Introduction
Bromocresol green (BCG) is a dye of the triphenylmethane family (triarylmethane dyes). It belongs to a class of dyes called sulfonephthaleins.[4] It is used as a pH indicator in applications such as growth mediums for microorganisms and titrations. In clinical practice, it is commonly used as a diagnostic technique. The most common use of bromocresol green is to measure serum albumin concentration within mammalian blood samples in possible cases of kidney failure and liver disease. In chemistry, bromocresol green is used in Thin-layer chromatography staining solutions to visualize acidic compounds.
A large number of low molecular weight compounds, including drugs, bind reversibly to human serum albumin (HSA), which functions as the major transport protein in the blood. The strength of the binding can affect the drug distribution and the intensity of drug effects.
Several methods like equilibrium dialysis (Rosenberg and Klotz 1960) and fluorescent probe techniques (Daniel and Weber 1966) have been widely used to obtain information about the strength of the binding of drugs onto serum albumin. However, the versatility of the spectrophotometric technique prompted us to apply this technique in the present study for which we made use of a new spectrophotometric probe, namely, bromocresol green (BCG). Hitherto, only 2-(4′-hydroxyphenylazo)-benzoic acid has been reported as a spectrophotometric probe for determining the relative affinities of drugs onto bovine serum albumin (Zia and Price 1975; Zia and Kamali 1976).
Abstract
Bromocresol green (BCG) has been employed as a new spectrophotometric probe to characterise the binding regions of human serum albumin (HSA) and bovine serum albumin (BSA). BCG binds with greater affinity onto BSA than onto HSA. Based on the abilities of ligands Naproxen and 1-anilino-8-naphthalenesulphonic acid (ANS) to displace BCG from the serum albumins by competitive or non-competitive mechanism, binding regions were identified for these ligands.
It has been found that both Naproxen and ANS share common binding sites with BCG in HSA with the relative ability of Naproxen > ANS on binding to HSA. In the case of BSA, ANS competes with BCG for the same binding sites, whereas Naproxen exhibits non-competitive binding. The highaffinity sites of Naproxen coincide with BCG binding sites while the low-affinity sites occur at sites distinct from the BCG binding region.
PRINCIPLE
The method is based on the specific binding of bromocresol green (BCG), an anionic dye, and the protein at acid pH produces a color change of the indicator from yellow –green to green –blue with the resulting shift in the absorption wavelength of the complex. The intensity of the color formed is proportional to the concentration of albumin in the sample.
1. CLINICAL SIGNIFICANCE
One of the most important serum proteins produced in the liver is albumin. This molecule has an extraordinarily wide range of functions, including nutrition, maintenance of oncotic pressure and transport of Ca ++, bilirubin, free fatty acid, drugs and steroids . Variation in albumin levels indicate liver diseases, malnutrition, skin lesions such as dermatitis and burns or dehydration. Clinical diagnosis should not be made on a single test result ; it should integrate clinical and other laboratory data.
2. ANALYTICAL METHODS FOR SOLID-PHASE SYNTHESIS
Points of Interest
- An indicator that would be deprotonated by free amino groups on resin and whose ionized form is deeply colored was developed. Among the several indicators tested, bromophenol blue(3′,3″,5′,5″-tetrabromophenolsulfonphthalein) displayed the best properties: the color changes dramatically from yellow (λmax 429 nm) to dark blue (λmax 600 nm) and the sensitivity is high (extinction coefficient ∈600 91,800 M−1 cm−1). Bromocresol green and bromocresol purple also display satisfactory properties.
- Reactions:Can be monitored continuously even in a parallel format (a solution of bromophenol blue is simply added to the acylating agent or to the last washing solvent before coupling) or discontinuously (a sample of the resin is withdrawn from the reactor and treated with bromophenol blue). The test is not destructive and the excess bromophenol blue can be washed out by a solution of a base.
- The number of free amine groups were determined quantitatively by the bromophenol blue test and provided results that were consistent with quantitative ninhydrinand picric acid
- The bromophenol blue test is highly sensitive; consequently triethylamine(TEA) is not compatible with the monitoring method due to significant background coloring. On the other hand diisopropylethylamine (DIEA) does not cause a significant side reaction.
