Biuret

The biuret method is based on the fact that proteins (and, as a rule, all substances containing two or more peptidic bonds) react with copper to form a colored complex whose absorption (λmax=454nm), in the presence of excess copper, is proportional to the amount of protein present.

From: Encyclopedia of Analytical Science (Second Edition) , 2005

PROTEINS | Physiological Samples

A. Bianchi-Bosisio , in Encyclopedia of Analytical Science (Second Edition), 2005

The biuret method

The biuret method is a colorimetric technique specific for proteins and peptides. Copper salts in alkaline solution form a purple 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 0.1–0.15   g   l−1. The use of bovine or human serum albumin to standardize the biuret method is well established. High-purity albumin contains only amino acids; its nitrogen content is a constant fraction of its molecular mass and the number of peptide bonds per molecule is known. Since the peptide bond is the biuret-reacting unit in all proteins and the number of peptide bonds determines the absorbance of the colored product, albumin is a reasonable peptide bond standard for all proteins in the mixture.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B0123693977004945

Spectrophotometry | Biochemical Applications☆

F. Sánchez Rojas , ... J.M. Cano Pavón , in Encyclopedia of Analytical Science (Third Edition), 2019

Derivatization

There are three prime methods for quantification of proteins based on derivatization reactions: the biuret, Lowry, and Bradford methods.

The biuret method is based on the fact that proteins (and, as a rule, all substances containing two or more peptidic bonds) react with copper to form a colored complex whose absorption (λ max=   454   nm), in the presence of excess copper, is proportional to the amount of protein present. The reagent is obtained by dissolving 1–5   g of copper(II) sulfate and 6   g of sodium potassium tartrate tetrahydrate in 3% sodium hydroxide. Bovine serum albumin is used as standard. The most serious drawback of this method is its poor sensitivity.

The Lowry method, more sensitive than the biuret method, affords the determination of protein at the microgram per milliliter level. The procedure involves two reactions: that of the protein with alkaline copper solution (the biuret reaction) and reduction of the Folin–Ciocalteau reagent by tyrosine and tryptophan residues of the protein. The Folin–Ciocalteau reagent is prepared by boiling a solution containing sodium tungstate, sodium molybdate, and phosphoric and hydrochloric acids, which produces molybdophosphate and tungstophosphate ions. The absorbance is measured at 750   nm. The Lowry method has also been applied to proteins in whole cells. Like the biuret method, it is subject to some limitations and interferences (particularly those from ammonium salts, glycine, and mercaptans). In addition, variations in the tyrosine and tryptophan contents from protein may introduce some uncertainty in the determinations, so the method is more practical for determining concentration changes than absolute protein concentrations. The Lowry method has also been applied by exploiting solid-phase spectrophotometric techniques.

The Bradford method is based on the absorbance of Coomassie Brilliant Blue on the protein to be determined, which results in a spectrum shift of the dye from 465 to 595   nm. The absorbance at 595   nm is proportional to the protein concentration. Bovine serum albumin is used as standard. The reaction is susceptible to interferences from surfactants and alkalinity of the solution. The main advantage of the method is rapidity, which enables efficient application to many samples.

A micromethod has been developed using the Bradford method for studying the proteins present in cell membranes.

Proteins can also be determined by 1% bicinchoninic acid solution in alkaline medium, to produce a red compound. The absorbance is measured at 562   nm against a reagent blank. Proteins can be determined in the interval 0.5–10   μg   ml−   1. The method offers a one-step alternative to the methods of Lowry and Bradford and is less subject to interferences.

In recent years, diverse chelating agents have been introduced for proteins quantification: Arsenazo III, fuchsine acid, methyl blue, bromophenol blue, methylene blue, etc.

Another common determination is that of haemoglobin, a major component of red blood cells. The method involves oxidation of haemoglobin with hexacyanoferrate(III) to form methaemoglobin, and further reaction with cyanide ion to form cyanmethaemoglobin that presents maximal absorptivity at 540   nm (ε=   4.4×104  l   mol−   1  cm−   1). The procedure is carried out by using Drabkin's reagent, a solution containing potassium hexacyanoferrate(III), potassium cyanide, sodium phosphate, and a surfactant (e.g., Triton X-100). The method can also be applied to whole blood samples.

