Colorimetric methods proteins, concentration

Calculate the protein concentration in the final preparation using its absorbance at 280nm or a colorimetric method, such as the Coomassie assay. (Note The presence of hydrazine or hydrazide groups on the protein will interfere with the BCA assay for total protein concentration.)... 

If analytical methods are at the heart of biopharmaceutical development and manufacturing, then protein concentration methods are the workhorse assays. A time and motion study of the discovery, development, and manufacture of a protein-based product would probably confirm the most frequently performed assay to be protein concentration. In the 1940s Oliver H. Lowry developed the Lowry method while attempting to detect miniscule amounts of substances in blood. In 1951 his method was published in the Journal of Biological Chemistry. In 1996 the Institute for Scientific Information (ISI) reported that this article had been cited almost a quarter of a million times, making it the most cited research article in history. This statistic reveals the ubiquity of protein measurement assays and the resilience of an assay developed over 60 years ago. The Lowry method remains one of the most popular colorimetric protein assays in biopharmaceutical development, although many alternative assays now exist. 

The colorimetric methods depend on a chemical reaction or interaction between the protein and the colorimetric reagent. The resulting generation of a chro-mophore, whose intensity is protein-concentration dependent, can be quantified using a spectrophotometer. Beer s Law is employed to derive the protein concentration from a standard curve of absorbances. Direct interaction of the protein with a chromogenic molecule (dye) or protein-mediated oxidation of the reporter molecule generates a new chromophore that can be readily measured in the presence of excess reagent dye. 

Finally, the protein assay for the drug product will also be used for realtime and accelerated stability testing if it has been validated to be stability indicating. A stability-indicating protein concentration method usually translates to a method that can reveal how much protein can be recovered from the dosage form. Many protein instabilities result in precipitation of the protein and adsorption to the container. An instability that results in only a modification of the protein structure but not in loss of protein from solution will not be detected by a sequence-independent protein assay such as a colorimetric assay. 

The first assay to be employed for protein concentration is the Bradford assay, a commercially available colorimetric assay used to quantitate the total extracted protein. Amb a 1 is approximately 1% of the total protein extracted from ragweed pollen hence the Bradford assay does not reflect Amb a 1 concentration. However, at this step of the production process, the protein concentration is used to calculate final yields and not to make time-dependent or expensive decisions. Hence the nonspecific Bradford assay is ideal. A simpler direct absorbance method is not suitable due to the presence of a nonprotein chromophore in the ragweed extract. 

As mentioned earlier, the response of each protein will vary. This is especially apparent with colorimetric assays or derivatization methods requiring a chemical reaction. These protein-to-protein reactivity differences mean that a protein assay suitable for one protein may not be suitable for another. Even for a given protein and a specific protein determination method, results may still vary based on limitations of the assay. Methods requiring extensive sample preparation including protein concentration, buffer exchange, and time-sensitive reactions are liable to be less reproducible than direct measurement techniques, which have fewer variable parameters. The application will determine the suitability of the method. 

Colorimetric assays are commonly used in molecular biology and biotechnology laboratories for determining protein concentrations because the procedures and their instrumentation requirements are simple. Two forms of assays are used. The first involves reactions between the protein and a suitable chemical to yield a colored, fluorescent, or chemiluminescence product. Second, a colored dye is bound to the protein and the absorbance shift is observed. Disadvantages of both these methods include limited sensitivity at below 1 pg/mL, interferences from buffers, and unstable chromophores (Jain et al. 1992). 

In order to demonstrate the viability of the approach, protein phosphatase inhibition was first performed with the enzyme in solution and detected by colorimetric methods. Two microcystin variants, microcystin-LR and microcystin-RR, were used. Both enzymes were inhibited by these toxins, although to a different extent. The 50% inhibition coefficients (IC50) towards microcystin-LR were 0.50 and 1.40 pgL 1 (concentrations in the microtitre well) for the Upstate and the GTP enzymes, respectively. Hence, the Upstate enzyme was more sensitive. The IC50 towards microcystin-RR were 0.95 and 2.15 pgL-1 for the Upstate and the GTP enzymes, respectively. As expected, microcystin-LR was demonstrated to be a more potent inhibitor. 