3. Advances in clinical pathology and diagnostic medicine
Elizabeth Marie , … • Rush Hillar Klandorf, in Current Therapy in Avian Medicine and Surgery, 2016
Proteins
Among protein measurement techniques, electrophoresis and the Biuret technique can be used, but the dye-binding methods with bromocresol are unreliable19,24,124; in birds, bromocresol green typically results in lower total protein values compared with bromocresol purple because of a weaker binding to avian albumin, although both are considered unreliable.23 To date, no veterinary biochemistry analyzer has the ability to perform automated albumin measurement by electrophoresis despite routine reporting of albumin as part of the biochemistry panel.
However, all common biochemistry evaluations use the Biuret technique for total protein measurement.
1 Refractometry is used to measure total solids, the value of which is consistently higher than the total protein value in birds and should not be interpreted as an evaluation of total proteins, as the correlation between the two is weak.19 Different types of equipment are available to perform plasma protein electrophoresis, including semiautomated agarose gel systems,125 high-resolution agarose gel,26 and an automated capillary system.126 Compared with the traditional semiautomated agarose gel system, the automated capillary system has been shown to have superior repeatability and reproducibility but requires a much higher sample volume (e.g., 14 times larger volume).
This higher volume requirement makes it impractical for use in many species. Reference intervals need to be established for this technique.
4. Uses
It is used as a pH indicator and as a tracking dye for DNA agarose gel electrophoresis. It can be used in its free acid form (light brown solid), or as a sodium salt (dark green solid). It is also an inhibitor of the prostaglandin E2 transport protein. Additional applications include use in sol-gel matrices,[8] the detection of ammonia,[9] and the measurement of albumin in human plasma and serum.[10]
5. Calcium: Properties and Determination
L.J. Harvey, in Encyclopedia of Food and Health, 2016
KMnO4 Titrimetric Method for Calcium in Wheat Flour (Method from the Association of Official Analytical Chemists)
After dry ashing and dilution of the ash to a suitable volume with demineralized water, bromocresol green indicator is added along with enough 20% sodium acetate solution to change the pH to 4.8–5.0 (blue). The sample solution is covered and heated to boiling. The calcium is precipitated by slow addition (1 drop every 3–5 s) of 3% oxalic acid solution (w/v) until pH is 4.4–4.6, as indicated by a distinct green shade. The solution is then boiled for 1–2 min and allowed to settle overnight.
The supernate is filtered through quantitative paper, Gooch, or fine fritted glass filter, and the beaker and precipitate are washed with small portions of ammonium hydroxide (1 + 50). A mixture of 125 ml of water and 5 ml of sulfuric acid is added to the precipitate with heating to 80–90 °C. The solution is finally titrated at 70–90 °C with 0.01 M KMnO4 to a slight pink end point, 1 ml of permanganate solution equating to 1 mg of calcium…
6. VITAMINS | Fat-Soluble: Thin-Layer (Planar) Chromatography
W.E. Lambert, A.P. De Leenheer, in Encyclopedia of Separation Science, 2000
Detection
Quenching the fluorescence of the indicator fixed on the thin-layer plate itself (F254) is a very common and nondestructive way to localize spots on a TLC plate. Of course, this can also be applied to vitamin A compounds. Retinol and retinyl esters on the other hand can be identified by the yellow-green fluorescence they exhibit under 366 nm UV light. Other techniques to visualize vitamin A compounds include absorption of iodine vapours (with the formation of brown spots) or destructive procedures such as spraying with sulfuric acid.
Other spray reagents include SbCl3 or SbCl5 solutions in chloroform, a 5% solution of phosphomolybdic acid in ethanol and a mixture of equal volumes of a 1% aqueous solution of potassium permanganate and a 5% aqueous solution of sodium carbonate. After heating the plate coloured spots appear for vitamin A. The same reagents are often applied to visualize vitamins D and E.
As an alternative a large array of dyes has been evaluated as visualizing agents for fat-soluble vitamins, including vitamin A.
7. Petroselinum crispum: a Review
- Agyare, … J.A. Apenteng, in Medicinal Spices and Vegetables from Africa, 2017
7 Toxicity
The toxicity of P. crispum and its essential oil has not been thoroughly investigated. In ethnomedicine, it has been claimed that P. crispum is abortifacient. Photodermatitis due to furocoumarins particularly 55 are responsible for its contact photodermatitis activity in pigs exposed to P. crispum (Chaudhary et al., 1986). Eighteen sows of mixed age from an outdoor herd of 400 sows and boars were put in a field of parsley for 4–5 days and after this period, vesicles were noted on the snouts with erythema and skin fissures.
