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PubMed1984

Postcolumn detection of serum proteins with the biuret and Lowry reactions.

The Lowry and biuret reactions have been adapted for the selective detection of chromatographically resolved proteins, specifically proteins separated by high-performance liquid chromatography. The protein reagents are continuously added to the column effluent and produce the characteristic chromophores with both proteins and peptides. The reaction chemistries are compatible with ion-exchange, steric exclusion, and reverse-phase chromatography. Detection limits for proteins resolved by ion-exchange are about 5 to 10 micrograms with the Lowry reaction. Peptides containing tyrosine can be detected at the 100-ng level when chromatographed on reverse-phase columns. The biuret reaction is about 8 times less sensitive for proteins and not very effective for peptides. Reaction detection can be combined with direct absorbance detection in the uv to distinguish proteinaceous peaks from other peaks containing uv-absorbing compounds.

Biuret Reaction↗
PubMed1986

[Protein determination in urine--a critical review].

Seven methods for protein determination in urine were systematically checked, viz. turbidimetric assay with benzethonium chloride, turbidimetry with trichloroacetic acid of various concentrations, micellar complex formation with Coomassie Brilliant Blue G-250, formation and gel filtration of protein complexes with copper, reaction of protein tannin compounds with iron, and biuret reaction after different precipitation procedures. The evaluation was mainly based on the following criteria: identical reaction with various proteins, stable and linear absorbance with concentration, complete recovery, and greatest possible freedom from interferences. On this basis, the biuret reaction must now be regarded as the method of choice. Albumin and gamma-globulins are reliably estimated after precipitation by perchloric acid or by an ethanol-diethylether mixture, whereas all proteins, including uromucoid, may be measured after precipitation by Tsuchiya's reagent. Interference from urinary pigments can be avoided by reading versus a sample blank.

Benzethonium↗
PubMedDate not supplied

The measurement of total serum proteins by the Biuret method.

The biuret reaction for proteins provides a simple and precise method for measuring serum proteins; Beer's law is obeyed to at least 10 g per dl. Several stable biuret reagents are available. Hemoglobin is the only important cause of interference which cannot be minimized by use of a sample blank. The mechanism of the biuret reaction is described and attention is drawn to the heterogeneity of the serum proteins and to the use of a certified albumin standard.

Biuret Reaction↗
PubMed1975

Standards for total serum protein assays--a collaborative study.

We have studied the standardization of total serum protein assay with the biuret reaction. Standard solutions were prepared from lyophilized preparations of human serum albumin and bovine serum albumin, with corrections made for volatile material and ash contents. These solutions and a solution of crystalline albumin standard were analyzed with a new stable biuret reagent, to establish absorptivity values (values for the absorbance of a 1 g/liter final reaction mixture). The mean values obtained were 0.302, 0.292, and 0.290 for human serum albumin, bovine serum albumin, and the crystalline albumin, respectively. We believe that the established absorptivity value will improve the accuracy of serum protein determinations. We studied the linearity of the relation between color produced and protein concentration, with use of the solutions described above and a serum pool. The color adheres to Beer's law up to the highest concentration tested: 3 g/liter for HSA and BSA, and 2.8 g/liter for serum in the final reaction mixture. The new biuret reagent has been stable for one year at room temperature. We recommend the use of bovine serum albumin as a primary standard for serum protein assays. It is inexpensive, easily available, and exhibits the best linearity in the biuret reaction.

Animals↗
PubMed1986

Comparison of five techniques for the determination of protein content in mixed human saliva.

This study was conducted to assess the relative accuracy of five different assay techniques for the determination of protein concentration in human mixed saliva. The protein concentration of paraffin-stimulated saliva from 20 individuals was determined using the biuret reaction, the Lowry assay, a modified Lowry technique using bicinchoninic acid, and two dye-binding assays. Using bovine serum albumin as the standard, mean values ranged from 0.67 to 2.37 mg/ml. The use of bovine serum albumin, trypsinogen, lysozyme, bovine pancreatic ribonuclease, and poly-L-lysine as standards with the five different assay techniques to measure protein concentration of pooled mixed saliva from the above subjects produced results ranging from 0.74 to 65.5 mg/ml. The protein concentration obtained for this saliva sample by amino acid analysis was consistent with the value obtained for the biuret reaction using any of the five different standard proteins. Thus, the protein concentration obtained for human saliva depends upon both the technique used and the protein standard.

