Protein Detection and Quantitation (BIOC3570 F24 02) PDF

# Protein detection and quantitation (1) ## Colorimetric Assays ### Protein quantitation: applications - Quantification of protein in foods (protein content is noted on all food labels) - Medical diagnosis: e.g., measurement of total protein in urine is important for diagnosing kidney dysfunction...

# Protein detection and quantitation (1) ## Colorimetric Assays ### Protein quantitation: applications - Quantification of protein in foods (protein content is noted on all food labels) - Medical diagnosis: e.g., measurement of total protein in urine is important for diagnosing kidney dysfunction (proteinuria) - Protein therapeutics need exact quantitation of dosage, e.g., insulin, HGH, therapeutic antibodies. - Biochemistry/molecular biology reagents. - Protein biochemistry applications require accurate protein quantitation (e.g., activity measurements are proportional to protein quantity) ### Protein quantitation: Analytical challenges - Proteins are challenging reagents to quantify because: - Each protein has a unique amino acid composition. - Unless the detection method is equally sensitive to all residues, quantitation will be inaccurate unless corrected for composition. - Non-peptide components (e.g., glycosylation, co-factors) can contribute to protein mass (and possibly signal). - Proteins can show microheterogeneity (e.g., apo- vs holoproteins, splice variants, alleles, variable glycosylation). - Highly purified reference materials are generally not available. - Diverse excipients are usually present in the same solution - e.g., buffers, detergents, reducing agents, salts etc. ### Deliberate, criminal efforts to subvert analysis - The 2008 Chinese milk scandal was a food safety incident in China. - The assays that test protein levels in food products essentially measure organic nitrogen. - Milk and infant formula can be made cheaper by removing protein, so this is monitored. - Some "enterprising" Chinese companies started adding melamine to baby formula and milk to add back missing nitrogen. - Melamine causes kidney stones and other kidney damage. - China reported an estimated 300,000 victims - An estimated 54,000 babies were hospitalized, and 11 infants died. ### Protein quantification - common techniques - Total nitrogen content. - Gravimetry. - Hydrolysis -> amino acid analysis. - UV spectrophotometry (A280). - Colorimetric assays: - BCA. - Bradford. ### Bulk methods for food analysis - Estimate protein content of complex biological materials ### Research labs - More accurate protein amount from pure samples. ### Protein quantitation by UV absorbance (A280) - Some amino acids absorb light at 11 = 280 nm, this can be used to quantify proteins. - However, many other analytes (e.g., DNA) absorb UV light, causing potential false positive signals. - UV quantitation is only applicable to relatively pure protein samples. - You need to know the protein sequence to predict the absorbance accuracy. - However, under the right conditions, UV absorbance can achieve **much better accuracy** than the Bradford or BCA assays. - We will discuss this method later in the course as part of UV-vis spectroscopy ### Chromogenic assays in biochemistry - Many analytes of interest (including polypeptides and most proteins) do not absorb visible light. - However, we can exploit chemical reactions that will generate substances that do. - These are called "colorimetric" or "chromogenic" assays. - Most of the common protein assays work this way. ### Colorimetric Chromogenic assays in biochemistry - Colorimetric assays have also been developed for many other classes of biomolecules, e.g.: | Analyte | Reagent | |---|---| | Amine groups (Lys, N-term) | ninhydrin | | DNA | diphenylamine | | RNA | Bial's reagent | | Thiols (cysteine, CoA) | Ellman's reagent | | Glucose | glucose oxidase/peroxidase system | ### Triarylmethane Dyes - Triaryl methane dyes share a common core structure. - These molecules have an intense blue or purple colour. - Gram stain which labels the peptidoglycan of bacteria is an important reagent in this molecular class. ### Triaryl methane dyes: colour shifts - These molecules have a triphenyl methane group with a ternary amine at the p-position. - The amines will accept protonation in a pH-dependent manner. - The change in the electronic structure causes a color change. - The non-polar triphenyl readily binds non-polar surfaces. - Importantly, the pKa is shifted in a non-polar environment. ### Coomassie Blue (synthetic triarylmethane dye) - Coomassie is a modified version of the basic triarylmethane structure (note the additional sulfophenyl and phenolic groups). - Originally, Coomassie blue waS invented as a wool dye (keratin is a protein). - Biochemists found it worked well as a protein stain (see electrophoresis), and later adopted it for protein quantitation. ### Coomassie binds the hydrophobic interior of acid unfolded proteins. - The protein should have been unfolded due to excess acid/increase in pH as Coomassie needs to be protonated. - **Protonation**: Phosphoric acid protonates free coomassie which is then red-brown in colour. Acid denatures the protein. - **Denaturation**: Coomassie binds in the exposed, hydrophobic protein interior. pKa is shifted towards neutral. Coomassie loses proton(s). Protein