83, 185-188



Detection of Haloacetyl and Haloketone Labels on Paper Chromatogramsl



of Tennessee and Biology

-Oak Ridge Graduate School Division, Oak Ridge National Oak Ridge, Tennessee 37830

of Biomedical Laboratory,3


Received May 9, 1977; accepted July 19, 1977 Thiol-reactive substances on chromatograms can be detected conveniently with a single spray reagent composed of Ellman’s reagent and reduced glutathione. Data pertaining to the visualization of reactive halogen compounds are presented.

Affinity labeling-the use of active-site-directed irreversible inhibitors of enzymes-has become a prevalent technique in recent years (l-4). Two widely used types of affinity labels are compounds that contain haloacetyl (5) or haloketone (6) groups. A facile and general method to detect these compounds on chromatograms should be useful to investigators who design new affinity-labeling reagents, as current detection methods are rather inconvenient (7,8). We have developed a single spray reagent prepared with Ellman’s reagent (9) which will detect thiol-reactive compounds on paper chromatograms. MATERIALS


3-Bromo-1,4-dihydroxy-2-butanone 1 ,Cbisphosphate; 3-chloroacetol phosphate; 3-iodoacetol phosphate (and their respective ketals); and N-bromoacetylethanolamine phosphate were synthesized by published procedures (lo- 12). Iodoacetic acid, iodoacetamide, and bromoacetic acid were purchased from Aldrich Chemical Co.; chloroacetic acid and bromoacetamide were purchased from Eastman Kodak Co. and Chemicals Procurement Laboratories Inc., respectively. ’ The U.S. Government’s right to retain a nonexclusive royalty-free license in and to copyright covering this paper is acknowledged. 2 Predoctoral Fellow supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health. 3 Operated by the Union Carbide Corporation for the Energy Research and Development Administration. 185 Copyright All rights

Q 1977 by Academic Press. Inc. of reproduction in any form reserved.

ISSN 0003.2697





Compound (1

Time for visualization (min)

Iodoacetol phosphate Iodoacetamide Bromoacetamide Iodoacetic acid N-bromoacetylethanolamine phosphate Bromobutanone bisphosphate Bromoacetic acid Chloroacetol phosphate Chloroacetic acid

0.2 2 3 5 5 5

10 10 120

a One application (2.5 pi) of a 5 mM solution was made to Whatman No. 1 filter paper. The paper was sprayed with the spray reagent described in Materials and Methods and allowed to air-dry. All of the above compounds were visualized within 1 min if the paper was placed in an oven at 110°C after spraying.

An aqueous solution consisting of 0.1% (3.2 mM) reduced glutathione (Sigma Chemical Co.), 0.25% (6.3 mM) Ellman’s reagent (Aldrich Chemical Co.), 2% (0.24 M) sodium bicarbonate, and 0.05% (1.2 mM> tetrasodium EDTA was used to spray chromatograms. Components were visualized by air-drying the chromatograms or by heating them in an oven at 110°C. RESULTS AND DISCUSSION

The spray solution detects thiol-reactive compounds as follows: The glutathione (present in limiting molar amounts) reduces a portion of Ellman’s reagent [5,5’-dithiobis(2-nitrobenzoic acid)] to 5-thio-2-nitrobenzoic acid, which exists predominantly in the highly reactive, highly chromophoric (E = 13,600 at 412 nm) (9,14) thiolate anionic form at pH 8 (maintained by the excess bicarbonate). Any compound on a chromatogram that will react with and therefore consume the thiolate will appear as a white spot on a yellow background. Other reducing agents (i.e., 2mercaptoethanol and dithiothreitol) can be used instead of glutathione with comparable results. Times required to visualize several compounds after spraying with reagent are given in Table 1. The ketals (see Materials and Methods) tested were not visualized within 24 hr at room temperature or 1 hr at 110°C using the conditions specified in Table 1. Spots developed from the haloketones tended to fade after several hours, while spots from the haloacetyl compounds remained visible for days. Optimal detection of compounds on paper chromatograms was ob-











12.5 25 50 100 200 400 N-bromoacetylethanolamine phosphate (nmol) FIG. 1. (A) N-Bromoacetylethanolamine phosphate was subjected to descending chromatography on Whatman No. 1 paper for 16 hr with butanokacetic acid:water (7:2:5) as solvent. The chromatogram was sprayed with the reagent of Hanes and Isherwood (13), dried, and exposed to uv light. (B) Same as (A), except that the: spray for detection of thiol-reactive compounds (see Materials and Methods) was used . (C) Same as for (A), except that N-bromoacetylethanolamine phosphate was first visu,alized with the spray for detection of thiol-reactive compounds; the same chromatogran 1 was then oversprayed with the reagent for phosphate esters.



tamed with a 100-nmol application or greater. Figure 1 shows localization of N-bromoacetylethanolamine phosphate on a paper chromatogram with the spray that detects thiol-reactive compounds and the spray reagent of Hanes and Isherwood (13) for phosphate esters. Both sprays can be used on the same chromatogram if the spray for thiol-reactive compounds is used first (Fig. 1C). We have also used the spray solution on thin-layer chromatograms with good results. While the data presented include only detection of reactive halogen compounds, it is assumed that other thiol-reactive compounds (e.g., epoxides, nitrogen mustards, oxidizing agents, maleimides, acid halides) could also be visualized. REFERENCES 1. Shaw, E. (1970) Physiol. Rev. 50, 244-296. 2. Shaw, E. (1970) in The Enzymes (Boyer, P. D., ed.), 3rd ed., Vol. 1, pp. 91-146, Academic Press, New York. 3. Baker, B. R. (1967) Design of Active-Site-Directed Irreversible Enzyme Inhibitors, Wiley, New York. 4. Singer, S. J. (1%7) Advan. Protein Chem. 22, l-54. 5. Naider, F., Becker, .I. M., and Wilchek, M. (1974) Zsr. J. Chem. 12, 441-454. 6. Hartman, F. C. (1977) Methods Enzymol. (in press). 7. Hashmi, M. H., and Cullis, C. F. (1956) Anal. Chim. Acta 14, 336-338. 8. Baker, B. R., Santi, D. V., Coward, J. K., Shapiro, H. S., and Jordaan, J. H. (1966) J. Heterocycl. Chem. 3, 425-434. 9. Ellman, G. L. (1959) Arch. Biochem. Biophys. 82, 70-77. 10. Hartman, F. C. (1975) J Org. Gem. 40, 2638-2642. 11. Hartman, F. C. (1970) Biochemistry 9, 1776-1782. 12. Hartman, F. C., Suh, B., Welch, M. H., and Barker, R. (1973) J. Biol. Chem. 248, 8233-8239.

13. Hanes, C. S., and Isherwood, F. A. (1949) Nature (London) 14. Silverstein, R. M. (1975) Anal. Biochem. 63, 281-282.

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Detection of haloacetyl and haloketone affinity labels on paper chromatograms.

ANALYTICAL 83, 185-188 BIOCHEMISTRY (1977) Detection of Haloacetyl and Haloketone Labels on Paper Chromatogramsl Affinity JOHN V. SCHLOSS~ AND F...
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