Specific Proteins Flashcards
Separates protein according to their electrical charges (usually at pH 8.6)
Protein electrophoresis
Major proteins in plasma that contribute to the electrophoretic pattern are:
Albumin Alpha1 anti trypsin Alpha2 macro globulin Haptoglobin Beta lipoprotein Transferrin Complement C3 Fibrinogen Immunoglobulin
Plays several roles, including maintaining oncotic pressure, transporting small molecules and promoting or inhibiting inflammatory reactions
Plasma proteins
The major clinical of serum and urine protein electrophoresis is to screen for:
Monoclonal gammopathies
Methods to quantitate and fractionate proteins:
Turbidimetry Colorimetry Absorption spectrophotometry Dye binding Column chromatography Electrophoresis Immunoassays
Process of unfolding the protein (using heat, pH or chemical compounds)
Denaturation
The final shape of protein, has the lowest free energy possible
Conformation
Protein shape determined by amino acid sequence
Protein folding
Consist of repeating sequence of amino acids
Polypeptide backbone
Important numbers: Proteins
16% nitrogen which differentiates it from CHO and lipids
15% make up of the cell
20 amino acids linked by peptide bonds
Simple protein: symmetrical, compactly folded polypeptide chains (albumin)
Globular
Simple protein: elongated, asymmetrical polypeptide chains (troponin and collagen)
Fibrous
Conjugated proteins: with a metal prosthetic group
Metalloproteins
Conjugated proteins: lipid prosthetic group
Lipoprotein
Conjugated proteins: with 10-40% CHO attachment ( haptoglobin)
Glycoproteins
Conjugated proteins: with >40% CHO attached (mucin)
Mucoprotein
Conjugated proteins: with DNA or RNA attached (chromatin)
Nucleoproteins
The A.A are linked to each other through covalent peptide bonding in a specific sequence to form polypeptide chain
Primary Structure
Hydrogen bonds in peptide backbone; forms alpha and beta pleated sheets
Secondary structure
Non covalent interaction; coiled polypeptide chain folds upon itself to form a 3 dimensional structure
Tertiary structure
Two or more folded polypeptide chains bind to each other through hydrogen bonds and electrostatic interactions to form a functional protein
Quaternary structure
Separated proteins dissolved in an electrolyte solution by application of an electric current through u shaped quartz tube
Electrophoresis
Separation of the protein fractions into discrete bands or zones due to introduction of filter paper as an anti convection support medium
Zonal electrophoresis
The buffer flow which carries the proteins with mechanical flow, not by charge (agar)
Endosmosis/ electro-osmosis
Principles of endosmosis/ electro-osmosis
Positively charged ion - free to move under electromotive force - flow of buffer towards the CATHODE
Negatively charged ion - tends to move it towards the ANODE
Determines the amount of current and the movement of the proteins for a fixed voltage
Ionic strength of the buffer
Principles of Ionic strength of the buffer:
If low, more current is carried by the charged proteins
If high, less current is carried by the proeins, shorter distance reached
Support medium for ionic strength:
Cellulose acetate membrane
Agarose gel
Starch gel
Polyacrylamide gel
Devised to resolve albumin and globulins into 2 or more fractions that can be measured for protein content
Precipitation
Globulins precipate through addition of:
Na sulfate
Na sulfite
ammonium sulfate
Methanol
Used extensively as it accentuates abnormalities in serum protein composition, which involves depression of albumin and elevation of more globulin fractions
A/G ratio
Simplest and mildest of all chromatography techniques, separation is based on differences in size
Size exclusion chromatography / SEC
Principles of SEC:
Size matters: Large proteins will elute first and the smallest ones will elute last
All CHON species continuously move through gel filtration columns all at the same time but at different rates
Based on the reversible interaction between a charged protein and an oppositely charged chromatography medium
Ion exchanged chromatography (IEC)
Proteins are usually applied at basic pH 8.6 at which they may negatively charged or may have no net charge
Anion Exchange chromatography
Principles of anion exchange chromatography:
Neutral CHON pass through an anion exchange column
Anionic proteins stick to the (+) charged column matrix
Begins at aid pH, with proteins having positive charge and adhering to a negatively charged column matrix
Cation exchange chromatography
Samples are applied at high salt and are eluted with low salt. The support medium interacts with proteins with hydrophobic nature
Hydrophobic chromatography
Specific binding between a protein of interest and another protein that has been covalently linked to a solid support medium of column
Affinity chromatography
The most common use of affinity chromatography is for:
Purification of recombinant proteins
Based on the flow through a capillary tube that can be tailored to resolution of different molecules based on size, hydrophobicity or stereospecificity
Capillary electrophoresis
The ultimate reference method for determining concentration of proteins
Nitrogen analysis
Consists of acid digestion to release ammonium ions from nitrogen containing compounds
Kjeldahl technique ( the reference method)
In which double iodides (K and Hg) forms a colored complex with ammonia in an alkali medium
Nesslerization
Used to estimate specific gravity and by interference, protein content
Cooper sulfate solution
Protein in solution absorbs ultraviolet light at 280 nm
Absorbance
Detects turbidity produced by precipitation of a reagent antibody with its target protein in a serum sample
Nephelometry
Measures major serum proteins in automated immunochemical analyzers
Nephelometry
Immunologic method that detects proteins in lower concentration
radioimmunoassay (RIA) and ELISA
Standard dyes for electrophoresis:
Coomassie brilliant blue
Ponceau S
Amido black
Possible by virtue of the specific binding of albumin to certain dyes. Dyes bound to albumin absorb maximally at slightly different wavelengths
Quantification of albumin
Dyes used for quantitation of albumin:
Bromphenol blue Methyl orange Hydroxybenzenebenzoic acid Bromcresol purple Bromcresol green
Protein forms precipitate on the addition of trichoroacetic acid and sulfosalicylic acid but not specific to proteins
Turbidimetric methods
Highly specific for proteins and peptides that uses biuret method in which copper salts in alkaline solution forms a purple complex with substances containing 2 or more peptide bonds
Colorimetric technique
Oxidizes phenolic compounds so that a greater sensitivity can be obtained giving a deep blue color
Folin ciocalteu reagent
Uses biuret method plus phenol reagent to enhanced color formation
Lowey assay