Amino Acids↗
PubMed1982

Six methods for urinary protein compared.

Six assays for protein in urine were compared for linearity, ease of standardization, precision, comparability of assay values, technical ease of assay, and current cost. The assays investigated were three dye-binding techniques, a recent turbidimetric technique, the trichloroacetic acid--biuret reaction, and a tannic acid protein precipitation reaction with ferric chloride. All assays suffered from standardization problems, although the biuret method showed the best analytical recovery of albumin and gamma-globulin. The tannic acid/ferric chloride method is dependent on sample pH. The turbidimetric assay exhibited the greatest imprecision; i.e., CVs were 19.5% at a protein concentration of 0.13 g/L and 6.0% at a protein concentration of 1.3 g/L. On the basis of all the factors assessed, we conclude that the Pesce/Strande Ponceau-S and the Bio-Rad Coomassie Brilliant Blue dye-binding techniques offer certain advantages over the other assays studied.

Azo Compounds↗
PubMed1981

A candidate reference method for determination of total protein in serum. I. Development and validation.

We developed a candidate Reference Method for measuring total serum protein by use of the biuret reaction. The method involves a previously described biuret reagent (Clin. Chem. 21: 1159, 1975) and Standard Reference Material (SRM) 927 bovine albumin (National Bureau of Standards) as the standard. At 25 degrees C, color development for 30 or 60 min provides identical serum protein values. Glucose (up to 10 g/L) and bilirubin (up to 300 mg/L) do not interfere. Hemoglobin, at 3 g/L, increases apparent serum protein by 0.4 g/L. The presence of dextran in serum causes easily detected turbidity, but this interference can be eliminated by centrifuging the reaction mixture. Therapeutic concentrations of ampicillin, carbenicillin, penicillin, oxacillin, nafcillin, chloramphenicol, cephalothin, and methicillin in blood do not interfere, nor do triglycerides up to 10 g/L. Within-run and day-to-day standard deviations of the method are 0.1 and 0.4 g/L, respectively.

Bilirubin↗
PubMed1985

Measurement of protein using bicinchoninic acid.

Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.

Hydrogen-Ion Concentration↗
PubMed1984

Prevention of interference by dextran with biuret-type assay of serum proteins.

In assay of serum proteins by use of the biuret reaction, dextran can cause turbidity by formation of an insoluble complex of dextran with copper and tartrate (or EDTA) in strongly alkaline solution. Whether or not the turbidity occurs depends on the tartrate concentration: turbidity is maximal at about 10 g/L, absent at 20 g/L or more, and only slight and delayed at 4 g/L. Two biuret reagents, containing respectively 5.6 and 22.5 g of tartrate per liter, obviate the interference, but the former is suitable only when a short (5 min) incubation is used. Both reagents show linear calibration curves and yield virtually identical results.

Biuret Reaction↗
PubMed1994

Measuring urinary protein with the new BioRad reagent kit: evaluation and comparison with five other methods.

Total urinary protein was measured by five methods: BioRad Total Protein Test (TPT), pyrogallol red, benzethonium chloride, sulfosalicylic acid, trichloroacetic acid, and the results compared to those obtained by a method combining preparative ultrafiltration and the biuret reaction. TPT was linear to 1.5 g protein/l, the detection limit 0.0135 g/l, and it was 3-5 times more sensitive than the other methods. Within-day precision (CV) was 4.3%, (0.60 g/l), the day-to-day precision was 4.5%. The protein contents of 35 selected urine samples assigned to one of five groups according to their electrophoretic pattern were assayed by the five methods. No method accurately measured physiological proteinuria, but the values for light chain (Bence Jones), glomerular, tubular and overload proteinurias measured by TPT did not differ significantly from the biuret value. The other methods differed significantly for at least three groups. Alpha 1 acid glycoprotein slightly inhibited TPT, but peptones, amino acids, antibiotics or normal urine constituents had little or no effect. The TPT method has been automated (Kone Progress); normal 24-h urinary protein excretion was 36 mg/day (range 12-114), the protein creatinine ratio was 34 mg/g (12-106 mg/g).

Autoanalysis↗