Calcofluor White

Determination of the Calcofluor White Molar Extinction Coefficient Value in the Absence and Presence of -Acid Glycoprotein... 

Calcofluor White is commonly used to study the mechanism by which cellulose and other carbohydrate structures are formed in vivo and is also widely used in clinical studies (Andreas et al. 2000 Green et al 2000 Srinivasan 2004 Doctor Fungus website, Candida Endoph thalmitis). 

The interaction between Calcofluor White and carbohydrate residues of a -acid glycoprotein depends on the secondary structure of the carbohydrate residues, with the fluorescence parameters of Calcofluor being sensitive to this spatial secondary structure. 

When dissolved in water, the fluorescence maximum of Calcofluor White is 435-438 nm, while in alcohol such as isobutanol or when bound to human serum albumin, it fluoresces at 415 nm. In the presence of -acid glycoprotein, the fluorescence maximum of Calcofluor shifts toward 439 nm when the fluorophore is at low concentrations and toward 448 nm when it is present at high concentrations. The shift, compared to water and observed in the presence of a -acid glycoprotein, is the result of Calcofluor binding on the carbohydrates (40% by weight) of the protein (Albani and Plancke 1998,1999). 

To the sample cuvette, add with a pipette Pasteur very small quantity of Calcofluor White powder, then mix slowly. 

Plot absorption spectrum of Calcofluor White from 190 to 450 nm. 

Compare the absorption spectra for Calcofluor and oq -acid glycoprotein what do you notice Do you have identical spectra, and can you explain the result At which wavelength(s) do you have to determine the value of e of Calcofluor White, and why ... 

Determination ofe value of Calcofluor White free in PBS buffer... 

Prepare in PBS buffer a stock solution of Calcofluor White equal to 4.24 mg ml-1. To a cuvette containing 1 ml of buffer, add aliquots of 5 /xl from the Calcofluor White stock solution. After each addition, mix the solution slowly and measure the optical density at the wavelength you have chosen from the results you obtained in Section 2.3.1. Add at least 10 aliquots, and repeat the experiment twice. 

Once you finished the measurements, plot the optical density as a function of Calcofluor White concentration expressed in mg ml-1. What type of plot do you obtain, and why How do you expect to calculate the value of s from the plot you obtained The molecular weight of Calcofluor is equal to 942.7. Can you give the value of s in M-1 cm-1 ... 

Determination of Calcofluor White e value in the presence of oq -acid glycoprotein... 

Repeat the experiment described in Section 2.3.2 in the presence of 10 ptM of oq-acid glycoprotein (the e of the protein at 278 nm in PBS buffer, determined from the same method you are using to calculate the e of Calcofluor White, is equal to 29.7 mM-1 cm-1). 

Determination of Calcofluor White Molar Extinction Coefficient Value... 

In presence of 10 //M 04 -acid glycoprotein, a value of s equal to 4.15921 g-1 1 cm-1 or 3920 M-1 cm-1 is obtained. The value of e for bound Calcofluor White is around 9% lower than that of free Calcofluor in solution. 

Figure 2.5 Optical density plots of calcofluor white at 352.7 nm as a function of its concentration in (a) the absence and (b) the presence of 10 p.M -acid glycoprotein. In the absence of protein, s = 4.65443 g 1 I cm-1 = 4387.76 M 1 cm 1. In the presence of the protein, s = 4.15921 g-1 I cm 1 = 3920 M cm-1. Values of e are not the same the value of e for bound calcofluor white is around 9% lower than that of free calcofluor in solution.

Modification in the value of e is the result of structural reorganization in the vicinity of Calcofluor White. The presence of a protein alters the electronic distribution within the ligand, inducing a modification of absorption spectrum properties. 

Albani, J.R. and Plancke, Y.D. (1998 and 1999) Interaction between Calcofluor White and carbohydrates of ai-acid glycoprotein. Carbohydrate Research 314, 169-175 and 318, 194—200. 

Albani, J.R., Sillen, A., Coddeville, B., Plancke, Y.D. and Engelborghs, Y. (1999) Dynamics of carbohydrate residues of ai-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and emission anisotropy studies of Calcofluor White. Carbohydrate Research 322,87-94. 

Endophthalmitis.htm + clinical + studies, + Calcofluor + white hl = fr [accessed on 20 March, 2006]. 

Green, L.C., LeBlanc, P.J. and Didier, E.S. (2000) Discrimination between viable and dead encephalitozoon cuniculi (microsporidian) spores by dual staining with Sytox Green and Calcofluor White M2R. Journal of Clinical Microbiology 38, 3811-3814. 

We will show here the example of an analysis method concerning the fluorescence emission of Calcofluor. First, with a Pasteur pipette, take a small amount of powder of Calcofluor White and dissolve it in 1 ml of phosphate buffer present in the fluorescence cuvette. Then, measure the OD at 352.7 nm and calculate the fluorophore concentration in the cuvette. The value of s at this wavelength is 4388 M-1 cm-1. In principle, you should have a Calcofluor solution concentration of 150-200 ptM in the fluorescence cuvette. 

Other vital stains take advantage of different cellular properties which can be correlated with cellular physiology Propidium Iodide, Ethidium Bromide, Ethidium Monoazide, Calcofluor White have been widely used to indicate the presence of dead eukaryotes or prokaryotes cells. 2-(p-iodophenyl-)3)(p-nitro-phenyl)-5-phenyl tetrazolium chloride (INT) belongs to a class of stains which can be used to determine if a cell or hyphal compartments [180] can maintain an internal reducing environment (Fig. 20a). There are, however, still a large debate about the reliability of those techniques, depending upon the cells under consideration [181]. Calcofluor (Aex = 380 nm, Aem 420 nm) is a specific cell wall stain which enables to counts buds scars on Saccharomyces cerevisiae [29] to estimate the age of a cell. 

The fluorochromes, Calcofluor White and Congo red bind to chitin and cellulose, while acridine orange binds to nucleic acids of the fungi. These dyes may be helpful when there is a lack of fungal elements (blastospores, hyphae or pseudohyphae) in the specimen, as even few elements are visible under the fluorescence microscope. By using specific antibodies conjugated with the fluorochromes, it should be possible to identify specific fungi. 

The values of of a cliromophore can change whether it is free in solution or bound to a protein. This change can be important or non-significant. For example we determined the value of of calcofluor white free in solution and bound to ai-acid glycoprotein which contains 40% carbohydrate by weight. Calcofluor White binds specifically to the hydrophilic carbohydrate residues (Albani and Plancke, 1998 1999), although interaction with hydrophobic pockets of some proteins such as serum albumin occurs also (Albani and Plancke, 1998 1999). 

Figure 1.14. Optical density plots of calcofhior white at 3S27 iwn as a lunclion of its codcentiatian in absence (a) and in presence (b) of 10 pM o -acid glycoprotein, tn the absence of protein, e — 4.6S443 g I cm 4387.76 M crni. In pieseiKe of the prolebu e — 4.15921 g I cm =3920 M cm. Wenoticelhatthevaliiesofcarenotlhesame.lhevalueofcfa bouid calcofluor white is around 9 % lower than that of tree calcofhjor in so ulion.

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