Haemoglobin can also be determined after complex formation with inositol hexaphosphate that is quantified by differential spectrometry to avoid the strong absorbance of the protein.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780124095472005011

UV-Visible Absorption Spectroscopy, Biomacromolecular Applications

Alison Rodger , Karen Sanders , in Encyclopedia of Spectroscopy and Spectrometry (Third Edition), 2017

Folin–Ciocalteau or Lowry method

While the biuret method is sensitive in the range 0.5 to 2.5  mg protein per assay, the Lowry method is 1 to 2 orders of magnitude more sensitive (5 to 150   μg). The main disadvantage of the Lowry method is the number of interfering substances; these include ammonium sulfate, thiol reagents, sucrose, EDTA, Tris, and Triton X-100.

The final colour in the Lowry method is a result of two reactions. The first is a small contribution from the biuret reaction of protein with copper ions in alkali solution. The second results from peptide-bound copper ions facilitating the reduction of the phosphomolybdic-tungstic acid (the Folin reagent) which gives rise to a number of reduced species with a characteristic blue colour. The amino acid residues which are involved in the reaction are tryptophan and tyrosine as well as cysteine, cystine and histidine. The amount of colour produced varies slightly with different proteins. In this respect it is a less reliable assay than the biuret method, but it is more reliable than the absorbance method since A 280 may include contribution from other species, and also the absorption of a given residue is dependent on its environment within the protein.

Two solutions are required for the Lowry method. For the alkaline copper solution, mix 50   cm3 Na2CO3 (2% w/v) in NaOH (0.1   M) with 1   cm3 of CuSO4.5H2O (0.5% w/v) and 1   cm3 of sodium potassium tartrate (1% w/v). This solution must be discarded after 1 day. The Folin reagent (phosphomolybdic-tungstic acid) may be made by diluting the concentrated Folin reagent obtained from, for example Sigma with an equal volume of water so that it is 1   N (i.e. 1   M H+).

To perform an assay add x  cm3 of sample (where x  <   0.6) containing 5–100   μg of protein as required to (0.6– x)   cm3 of water. Then add 3   cm3 of the alkaline copper solution. The solutions must then be mixed well and allowed to stand for 10   min at room temperature. Add 3.0   cm3 of Folin reagent and after 30   min, determine the absorbance at 600   nm.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780128032244001084

Amino Acids, Peptides, and Proteins

Dennis J. Dietzen , in Principles and Applications of Molecular Diagnostics, 2018

Variables Affecting Measured Protein Concentrations

Calibration of biuret methods commonly uses bovine or human albumin. Protein mixtures with specific albumin-to-globulin ratios often have been recommended for calibration of other methods. Using these calibration schemes, the total protein concentration of plasma obtained from healthy ambulatory adults is typically 6.5 to 8.5 g/dL. Serum usually contains a protein concentration about 0.3 g/dL less because of the content of fibrinogen and other proteins removed during clotting to form serum. Hemoconcentration and relative hyperproteinemia, with increased concentrations of all plasma proteins, occur with inadequate water intake or excessive water loss as in severe vomiting, diarrhea, Addison's disease, or diabetic ketoacidosis. Some hemoconcentration also occurs with standing (reduced intravascular volume) or prolonged tourniquet time during blood collection. Hemodilution and relative hypoproteinemia, with decreased concentrations of all plasma proteins, occur with water intoxication or salt retention syndromes, during massive intravenous infusions, and physiologically when a recumbent position is assumed. A recumbent position decreases total protein concentration by 0.3 to 0.5 g/dL and many individual proteins, including albumin, by up to 10%. This reflects the redistribution of extracellular fluid from the extravascular space to the intravascular space and therefore dilution of a constant amount of plasma protein in a larger volume.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780128160619000138

Six-membered Rings with Two or More Heteroatoms and Fused Carbocyclic Derivatives

Gottfried Maerkl , Peter Kreitmeier , in Comprehensive Heterocyclic Chemistry II, 1996

6.26.6.2 Rings with Arsenic, Oxygen and Nitrogen Atoms

6.26.6.2.1 1,3,5,2-Dioxazarsinanes and 1,3,2,6-oxazadiarsinanes

The transamidation reaction of biuret ( 318 ) with bis(dimethylamino)phenylarsine produces the 4,6-diimino-2-phenyl-1,3,5,2-dioxazaarsinane ( 319 ). The related compound ( 320 ) is formed using trisdimethylaminoarsine (Scheme 56) 〈70ZAAC(378)310〉.

