Spectrophotometric determination of disulfides, sulfinic acids, thio ethers, and thiols with the palladium (II) ion

ANALYTICAL

BIOCHEMISTRY

8, 223-228 (1964)

Spectrophotometric Acids,

Determination Thio

Ethers,

and

Palladium STIG

of Thiols

(II)

AKERFELDT

AND

Disulfides, with

the

Ion

GUNN

LWGREN

From the Research Institute of National Defence, Department Sundbyberg, Sweden Received November

Sulfinic

1,

1, 1963

The interaction of palladium(I1) ions with compounds containing sulfur has been the subject of extensive studies at this Institute. The aim for these investigations has been to obtain analytical methods for certain sulfur-containing phosphorus compounds. A recent report by Haglund and Lindgren (1) thus describes the use of palladium(I1) ions in the determination of a series of phosphonothioates. In addition, these authors report the use of palladium(I1) ions for the quantitative estimation of cysteine and some related thiols. The absorption of the formed thiol-palladium complexes was measured in the ultraviolet. The investigation by Haglund and Lindgren has now been extended, and it has been shown that a number of sulfur-containing compounds of biochemical interest, such as disulfides, sulfinic acids, and thiols give yellow-colored complexes with palladium (II) ions, which can conveniently be determined in the visible region of the spectrum. The method developed for quantitative determination of these sulfur compounds is extremely simple and sensitive. It should be especially valuable for the estimation of disulfides and sulfinic acids, since simple calorimetric methods for the determination of these compounds are lacking. RECOMMENDED

METHOD

To 0.1 ml of sample (containing O-l pmole of sulfur compound) was added 0.5 ml of reagent solution (containing 20 ~moles/ml of ammonium chloropalladite, (NH,),PdCl,, in 1 M HCl), followed by 2.4 ml of 1 iIf HCl. The color was allowed to develop for 5-60 min depending on the nature of sulfur compound present. Thiols and sulfinic acids react momentarily, and readings were usually performed after 5 min, whereas disulfides require a longer time for full color development (60 min). The colors obtained are stable. For choice of wavelengths see Table 1. 223

MOLAR

EXTINCTION COMPLEXES

TABLE 1 (e) FOR THE PALLADIUM-SULFUR AND A LIST OF NONREACTIVE

COEFFICIENTS’ INVESTIGATED

Compound

Abs max. nlp

-

Thiols Cysteamine L-Cysteine L-Cysteine ethyl ester L-Ergothioneine Glutathione nn-Homocysteine Mercaptoacetic acid 2-Mercaptoethanol L-2-Mercaptohistidine Mercaptosuccinic acid nn-Penicillamine Disulfides Cystamine n-Cystine Glutathione disulfide 2,2’-Dithiodibensoic acid Sullinic acids Benzenesullinic acid Cysteinesulfinic acid Hypotaurine Thio ethers DL-( +)-Allocystathioneine S-Ethyl-n-cysteine L-(+)-Mesolanthionine n-Methionine Other reactive sulfur compounds S-Acetylglutatione N-Acetylhomocysteine thiolactone Homocysteine thiolactone Sodium bisulfite Sodium thiophosphate Sodium thiosulfate Nonreactive compounds Adenosine Adenosine 5’-triphosphate n-Alanine n-Arginine n-Aspartic acid Choline n-Cysteic acid Cytosine Ethylenediaminetetraacetate

c. 10-a

410 350 360 415 380 375 385 415 415 415 415

2.4 2.6 3.3 8.0 1.5 2.2 2.6 2.8 17.2 1.9 9.5

415 415 415 415

2.4 5.8 1.3 7.1

415 415 415

1.6 1.8 1.0

415 410 410 410

0.62 0.80 0.75 0.80

400 400 400 380 390 390

0.8 0.8 0.7 2.4 8.0 11.0

L-Histidine nn-5-Hydroxytryptophan Hypoxanthine Inosine on-Methionine sulfone on-Methionine sulfoxide Potassium cyanate L-Proline on-Serine Sodium phosphate Taurine Tris(hydroxymethyl)aminomethane

Glycine Guanosine 0 Approximate

COMPOUND COMPOUNDS

values. 224

DETERMINATION

OF

SULFUR

225

COMPOUNDS

For the recording of spectra a Beckman DB recording spectrophotometer was used. Standard curves (Fig. 2) were obtained with the aid of a Beckman B spectrophotometer. All chemicals used in this investigation were of the highest available purity. RESULTS

