ビウレット 反応。 アミドが塩基性を示さない理由

第126章 実験−タンパク質

ビウレット 反応

が3つ以上つながった(トリペプチド以上の)は、に似た構造を持ち、溶液中で銅(II)イオンに配位し、赤紫色から青紫色に呈色する。 この反応は1833年にドイツで発見されたが 、1857年にそれとは別にポーランドの者、G. ピオトロウスキーによって再発見された。 タンパク質がを多く含むほど強く呈色し、さらに、タンパク質のグラムあたりに現れるペプチド結合の数はほぼ同じであるため、タンパク質のおおよそのを測定するのにもビウレット反応は有効である。 この反応で使う液体を ビウレット溶液 Biuret solution という。 ただし、ビウレット反応という名前だが、は生成しない。 104, pages 132-142, :• 24, p. 335-337. - Thermo Scientific Protein Methods Library• Fenk, C. ; Kaufman, N. ; and Gerbig, D. Chem. Educ. 2007, 84, 1676-1678.

次の

ビウレット反応なんですが、どのような仕組みでトリペプチド...

ビウレット 反応

Most colorimetric protein assay methods can be divided into two groups based on the type of chemistry involved: those involving protein-copper chelation with secondary detection of the reduced copper and those based on protein-dye binding with direct detection of the color change associated with the bound dye. Most commercial protein assay reagents are well-characterized, robust products that provide consistent, reliable results. Nevertheless, each assay reagent has its limitations; having a basic understanding of the chemistries involved with each type of assay is essential for selecting an appropriate method for a given sample and for correctly evaluating results. This became known as the biuret reaction because it is chemically similar to a complex that forms with the organic compound biuret NH2-CO-NH-CO-NH2 and the cupric ion. Biuret, a product of excess urea and heat, reacts with copper to form a light blue tetradentate complex. Single amino acids and dipeptides do not give the biuret reaction, but tripeptides and larger polypeptides or proteins will react to produce a light blue to violet complex that absorbs light at 540 nm. One cupric ion forms a colored coordination complex with four to six nearby peptides bonds. The intensity of the color produced is proportional to the number of peptide bonds participating in the reaction. Thus, the biuret reaction is the basis for a simple and rapid colorimetric reagent of the same name for quantitatively determining total protein concentration. The was introduced by Smith, et al. , in 1985. Since then it has become the most popular method for colorimetric detection and quantitation of total protein. One particular benefit is that, unlike other methods available at that time e. In addition, the BCA Assay responds more uniformly to different proteins than the Bradford method. Thus, two steps are involved. First is the biuret reaction, whose faint blue color results from the reduction of cupric ion to cuprous ion. Second is the chelation of BCA with the cuprous ion, resulting in an intense purple color. The purple colored reaction product is formed by the chelation of two molecules of BCA with one cuprous ion. The BCA reagent is approximately 100 times more sensitive lower limit of detection than the biuret reagent. However, studies performed with di- and tripeptides indicate that these produce more color than can be accounted for by the four individual BCA-reactive amino acids. In other words, the peptide backbone and thus the total amount of protein is the major contributor to the reduction of copper in the biuret reaction and color development in the BCA assay. Slight protein-to-protein variation in the BCA protein assay results from differences among proteins in composition with respect to these three amino acids. The binding of BCA to cuprous ion effectively removes the weakly chelated peptides of the biuret reaction. Those peptide groups are then free to bind another molecule of cupric ion. Therefore, if bicinchoninic acid and copper are present in large excess as they always are in BCA protein assay reagents , the protein assay does not reach an end-point. In addition, the rate of BCA color formation is dependent on the incubation temperature. Consequently, the key to obtaining accurate results with the BCA assay method is to assay standards and unknown samples simultaneously so that they both receive identical incubation time and temperature. Assuming that the assay is performed in this way, the assay characteristic enables one to speed development or wait longer for desired colored development as needed. Substances that reduce copper will also produce color in the BCA assay, thus interfering with the accuracy of the protein quantitation. Reagents that chelate the copper also interfere by reducing the amount of BCA color produced with protein. Certain single amino acids cysteine or cystine, tyrosine and tryptophan will also produce color and interfere in BCA assays. Tech Tips and specialized versions of BCA protein assay products address one or another of these sample- incompatibility issues. The latest advance in colorimetric protein assays is the which retains the high sensitivity and linearity of the traditional BCA assay, but provides ready-to-read results within 5 minutes with room temperature RT incubation. Like the conventional BCA assay, the Pierce Rapid Gold BCA Protein assay involves the reduction of copper by proteins in an alkaline medium biuret reaction to produce sensitive and selective colorimetric detection by a new copper chelator. The amount of reduced copper is proportional to the amount of protein present in the solution. The selective copper chelator forms an orange-gold—colored complex that strongly absorbs light at 480 nm. This representative data compares the performance of the conventional and newly adapted BCA protein assays. Figure 4. Protein concentration determination in lysates using the standard Pierce BCA Protein Assay and Pierce Rapid Gold BCA Protein Assay. The Lowry protein assay is named after Oliver H. Lowry, who developed and introduced it Lowry, et al. , 1951. It offered a significant improvement over previous protein assays and his paper became one of the most cited references in life science literature for many years. The uses a stable reagent that replaces two