In an adjoining paddock of parsley, 14 out of 18 gilts were affected with lesions, principally on their ears. In other paddocks, up to 16 out of 18 sows showed similar lesions; suckling sows and those about to furrow were most severely affected. History, clinical signs, and pathology were consistent with phytophotodermatitis (Griffiths and Douglas, 2000).
Awe and Banjoko (2013) reported that ethanol leaf extract of P. crispum exhibited hepatotoxic and nephrotoxic activities determined by colorimetric method using bromocresol green and urease cleavage (Berthelot’s reaction) at continued oral doses equal to or more than 1000 mg/kg, but no obvious toxicity when used at lower doses (Awe and Banjoko, 2013).
8. Meenakshi Maruthamuthu and S Kishore
BCG has been used in the determination of serum albumin in blood (Spencer and Price 1977) and has high affinity for HSA (Bowmer and Lindup 1980). It exists in phenolic form (see structure) or in quinonoid form depending upon the pH. As the pK, of BCG is 4-7,
Naproxen and a non-polar ligand 1-anilino-8-naphthalenesulphonic acid (ANS) as competing ligands for BCG in serum albumins. They are found to bind strongly to HSA (Kober and Sjoeholm 1980; Sudlow et al 1975) and to BSA (Meenakshi Maruthamuthu and Kishore 1987b; Jun et al 1975). From their relative abilities to displace BCG by competitive or non-competitive mechanism, it is our aim to characterise the binding regions for the above two ligands in HSA and in BSA.
1. Experimental
Human serum albumin (HSA) and bovine serum albumin (BSA) (both are fatty acid-free), 1-anilino-8-naphthalenesulphonic acid (ANS) and the dialysis membranes were obtained from the Sigma Chemical Co., USA. Pure samples of Naproxen (gift from Cipla Ltd.) and bromocresol green (BCG) (from Sisco-Chem Industries) were used without purification. All experiments were carried out at pH 7.4 using Na2HPO4-KH2PO4 buffer of 0.05 M concentration. The concentrations of HSA and BSA were expressed on the basis of their molecular weights (69,000). A set of typical spectrophotometric experiments of BCG binding to HSA was carried out as follows: To a fixed [BCG] = 28-0/zM, varying [HSA] were added in different sample tubes. HSA concentrations of 1.0, 2.0, 3.0, 4-0, 6.0 and 8.0/xM were used. The mixtures were maintained at 25~ for 30 min with constant shaking.
Then the absorbance of BCG and BCG-HSA mixtures were measured at 621 nm using a Carl-Zeiss UV-Vis Specord spectrophotometer. It was found that 10.0/zM of HSA was required for the complete binding of 28.0 tzM of BCG. The above mentioned set of experiments were also made in presence of either Naproxen or ANS and the absorption measurements were made as before.
[Naproxen] and [ANS] were varied from 5.0 to 30.0 t~M to study the effect of competing ligands on BCG binding to HSA. Similarly experiments were also performed for the binding of BCG to BSA both in the absence and the presence of the competitors. [BSA] of 2.0, 4.0, 6-0, 8.0, 10-0 and 12.0 p~M were used.
Human serum albumin (HSA) and bovine serum albumin (BSA) (both are fatty acid-free), 1-anilino-8-naphthalenesulphonic acid (ANS) and the dialysis membranes were obtained from the Sigma Chemical .
2. Equilibrium dialysis experiments
In order to ensure that the change in physical property, i.e., absorbance of BCGalbumin, A l, on addition of the competitor is due only to displacement of BCG, an independent technique, namely, equilibrium dialysis was carried out as mentioned in the literature (Rosenberg and Klotz 1960). For the representative experiments, [BSA] of 4.0, 6.0 and 10-0 ~M were employed. The albumin solution was inside the dialysis membrane and equilibrated against 28-0 ~M of BCG without and with 10-0 ~M of ANS as competitor (both ligands taken outside) at 25~ for 48 h.
The binding of BCG onto the membrane was found to be negligible. After equilibration, the free [BCG] outside the membrane was determined by measuring ~ts absorbance at 621 nm. From this, CB and hence percentage of BCG bound in the absence and in the presence of ANS were determined
3. Results and discussion
When varying [serum albumin] is added to a constant [BCG], the environment around the chromophore is perturbed resulting in the decrease of absorbance without change in Amax as shown in figure 1A for the HSA-BCG system.
This suggests that the displacement of monomer-polymer equilibria of the protein (Sheppard and Geddes 1945) or stacking of bound dye molecules (Bradly and Wolf 1959) is not involved in causing the observed spectral changes.
