive force to which it is subjected tends to move it toward the _______.
Negatively charged ion. Anode.
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
Ionic Strength of the Buffer: If ____, less current is carried by the proteins, which move a shorter distance.
High
Ionic Strength of the Buffer: If _____, relatively more current is carried by the charged proteins.
Low
Rate of migration factors: _______ on the particle.
Net charge
Rate of migration factors: ______ & ______ of the particles.
Mass. Shape.
Rate of migration factors: ____ & ____ of the medium
pH & Temperature
Rate of migration factors: ______ of the electric field(buffer system)
Strength
Rate of migration factors: _______ of supporting medium
Properties
Support media
Polyacrylamide gel, Agarose gel, Cellulose acetate membrane & Starch gel “PACS”
Have predominated in the clinical laboratory because of the ease of use, low cost and commercial availability.
Cellulose acetate & Agarose
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
Globulins can precipitate through the addition of
Methanol, Aluminum Sulfate, Sodium Sulfite & Sodium Sulfate “MASS”
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.
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
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
Simplest & mildest of all chromatography (column separation) Separates molecules based in difference in size.
Gel Filtration
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
Gel Filtration: _______ doesn’t enter gel pores and elute rapidly from the column in the void volume.
Large proteins
Gel Filtration: _________ repeatedly enter and leave pores of the gel thus remain longer in the column.
Smaller proteins
Gel Filtration: All protein species continuously move through a gel filtration column all at the same ______ but at different ______.
Time. Rates.
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.
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
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
Keep positively charged cations and shows negatively charged
Cation Exchange Chromatography
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
Anion Exchange Chromatography: are considered as anions
Albumin, A1, A2 & B globulins
Anion Exchange Chromatography: No net charge
Y globulins
Samples are applied at high salt; eluted with low salt. The support medium interacts with proteins with a hydrophobic nature.
Hydrophobic Chromatography
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
The most common use affinity chromatography is for the
Purification of recombinant proteins
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
Solvent is pumped through a column that retains/passes solutes according to chemical interactions
High Performance Liquid Chromatography (HPLC)
Very basic. Ultimate reference method for determining concentration of proteins. Has the basic principle.
Nitrogen Analysis
Acid digestion release of ammonium ions. Forms a nitrogen containing compound. Highly accurate but not commonly used in the lab.
Kjeldahl Technique
Wherein double iodides (potassium & mercuric) forms a colored complex with ammonia in an alkali medium
Nesslerization
Kjeldahl Technique: ammonium is quantitated by conversion into _________ and titration as a base or by nesslerization.
Ammonia gas
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
Protein concentration is estimated from the __________ of the copper sulfate solution in which the drop remains stationary.
Specific gravity
Forms to prevent dissolution as a drop falls to the bottom and remains stationary or rises to the top
Copper sulfate capsule
Protein in a solution absorbs ultraviolet light at 280nm
Absorbance
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.
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
Used the Biuret method plus phenol reagent equals greatly enhanced color formation. Used for consistently accurate determination of protein concentration.
Lowry Assay
Reacts with the Biuret complexes involving all peptide bonds
Phenol
Standard dyes for Electrophoresis
Ponceau S, Amido black & Coomasie Brilliant Blue “PAC”
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
Immunologic method that detects proteins in lower concentration
Radioimmunoassay(RAI) & Enzyme-linked Immunosorbent Assay(ELISA)