unstable reagents described by Lowry. Essentially, the assay is an enhanced biuret assay involving copper chelation chemistry. Although the mechanism of color formation for the Lowry assay is similar to that of the BCA protein assay, there are several significant differences between the two. The exact mechanism of color formation in the Lowry assay remains poorly understood. The assay is performed in two distinct steps. First, protein is reacted with alkaline cupric sulfate in the presence of tartrate for 10 minutes at room temperature. During this incubation, a tetradentate copper complex forms from four peptide bonds and one atom of copper this is the "biuret reaction". Second, a phosphomolybdic-phosphotungstic acid solution is added. This compound called Folin-phenol reagent becomes reduced, producing an intense blue color. It is believed that the color enhancement occurs when the tetradentate copper complex transfers electrons to the phosphomolybdic-phosphotungstic acid complex. The blue color continues to intensify during a 30 minute room temperature incubation. It has been suggested that during the 30 minute incubation, a rearrangement of the initial unstable blue complex leads to the stable final blue colored complex which has higher absorbance Lowry, et al. 1951; Legler, et al. 1985. The final blue color is optimally measured at 750nm, but it can be measured at any wavelength between 650 nm and 750 nm with little loss of color intensity. It is best to measure the color at 750 nm since few other substances absorb light at that wavelength. For small peptides, the amount of color increases with the size of the peptide. With the exception of tyrosine and tryptophan, free amino acids will not produce a colored product with the Lowry reagent; however, most dipeptides can be detected. In the absence of any of the five amino acids listed above in the peptide backbone, proteins containing proline residues have a lower color response with the Lowry reagent due to the amino acid interfering with complex formation. Unlike in the BCA assay, the secondary binding step in the Lowry method does not involve detachment of the peptide-copper chelate. Therefore, the Lowry method is effectively an end-point assay. Although it is always best to include internal standards in any protein assay, it is possible to obtain reasonable protein estimations with this assay method by comparing to a previously-plotted standard curve. The protocol requires that the Folin phenol reagent be added to each tube precisely at the end of the ten minute incubation. At the alkaline pH of the Lowry reagent, the Folin phenol reagent is almost immediately inactivated. Therefore, it is best to add the Folin phenol reagent at the precise time while simultaneously mixing each tube. Because this is somewhat cumbersome, some practice is required to obtain consistent results. This also limits the total number of samples that can be assayed in a single run. The Lowry assay reagent forms precipitates in the presence of detergents or potassium ions. When potassium ions are the cause, the problem can sometimes be overcome by centrifuging the tube and measuring the color in the supernatant. Most surfactants cause precipitation of the reagent even at very low concentrations. Chelating agents interfere by binding copper and preventing formation of the copper peptide bond complex. Reducing agents and free thiols also interfere by reducing the phosphotungstate-phosphomolybdate complex, immediately forming an intensely blue colored product upon their addition. The Modified Lowry Protein Assay Reagent must be refrigerated for long-term storage, but it must be warmed to room temperature before use. Using cold Modified Lowry Protein Assay Reagent will result in low absorbance values. In the acidic environment of the reagent, protein binds to the Coomassie dye. The difference between the two forms of the dye is greatest at 595 nm, so that is the optimal wavelength to measure the blue color from the Coomassie dye-protein complex. If desired, the blue color can be measured at any wavelength between 575 nm and 615 nm. Development of color in Bradford protein assays is associated with the presence of certain basic amino acids primarily arginine, lysine and histidine in the protein. Van der Waals forces and hydrophobic interactions also participate in the binding of the dye by protein. The number of Coomassie dye ligands bound to each protein molecule is approximately proportional to the number of positive charges found on the protein. Free amino acids, peptides and low molecular weight proteins do not produce color with Coomassie dye reagents. In general, the mass of a peptide or protein must be at least 3000 daltons to be detectable with this reagent. In some applications this can be an advantage. For example, the Coomassie Protein Assay has been used to measure "high molecular weight proteins" during fermentation in the beer brewing industry. Coomassie dye binding assays are the fastest and easiest to perform of all protein assays. The assay is performed at room temperature and no special equipment is required. Standard and unknown samples are added to tubes containing preformulated Coomassie assay reagent and the resultant blue color is measured at 595 nm following a short room temperature incubation. The Coomassie dye-containing protein assays are compatible with most salts, solvents, buffers, thiols, reducing substances and metal chelating agents encountered in protein samples. The main disadvantage of Coomassie based protein assays is their incompatibility with surfactants at concentrations routinely used to solubilize membrane proteins. In general, the presence of a surfactant in the sample, even at low concentrations, causes precipitation of the reagent. In addition, the Coomassie dye reagent is highly acidic, so proteins with poor acid-solubility