Choice of Reaction Conditions. In neutral or slightly acid solution palladium(I1) ions give colored complexes with a number of compounds. In strongly acid solution, however, the reaction is much more specific for compounds containing sulfur (cf. Table 1). Therefore 1 M HCl has been chosen as a suitable solvent for the color development. The absorption spectra of the complexes between palladium(I1) ions and various sulfur compounds in 1 M HCl usually show two absorption maxima between 360 and 415 rnp. In addition, a maximum in the ultraviolet at 280 rnp was found for all complexes investigated. The molar extinction coefficient at absorption maximum in the visible region is of the order of lo3 to I@; at 280 rnp it is of the order of loj to 10’;. Since these investigations were carried out mainly with the aim of developing a method suitable for biochemical samples, the absorption of the palladium-sulfur compound complexes was investigated in the visible region of the spectrum. Interference by the numerous ultraviolet-absorbing compounds present in tissue extracts is thus largely avoided. Efiect of Concentration of Reactants. Figure 1 describes the effect of increasing concentration of palladium(H) ions on color formation with

l-l OO

I

I

I

I 5

I

I

I

MOLAR

FIG. 1. Effects of palladium(I1) (0.37 mM in 1 M HCI). Values 360 rnfi.

I

I R&O

I

I

I

I

Pd2&‘STE,N

ion concentration on color yield with cysteine are corrected for palladium(I1) absorbance at

226

AKERFELDT

AND

LijVGREN

cysteine. At a molar ratio Pd2+/cysteine of about 7, the maximum color available from the interaction has been obtained. Studies on other sulfur compounds have shown similar saturation curves, and the recommended molar ratio Pd?+/sulfur compound is 210. With the recommended molar ratio Pd2+/sulfur compound, Beer’s law is closely followed for all compounds investigated (Fig. 2).

p MOLES

PER

SAMPLE

FIG. 2. Calibration curves for cystine (CSSC, 415 mp), cysteinesuliinic acid (CSO,H, 350 mp), glutathione (GSH, 380 mp), and glutathione disulfide (GSSG, 415 ma).

Specificity of the Reaction. All tested compounds containing divalent sulfur react with the palladium reagent. Of these, compounds containing a thio ether bridge gave the lowest color yields. In addition, compounds containing easily oxidizable sulfur, like sulfinic acids and bisulfite, give highly colored palladium(I1) complexes. On the other hand, sulfur compounds exist which do not react with the palladium reagent. Examples are sulfonic acids (taurine and cysteic acid) and the sulfone and sulfoxide of methionine. Of a large number of tested nonsulfur compounds of biochemical interest, none has been found that gives a yellow complex with palladium(U) ions under the conditions of the method, which therefore seems to be highly specific for sulfur compounds. However, certain substances, like methionine sulfone, when present in large amounts, slightly decolorize the palladium reagent. A summary of the results is presented in Table 1. DISCUSSION

The fact that disulfides than the other investigated

react more slowly with palladium(I1) ions groups of sulfur compounds can be utilized

DETERMINATION

OF

SULFUR

227

COMPOUNDS

analytically. This is exemplified in Fig. 3, which demonstrates the separate quantitative determination of a thio ether (S-ethylcysteine) and a disulfide (cystine) in the same solution. The thio ether reacts momentarily with the palladium(H) ions, and the amount of this substance is determined by the absorbance immediately after the mixing of sample and reagent. At this moment, the disulfide present has not yet reacted with the reagent. After 60 min, however, all the disulfide present has reacted, and a measurement of the absorbance at this time gives the sum of the thio ether and the disulfide concentrations. A quantitative determination of the added substances is thus obtained (Fig. 3).

TIME, MN

FIG. 3. Determination of S-ethylcysteine same sample. For interpretation see text.

(EtSC)

and cystine

(CSSC)

in the

Although the method presented does not dis6inguish between the various fast-reacting sulfur compounds, it would seem possible to determine a pair of them in a mixture by applying the palladium method in combination with other methods. For instance, a thiol present can be separately determined by the method of Grunert and Phillips (2). Preliminary experiments have shown that the palladium(I1) ion momentarily splits the S-P bond in S-phosphorylated thiols, such as cysteamine S-phosphate (3, 4). A palladium thiolate and orthophosphate are thus formed. The thiol part of S-phosphorylated thiols can therefore be conveniently determined with the palladium method without previous hydrolysis of the substances. SUMMARY

Palladium(I1) ions are shown to give colored complexes with a variety of suIfur compounds. This reaction has been utilized for the quantitative determination of disulfides, sulfinic acids, thio ethers, and thiols. A

228

AKERFELDT

AND

LihREN

method is outlined for the separate determination of a disulfide another sulfur compound, like a thio ether, in the same solution. specificity of the reaction has been investigated.

and The

REFERENCES 1. 2. 3. 4.

HAGLUSD, GRUNERT, AKERFELDT, AKEFWELDT,

H., AND LINDGREN, I., Tahta, in press. R. R., AND PHILLIPS, P. H., Arch. Biochem. S., Acta Chem. Scud 13, 1479 (1959). S., Svensk Kern. Tidskr. 75, 231 (1963).

Biophys.

30, 217

(1951).