[ 4 ] S U L F 1 T E : F U C H S I N M E T H O D 15

[4] Sulf i te D e t e r m i n a t i o n : F u c h s i n M e t h o d

By FRANZ-JOSEF LEINWEBER a n d KENNETH J. MONTY

The method of Grant for the colorimetric determination of sulfur diox- ide in extracts of animal and plant tissues ~ has been adapted to the study of microbial enzymes producing sulfite from substrates such as 3'-phos- phoadenosine 5'-phosphosulfate (PAPS), thiosulfate and cysteine sulfi- nate (CSA), both in oioo 2-4 and in vitro. 5,6 Grant's method was also ap- plied to the enzymatic determination of CSA. v,8 The method relies on the formation of a red chromophore with basic fuchsin (pararosaniline).

H,S()~ RCHO

Basic fuchsin" ," ~ SchiWs reagent \ , colored aldehyde addil ion product (colorless)

Fuchsin Reagent

Grant's fuchsin reagent is prepared by adding 1.6 ml of a 3% ethanolic solution of basic fuchsin (Fisher) to 93.6 ml distilled water containing 4.4 ml concentrated H2SO4. After thorough mixing, 0.4 ml 40% formaldehyde is added. The solution is decolorized by addition of 200 mg activated carbon. After repeated shaking over a period of 15 min, the solution is clarified by filtration. Fresh reagent is prepared daily.

Standard Procedure

To a 2.5 ml sample, add 0.5 m. 9f 1% alcoholic KOH, mix thoroughly, and add 1.0 ml of a saturated aqueous solution of HgCI> After mixing and centrifuging, 1.0 mi of the clear supernatant fluid is mixed with 4.0 ml fuchsin reagent. After 15 min at room temperature, the absorbance of each sample is measured at 580 nm. The color is compared with that produced from standardized samples of sodium bisulfite ranging from 80 to 480 nmol.

i W. M. Grant, Anal. Chem. 19, 345 (1962). 2 F.-J. Leinweber and K. J. Monty, Biochem. Biophys. Res. Commun. 6, 355 (1961). 3 F.-J. Leinweber and K. J. Monty, J. Biol. Chem. 238, 3775 (1963). 4 F.-J. Leinweber and K. J. Monty, J. Biol. Chem. 240, 782 (1965). 5 F.-J. Leinweber and K. J. Monty, Biochim. Biophys. Acta 63, 171 (1962). 6 j. Dreyfuss and K. J. Monty, J. Biol. Chem. 238, 3781 (1963). 7 F.-J. Leinweber and K. J. Monly, Anal. Biochem. 4, 252 (1962).

See this volume [31].

Copyrighl (~v 1987 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 143 All rights of reproduction in any form reserved.

16 SEPARATION AND ANALYSIS [ 4 ]

Expanded Sensitivity

The sensitivity of the assay may be increased to 5 nmol by the follow- ing modification. To a 0.25 ml sample, add 50/~1 1% aqueous KOH fol- lowed by 50/zl of a saturated solution of HgCI2 in absolute ethanol. Note the reversal of solvent for the two reagents between the two methods. Then combine 0.2 ml of the clear supernatant liquid obtained from that mixture with 1.0 ml fuchsin reagent, and measure the resulting color as described above.

Color Stability

Maximum color intensity is reached at approximately 15 min after addition of fuchsin reagent. Fading occurs slowly: after 60 min at room temperature approximately 85% of the color measured at 15 rain remains.

Specificity

The selectivity of the method depends on the removal of potential interfering compounds by precipitation with HgCl2.~ Both versions of the method have been used successfully without interference from PAPS (0.28 mM), CSA (2 raM), cysteic acid, hypotaurine, thiosulfate (all at 16 raM), or bacterial protein (14 mg/ml).

Preparing a Sulfite Standard

A standardized solution of sodium bisulfite may be prepared titrimetri- cally, using sodium thiosulfate as a primary standard. An iodine solution is titrated with a standard solution of thiosuifate. A parallel iodine sample is reacted with an aliquot of the bisulfite solution, and the remaining iodine is titrated with thiosulfate. The decreased volume of thiosulfate used in the second titration indicates the number of equivalents of bisul- rite introduced. Equations (i) and (2) explain the standardization:

3 1 _ , + 6S_,()~ ~ ~ 3 S ~ ( ) ~ , -~ + 6 1 ( l )

lz + S ( ) c ~ + H_,O ~ 2 HI + S()4 -~ (2)

Reagents

0.1 M Na28203 (primary standard; may be purchased as a standard solution or standardized against a true primary standard such as KIO3)

0.I M iodine (6.35 g crystalline iodine plus 20 g KI dissolved in distilled water, final volume 500 ml)

0.1 M NaHSO3 (1.0548 g in I00 ml water)

[ 5 ] S U L F I T E A N D T H I O S U L F A T E 1 7

Starch reagent (make a paste with 1 g soluble starch in 5 ml H20, pour into 200 ml boiling water, boil 1 min and cool; preserve with a few drops of chloroform)

Titrate a 50 ml aliquot of iodine solution with thiosulfate until the color turns pale yellow. Add 3 ml starch reagent. Continue the titration until the blue color just disappears, and record the volume of thiosulfate used as V~. To a second 50 ml aliquot of iodine solution, add 25 ml of the bisulfite solution to be standardized. Titrate as before with thiosulfate, and record the volume of thiosulfate as Vb. Calculate the molarity of the bisulfite solution as follows:

Molaritybi~ulfite = molaritythio~uif~te × (V~, - Vb)/50

The bisulfite solution is then diluted appropriately (e.g., 1 : 1000) for use as a standard in calibrating the fuchsin reaction.

[5] Sulf i te a n d Th iosu l f a t e : Us ing

S - ( 2 - A m i n o - 2 - c a r b o x y e t h y l s u l f o n y l ) - L - c y s t e i n e

By TOSH|HIKO UBUKA

Sulfite and thiosulfate are important metabolites of cysteine sulfur in animals.~ 3 The method described here utilizes S-(2-amino-2-carboxy- ethylsulfonyl)-L-cysteine (ACESC) as a reagent for converting inorganic sulfite and thiosulfate into organic cysteine derivatives. 4,5 ACESC was first prepared by Toennies and Lavine by oxidizing L-cystine in nonaque- ous media, and its structure was proposed to be cystine disulfoxide. 6

A. Meister, "Biochemistry of the Amino Acids," 2nd ed., Vol. 2, p. 757. Academic Press, New York, 1965.

2 A. B. Roy and P. A. Trudinger, "The Biochemistry of Inorganic Compounds of Sulphur," p. 289. Cambridge Univ. Press, London and New York, 1970. L. M. Siegel, in "Metabolic Pathways" (D. M. Greenberg, ed.), 3rd ed., Vol. 7, p. 270. Academic Press, New York, 1975.

4 T. Ubuka, M. Kinuta, R. Akagi, S. Kiguchi, and M. Azumi, Anal. Biochem. 126, 273 (19821. T. Ubuka, N. Masuoka, H. Mikami, and M. Taniguchi, Anal. Biochem. 1411, 449 ( 19841. G. Toennies and T. F. Lavine, J. Biol. Chem. 113, 571 (1936).

Copyright (c) 1987 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 143 All rights of reproduction in any torrn reserved.