cannot be assayed with this reagent. Finally, Coomassie reagents result in about twice as much protein-to-protein variation as copper chelation-based assay reagents. Figure 7. Absorbance spectra for protein standards in the Thermo Scientific. Notice that an inverse relationship between protein concentration and absorbance occurs below 525 nm maximum at 465 nm. The ready-to-use liquid Coomassie dye reagents should be mixed gently by inversion just before use. The dye in these liquid reagents forms loose aggregates within 60 minutes in undisturbed solutions. Gentle mixing of the reagent by inversion of the bottle will uniformly disperse the dye and ensure that aliquots are homogeneous. After binding to protein, the dye also forms protein-dye aggregates. Fortunately, these protein-dye aggregates can be dispersed easily by mixing the reaction tube. This is common to all liquid Coomassie dye reagents. Because these aggregates form relatively quickly, it is also best to routinely mix vortex for 2-3 seconds each tube or plate just before measuring the color. Introduced in 2008, the is a dye-based reagent that offers the same convenience as Coomassie-based assays while overcoming several of their disadvantages. In particular, the Pierce 660 nm Assay is compatible with most detergents and produces a more linear response curve. The detailed assay chemistry is proprietary, but the essential mechanism can be summarized as follows. The reagent contains a proprietary dye-metal complex in an acidic buffer. The dye-metal complex binds to protein in the acidic condition, causing a shift in the dye's absorption maximum, which is measured at 660nm. The reagent is reddish-brown and changes to green upon protein binding. Figure 8. Absorption maximum of the 660 nm Assay Reagent-metal complex shifts proportionally upon binding to BSA. The absorption spectra were recorded for the Pierce 660 nm Protein Assay Reagent from 340 to 800 nm using a spectrophotometer. Protein in the presence of the reagent-metal complex produces a significant absorbance shift at a wavelength of 660 nm. The color produced in the assay is stable and increases in proportion to a broad range of increasing protein concentrations. The color change is produced by the deprotonation of the dye at low pH facilitated by protein-binding interactions through positively charged amino acid groups and the negatively charged deprotonated dye-metal complex. The assay binds to proteins in a manner similar to Coomassie dye. Thus, it has similar protein-to-protein variability to Coomassie Bradford assay methods. However, unlike Coomassie-based assays, the Pierce 660 nm Protein Assay is fully compatible with nonionic detergents typically used in protein samples. In fact, when used with the Ionic Detergent Compatibility Reagent IDCR , the Pierce 660 nm Assay is also compatible with sample containing Laemmli SDS sample buffer with bromophenol blue and other buffers containing common ionic detergents. Fluorescence-based protein quantification detection methods provide superior sensitivity, which means that less protein sample is used for quantitation, leaving more samples available for experiments. For the assays described below, few steps are required and timing is not critical, as signal duration is typically hours, so the assays can be adapted for automated handling in high-throughput applications. The fluorescence signal can be detected using a fluorometer or microplate reader. Fluorescent protein assays typically provide researchers with greater sensitivity than what can be measured with colorimetric protein assays. The are based on the dye molecule binding to detergent coating on proteins and hydrophobic regions of proteins, and resulting in fluorescence while unbound dye is non-fluorescent. The first two produce a quasi-linear standard curve from 0. For detection of lipoproteins or proteins in a complex lipid environment, the CBQCA Protein Quantitation Kit may be utilized. These representative data show a typical standard curve produced using a fluorescent protein assay kit. This fluorescent dye is suitable for use with spectrofluorometers and microplate readers. For detection of lipoproteins or proteins in a complex lipid environment, check out our CBQCA Protein Quantitation Kit. The is a very sensitive assay for quantitating proteins in solution, capable of detection as low as 10 ng of protein per mL. Similar in sensitivity to our NanoOrange protein quantitation reagent N-6666 , CBQCA is better suited for accurate quantitation of proteins in the presence of lipids, membrane fractions or detergents, and for lipoproteins and small peptides. This assay is based on the reaction of the dye with primary amine groups in the presence of cyanide or thiols, causing it to become fluorescent. Unreacted dye remains non-fluorescent. Finally, the provides a fluorescence-based protein assay that facilitates fast quantitation of protein samples prepared for gel electrophoresis. are instruments that measure the intensity of the fluorescent signal from dyes attached to biological molecules as well as naturally fluorescent molecules based on signature excitation Ex and emission Em wavelengths. Fluorometers were designed to quantify, detect and monitor analytes and their reactions with a high degree of sensitivity and specificity. The is a benchtop device that accurately measures DNA, RNA, and protein using the highly sensitive Qubit quantitation assays. In conjunction with optimized algorithms, the Qubit 3 Fluorometer employs fluorescent dyes that only produce signal when bound to the target of interest, thereby minimizing the effects of contaminants— including degraded DNA or RNA—on experimental results. Popular• Popular• Most Popular Products• Most Popular Categories• Order Tools• Product Documentation• Other Product Information• Product Support• Educational Resources•