The coupling ratio is determined most easily with a dual beam spectrophotometer with solutions containing equal concentrations of unmodified and ribonucleoside conjugated keyhole limpet hemocya-nin in the reference and sample cuvettes, respectively. Protein concentrations can be determined by standard colorimetric assay (e.g., 18), and the concentration of coupled ribonucleoside can be calculated using published extinction coefficients (e.g., 19). An alternate method, based on the extinction coefficients of the carrier protein and of the coupled ribonucleoside, is described in ref. 20. 

The protein concentration in solution was measured by the Bradford protein assay using BSA as a standard (Bio-Rad). The Bradford colorimetric method cannot distinguish the BSA protein from enzyme protein. Therefore, no differentiation was possible between BSA and enzymes in solution other than by measuring the enzyme activity. The enzyme activities in solution including FPU and CBU were measured according to the methods described by Ghose [35]. 

Various colorimetric methods are also available, based on non-specific dye binding to polypeptide chains, one of the more common being the Bradford assay. One drawback with such methods is that the actual colour intensity (absorbance) developed is not absolute, but depends on the specific protein. Calibration can therefore be a problem if accurate concentrations are required. 

Quantitative analysis of proteins can be achieved by UV spectroscopy. The peptide bond has an absorption maximum around k = 205 nm, the aromatic rings on the amino acids Tryptophan and Tyrosine absorb strongly around k = 280 nm. Also commonly used are colorimetric assays, which contain reagents that specifically form coloured complexes with proteins. These quantitative methods usually measure the total protein concentration. Either the protein of interest has to be isolated prior to analysis, or a very specific method has to be found to quantify only the targeted protein. Very sensitive and specific analysis of antibodies and antigens can be achieved with bioassays (section 5.1) or biosensors (section 5.2). 

The biuret method The bimet method is a colorimetric technique specific for proteins and peptides. Copper salts in alkaline solution form a pruple complex with substances containing two or more peptide bonds. The absorbance produced is proportional to the number of peptide bonds that are reacting and therefore to the number of protein molecules present in the reaction system. Thus, the biuret reaction with proteins is suitable for the determination of total protein by spectrophotometry (at 540-560 nm). The method is used extensively in clinical laboratories, particularly in automated analyzers in which protein concentration can be measured down to 0.1-0.15gl. The use of bovine or human serum albumin to standardize the biuret method is well established. ITigh-purity albumin contains only amino acids its nitrogen content is a constant fraction... 

The standard protocol for measuring inorganic phosphate using variations of the Fiske and Subbarow method were described in Section 8.3. There have been a number of applications described recently that have adopted a plate-reader format (see, for example, 25-27). With careful planning most assays that use colorimetric reagents (e.g. those for measuring protein concentrations described in Section 4) can be modified to facilitate the use of a plate reader. 

All protein concentration methods have sources of error. Colorimetric methods rely on protein standards that may not be representative of the target product protein s response in the assay. AAA and absorbance methods are more direct than colorimetric methods, however, AAA does rely on amino acid standards, and absorbance methods rely on the correspondence of extinction coefficients of model compounds with those of certain amino acid residues within the target protein. Additionally, AAA may suffer from environmental contamination from the laboratory, whereas absorbance methods may suffer from contaminating compounds that absorb light at the wavelength(s) used. Colorimetric results can be influenced by contaminating protein or formulation excipients. 

Some proteins, such as collagen and HPr, do not contain tryptophan or tyrosine residues therefore they do not intrinsically absorb light at 280 nm. In these cases, it will be necessary use either AAA, refractive index ( ) detection, backbone absorbance (<240 nm), or a colorimetric method (see below). When using refractive index detection, the incremental change in n with concentration (dn/dc, a constant analogous to a protein s extinction coefficient) must be known. The determination of dn/dc requires the generation of a standard curve ( vs. concentration) the concentration must be previously determined by different method. It quickly becomes evident how circular the determinations can be, with one method relying on the outcome of another. 

There are many cases when it may be more convenient, or even necessary to use a colorimetric assay. These methods can be used when a protein s extinction coefficient is not known (or has no Trp/Tyr residues), or is not free of absorbing contaminants. These methods are also useful when trying to nonspecifically determine total protein concentration. In general, they all rely on an exogenous molecule forming a complex with the protein in solution, which leads to an absor-bance/fluorescence signal proportional to the protein concentration. 

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