bound coomassie is blue-purple. ### Bradford dye-binding assay - Coomassie Blue has two SO groups, making it anionic. - Under acidic conditions (8.5% phosphoric acid), dye binds to protein (especially to basic and aromatic a.a. residues). - Note: the protein is acid denatured under these conditions. - Binding shifts the protonation equilibrium of dye towards less-protonated forms (protein environment shifts the pKa). - This shifts the colour from red-brown to blue. - The degree of colour shift is directly proportional to the amount of protein. ### Bradford assay: running the assay - A dilution series of a standard protein (often bovine serum albumin) of known concentration is prepared. - These standard samples, as well as the unknown protein samples are then mixed with the assay reagent. - After incubation, the absorbance is measured using a spectrophotometer. - Absorbance is typically measured at 595 nm, the peak absorbance of the anionic (blue) species. - The standard samples are used to prepare a standard curve. - Sample protein concentrations are determined by comparing their assay responses to the standard curve. ### Protein assays: Data - Net absorbance is plotted against protein concentration for the protein standard (here BSA). - Protein concentration for the sample is read by reading the BSA concentration that gives the measured absorbance. - Note that the fit (red line) is only linear to ~700 g/ml. ### Assays: Dynamic - Any assay can be applied only within a certain dynamic range. - Test several dilutions of your sample. - Rely only on the results that are within the linear dynamic range of the assay. ### BSA: experimental accuracy and precision - Bradford assays are quite reproducible with good technique. - However, the Bradford assay response varies a great deal with different proteins. - Bradford is more sensitive to hydrophobic and basic residues. - Since you do not know a priori how a specific protein will respond this reduces the accuracy of the assay. - The accuracy of the Bradford assay is only ~± 20%. ### Bradford assay - limitations and uses - The Bradford assay is compatible with most common biochemical reagents - e.g., salts, chelators, reducing agents. - However, detergents will also bind the Coomassie dye, and give a false positive result. - The accuracy of this assay is also somewhat limited, and the dynamic range not great. - However, it is quick, cheap and easy, needs only standard lab equipment (spectrophotometer) and works well for measuring total protein concentrations (e.g., during a purification). ### BCA assay - BCA reagent solution contains BCA and CuSO4 at pH 11.25. - **Step 1**: protein reduces Cu2+ to Cu+. - **Step 2**: selective detection of Cu⁺ by BCA reagent results in a purple color. ### BCA assay - chemistry - Cysteine, Tyrosine and Tryptophan side chains react with copper, reducing it. - Copper is also reduced by the Biuret reaction with the peptide backbone. - The assay therefore detects all proteins, but is more sensitive to Tyr, Trp and Cys -rich proteins. ### The Chelate effect - In chelation a single ligand ligates a metal ion at multiple positions simultaneously. - Ligands pay an entropic cost (loss of conformational freedom) when binding a metal. - **Keq** is 109-fold more favourable for formation of the chelate complex, so a second ligand within the same complex is much lower entropic cost. More binding sites and a more rigid chelator enhance this effect for even tighter binding. ### Chelates (examples in biochemistry) - In nature: heme: porphyrin-iron chelate. - In the lab: EDTA: transition-metal scavenger. ### Bicinchoninic acid (BCA): - BCA: a sensitive, stable, and highly specific chelating ligand for Cu+. - **Cu++:** dº; prefers 5- or 6-coordinate square-planar geometry. - **Cu+: ** d¹º; prefers 4-coordinate tetrahedral geometry. ### BCA assay procedure - The basic procedure of the BCA assay is similar to the Bradford assay. - The reactions need to be heated as the Biuret reaction is inefficient at room temperature. - Heating to 60°C instead of 37°C will increase colour development roughly 8-fold - so much more sensitive. - Absorbance is measured at 562 nm (the colour is different than the Bradford assay). ### BCA assay - limitations and uses - The BCA assay is fast, reproducible, inexpensive and relies only on standard lab equipment. - **Reducing agents** will reduce Cu+, giving a false positive signal. - **Chelators** (e.g., EDTA) will compete with BCA for Cu+, weakening the signal (false negative). - Most other standard reagents, including detergents are compatible. - Because of the composition dependence, the BCA assay shows protein-to-protein variation, but less so than the Bradford assay. ### Colorimetric protein assays: summary - **Principle**: Protein reacts with reagents to give coloured complex. - **Application**: Quantitation of protein content in sample. **Strengths:** - **Little instrumentation required** (plate reader or spec). - **Good for comparing similar batches**, e.g., lysates of cells treated in an experiment. - **E.g., normalizing inputs for gels or western blots**. **Weaknesses:** - **Indirect assay**: absolute quantitation is poor (so not ideal if you need absolute enzyme concentration to figure out a kcat). - **Subject** to interferences from reagents. - **Limited dynamic range**.