次の

タンパク質の定量法 【ビウレット・BCA法やBradford法】

ビウレット 反応

概要: ビウレット法とは• ビウレット法の長所と短所• ビウレット法のプロトコール 広告 概要: ビウレット法とは ビウレット法は、ペプチド結合と銅イオン Cu II の反応を利用したタンパク質濃度の定量法である。 トリペプチド以上のペプチドまたはタンパク質を、Cu II を含む溶液とアルカリ条件下で混合すると、含まれる 原子がCu II を Cu I に還元する 2。 この Cu I の呈色を 540 nm 吸光度で測定する。 広告 ビウレット法の長所と短所 ビウレット法には、以下のような特徴がある 2。 ペプチド結合を検出するため、アミノ酸組成に影響を受けない。 この点を改良したのが であり、ローリー法をさらに改良したのが である。 トリス、アミノ酸、スクロース、アンモニアイオンが発色反応に影響を与える 2。 廃液に重金属である銅が含まれる。 広告 References• Amazon link: Cold Spring Harbor Laboratory Press. 鈴木 2018a Review. 総タンパク質の定量法. ぶんせき. コメント欄 各ページのコメント欄を復活させました。 スパム対策のため、以下の禁止ワードが含まれるコメントは表示されないように設定しています。 レイアウトなどは引き続き改善していきます。 「管理人への質問」「フォーラム」へのバナーも引き続きご利用下さい。

次の