This evidently also points to the presence of a single spectroscopic complex which implies that independent and identical sites were involved in the binding of BCG onto serum albumin. Figure 2 depicts typical double-reciprocal plots for HSA-BCG without and with [Naproxen] = 5.0 t~M to 20-0 p,M.
Linear regression analysis was adopted to determine the slope and intercept (i.e., 1/nK and 1/n), from which nK and n for the binding of BCG to serum albumins were determined.
Effect of competitors on the binding of BCG to serum albumin
Figures 1B and C show the effect of competing ligands ANS and Naproxen, respectively, on the binding of BCG to HSA. The addition of fixed [competitor] to serum albumin-BCG solution leads to the decrease of AA1 [see (1)].
This can be either due to displacement or change in the extinction coefficient of bound BCG. However, equilibrium dialysis experiments revealed that at fixed [BCG] = 28.0/xM and at varying [BSA] of 4.0, 6.0 and 10.0 txM, ANS of 10 txM was able to displace, respectively, 13.6%, 13.2% and 10.8% of bound BCG.
The corresponding values as obtained from spectrophotometric methods, respectively, were 14.1%, 13.0% and 11.3%. Hence the equilibrium dialysis experiment was found to corroborate the spectrophotometric method and thus confirm that the decrease in AA1 value in presence of the competitor was only due to displacement of BCG.
4. Acknowledgement
This work was supported by the Council for Scientific and Industrial Research, New Delhi. The authors wish to thank Cipla Ltd., India, for providing a pure sample of Naproxen.
5. References
Bowmer C J and Lindup W E 1980 Biochim. Biophys. Acta 624 260 Bradley D F and Wolf M K 1959 Proc. Natl. Acad. Sci. USA 45 944 Daniel E and Weber G 1966 Biochemistry 5 1893 EdsaU J T and Wyman J 1958 Biophys. Chem. 1 652 Jun H W, Luzzi L A and Joseph K H 1978
- Pharm. Sci. 64 493 Klotz I M, Walker F M and Pivan R B 1946 J. Am. Chem. Soc. 68 1486 Kober A and Sjocholm I 1980 Mol. Pharmacol. 18 421 Meenakshi M and Kishore S 1987 Proc. Indian Acad. Sci. (Chem. Sci.) 99 273 Muller W E 1978 Naunyn-Schmiedeberg’s Arch.
Pharmacol. 302 227 Rosenberg R M and Klotz I M 1960 in Analytical methods in protein chemistry (eds) P Alexander and R J Block (London: Pergamon) p. 133 Sheppard S E and Geddes A L 1945 J. Chem. Phys. 13 63 Spencer K and Price C P 1977 Ann. Clin. Biochem. 14 105 Sudlow G, Birkett D J and Wade D N 1975 Mol. Pharmacol. 11 824 Zia H and Kamali H 1976 Can. J. Pharm. Sci. 11 82 Zia H and Price J C 1975 J. Pharm. Sci. 64 1177
Question
1. What is the difference between bromocresol green and purple?
Background: The bromocresol green (BCG) assay is commonly used for measuring albumin (ALB), but is affected by α1- and α2-globulins, which are elevated in systemic inflammation. The modified bromocresol purple (mBCP) assay is another dye-binding method developed to overcome non-specific reactions.
2. What is the difference between bromocresol green and phenolphthalein?
Phenolphthalein turns from colourless to purple-pink at pH 8-10, and bromocresol green from yellow to blue at pH 4-5.5. The indicator colour was thus identical to bromocresol green’s until pH 9.135, when the phenolphthalein affected overall colour by turning purple-pink.29-Apr-2002
3. What Colours are bromocresol green?
Bromocresol green is a bio-based dye with a yellow-green to blue-green color. Bromocresol green turns yellow (λmax=435 nm, protonated form) when placed in acidic solution (e.g. pH=4.15), and turns blue in basic solution (λmax=615 nm, deprotonated form).
4. Why is bromocresol green a good indicator?
Bromocresol green (pKa = 4.66) is an acid-base indicator dye that exhibits a pH-dependent absorption spectrum. The only structural difference between the acid form (yellow) and the basic form (blue) of this dye is the protonation of a phenolic oxygen.15-Aug-2024
5. What is bromocresol green used for?
Bromocresol green serves as a pH indicator in biology and medicinal experiments. It is used widely as a vital stain to study blood-brain permeability.