Scheme 56.

The 5H,11H-6,12-epoxydibenzo[c,g][1,5,2,6]diazadiarsocine ( 322 ) can be considered as a derivative of tetrahydro-1,3,2,6-oxazadiarsinine. It is formed by the thermal dimerization of o-amino-phenyldichloroarsine ( 321 ) followed by hydrolysis of the Assingle bondCl groups (Scheme 57) 〈70JPR751〉.

Scheme 57.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780080965185001428

Veterinary Toxicology

W.K. Rumbeiha , D.B. Snider , in Encyclopedia of Toxicology (Third Edition), 2014

Nonprotein Nitrogen Compounds

Nonprotein nitrogenous sources include urea, biuret, and ammoniated feeds. These compounds are cheap sources of the nitrogen required by the animals for protein synthesis. Nonprotein nitrogen poisoning is a common problem and is often seen in animals not gradually introduced to feeds containing these compounds. Commonly known as urea poisoning, this is an acute fatal condition characterized by bloating, intense abdominal pain, ammonia breath, frequent urination, and frenzy. Often, several animals (or the entire herd) are affected simultaneously.

In ruminants, the rumen microflora normally convert urea to ammonia, and the ammonia is rapidly utilized by the liver for protein synthesis. However, in cases of excess ammonia production, blood ammonia concentration quickly builds up to toxic levels and induces central nervous system (CNS) derangement. Therefore, in addition to the gastrointestinal signs, the animals will show fulminating CNS signs. Treatment of this disease involves giving a weak acid such as vinegar and plenty of cold water orally. The rationale for giving cold water and acetic acid is to slowdown the action of urease, the enzyme responsible for converting urea to ammonia, which requires body core temperature and pH for optimal function. The cold water lowers rumen temperature and the acetic acid lowers the pH. Infusions of calcium and magnesium solutions are recommended to alleviate tetany.

Another source of nonprotein nitrogen (urea) poisoning in ruminants is the accidental ingestion of nitrogen-based fertilizers such as ammonium phosphate. Occasionally, cattle break into drums or bags of fertilizers containing these nitrogen-based compounds. The prognosis is grave in most cases if several animals are affected. If only a few valuable animals are affected, a rumenotomy can be performed. Although small ruminants (e.g., sheep and goats) have the same anatomical predisposition to nonprotein nitrogen poisoning, they are rarely poisoned.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780123864543004449

Hydrazine

Thomas F. DeRosa , in Advances in Synthetic Organic Chemistry and Methods Reported in US Patents, 2006

Experimental

1.

Chlorobiuret Sodium Salt

A 12.9% aqueous biuret slurry (0.287 moles) was placed in a 2-liter glass reactor, cooled to 5 °C, 12% sodium hypochlorite solution (0.287 moles) added, and the reaction temperature kept at 5 °C. Thereafter, the reaction product was analyzed by iodine titration method and liquid chromatography indicated the product yield was 98%.

2.

Hydrazodicarbonamide

The entire product from Step 1 was added to a 2-liter glass reactor, the temperature raised to 100 °C while adding liquid ammonia (8.8 moles), and the reaction temperature maintained at 100 °C 30 minutes. After the reaction was completed, unreacted ammonia was removed, the reaction solution filtered, and the product isolated in 95% yield.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9780080444741500396

The influence of plasma proteins on bone cell adhesion

Å ROSENGREN , S OSCARSSON , in Cellular Response to Biomaterials, 2009

21.3 Bicinchoninic acid (BCA) protein assay

The BCA assay [41 ] is a coloring assay that is used for protein quantification. The method is based on the Biuret reaction during which Cu 2+ is reduced to Cu+ by peptide bonds in proteins under alkaline conditions. The Cu2+ is then detected by a chelating reaction with BCA yielding an intense purple color. The production of Cu+ is a function of protein concentration and incubation time, allowing the protein content of unknown samples to be determined spectrophotometrically by comparison with known standards. The advantage of BCA assay over other assays is that it is relatively insensitive to detergents and denaturing agents. It is, however, more sensitive to sugars.

Read full chapter

URL:

https://www.sciencedirect.com/science/article/pii/B9781845693589500211