44
-..--.-
MOLES/ LITER X IO3 Figure 2. Changes in absorbance of 1.5 X 10-%i
I 0
I
10
A MINUTES
t W w
much slower, a final value being obtained a t 55 minutes which corresponded to the presence of 0.60 sulfhydryl groups per albumin molecule. This is comparable to the value of 0.71 obtained by amperometric titration (?') but lower than the value of 1.0 shown by niercurimetric determination ( 3 ) . The reaction of S-ethylmaleimide M ith myokinase, as judged by inactivation of the enzyme, also took place slowly at pH 7 . 5 (6). Variations of the method described should prove useful in a variety of biochemical studies.
N-ethylmaleimide as function of glutathione or cysteine concentration
Figure 3. Rates of reaction of cysteine and bovine plasma albumin with N-ethylmaleimide a t 25" C.
(1) Benesch, Reinhold, Benesch, R. E.,
0.1M phosphate buffer, p H 6.0
Points plotted from continuous curves of spectroDhotornetric record
Science 123, 981-2 (1956). ( 2 ) Boyer, P.D., J . Am. Cheni. SOC.7 6 ,
-Decrease a t 300 rnb ._.____._ Increase a t 248 rnb
tral characteristics a t these wave lengths (curve 6). Solutions of 1.5 X l O - 3 J f N-ethylmaleimide with increasing concentrations of cysteine up to 1.5 x 10-3M produced progressive decreases in absorbance a t wave lengths between 265 and 340 mp and increases between 238 and 265 mp (curves 2 to 5 are typical). There was an isomolar reaction between cysteine and 'V-ethylmaleimide. Similar results were obtained with glutathione. The results in Figure 2 show the changes in absorbance a t 300 and 248 mp as a function of the concentration of cysteine or glutathione. Linearity of response was obtained over the range tested,
with relative change greatest a t 300 mp. Similar plots could be made for the changes in absorbance a t other wave lengths. The slightly greater changes in absorbance obtained for glutathione than for cysteine at comparable molarities are probably due to small amounts of sulfhydryl inipurities in the sample of glutathione used. Results of kinetic experiments in which reaction rates of S-ethylmaleimide with cysteine and bovine plasma albumin were measured are shown in Figure 3. The stoichiometric interaction of cysteine and Y-ethylmaleimide was complete in 2 minutes after mixing, agreeing with a previous report (6). The reaction with bovine albumin was
LITERATURE CITED
Gutcho, hIarcia, Laufer, Louis,
4331-7 (1954). (3) Fridovich, Irtvin, Handler, Philip, ASAL. CHEJI.29, 1219-20 (1957). (a) Friedmann, E., Biochim. et Biophys. Acta 9, 65-75 (1952). ( 5 ) Friedmann, E., Marrian, D. H., Simon-Ruess, I., Brit. J . Pharmacol. 4, 105-8 (1949). (6) Gregory, J. D., J . A m . Chem. SOC.77, 3922-3 (1955). ( 7 ) Jensen, E. V., Hospelhorn, L-. D., Tapley, D. F., Huggins, Charles, J . Biol. Cheni. 185, 411-22 (1950). (8) Price, C. A., Campbell, C. W., Riochenz. J . 65. 512-16 (1957). ( 9 ) Tsou, Kmn-Ch&g, Barrnett, R. J., hligman, A. AI,, J . Ani. Chem. SOC.77, 4613-16 (1955). RECEIVED for review September 13, 1957. Acctpted February 25, 1958. Investigation supported by research grants C-2568 and C-3134 from the Sational Cancer Institute, C. S. Public Health Service.
Spectrophotometric Assay for S uIf hyd ryI Groups Using N-Ethylmaleimide NICHOLAS M. ALEXANDER Radioisotope Service, Veterans Administration Hospital, West Haven, Conn., and the Department o f Biochemistry, Yale University, New Haven, Conn,
b A rapid, simple spectrophotometric method for determining thiols with N-ethylmaleimide is presented. The reaction is observed a t 300 mp. Sulfhydryl solutions having concentrations as low as 0.0001M can b e assayed. The procedure has been successfully used to determine sulfhydryl concentrations in a tissue extract and in whole blood.
N
(SEIf) reacts rapidly with sulhydryl compounds a t neutral p H (4, 6). The rate -ETHYLhfALEIhlIDE
1292
ANALYTICAL CHEMISTRY
of reaction of equimolar amounts of X-ethylmaleimide and reduced glutathione has been followed spectrophotometrically by a decrease in absorption of the former a t 300 mp (5),but the reaction does not go to completion under these conditions. Roberts and Rouser ( 7 ) showed that the change in absorbance a t 300 mp is proportional to the concentrations of cysteine and glutathione when S-ethylmaleimide is present in excess. They used this method to determine the extent to which bovine serum albumin reacts with .Y-ethylmaleimide.
The present report also shows that, \Then present in excess, S-ethylmaleimide reacts stoichiometrically with sulfhydryl compounds. The decrease in absorption of this compound a t 300 nip can be used as an assay method for sulfhydryl groups. EXPERIMENTAL
Materials. Reduced glutathione (GSH) and S-ethylmaleimide were obtained from Schn-arz Laboratories, Inc.. J l o u n t Vernon, S . Y. Cysteine
Iiydrocliloi~ide ant1 nierc,:il)toetliaiiuI ivwc pui,clinsctl froin ICastnian Organic Chemicals, Rochester, S . Y. Thioglycolic acid wxs a product of Evans ('hemeticas, Iiiv., S e w York, S. Y., :inti was 1aI)c~lerias !H%,pure. IIoniostallinc egg alhuniin \vert bought froiii Kutritional Biochemicals Corp., Cleveland, Ohio. Ergothioneine and thiolhistidine were obtained from the California Foundation for Biochemical Research, Los Angeles, Calif. A lioileti aqueous extract of rat liver was prepared as dcscri1)ctI ( 1 ) . .~-l,;t,liyliii~il(,aiiii(~ :itid XIS prep:irecl 1)y :itl(ling 0.3 nil. of 0.51- sotlimn hyclroritle to 1 ml. of 0.01J1 lV-ethylmaleimide and allowing to stand for 5 minutes. Six milliliters of 0 . U phosphate buffer ( p H 6.8) were added to neutralize the solution before carrying out the reaction of glutathione with N-ethylmaleimide. N-Ethylmaleimide was stored in the refrigerator and the molar extinction coeficient was determined on a saniple d i i c h had been tlrice recrystallized from ethanol. Once this coefficient is k n o i ~ n ,the highly pure comnierc,ial .\'-cthyliiialeimide can he used directly, 1)ecniiw any slight contaminants in the solid material or the drgradation products fornicd after it is Iirouglit, into solution do not) react with thiol groups. All the solid thiol compounds were dried to constant weight in vacuo (5.5 nim.) a t 55' C.,just prior to use. Thc liquid compounds were diluted directly to a desired concentration, Prior to reacting with .Y-ethylmaleimide, the concentrations of the thiol solutions wcre determined by iodometric titration. The titrations sho\\-ed that' glutathione was 99.470 pure and that cysteine hydrochloride contained 101.7% cysteine. The value for cysteine hydrochloridc is explained by the fact that it loses hydrochloride ( 3 ) . Homocysteine was 98.5% piire. Thiolhistidine and ergothioneine had negligible reducing action in the acid iodonietric titration, in agreement with the earlier observation on er othioneiiie (10). Procedure. ie reaction is usually carried out in 0.1111 phosphate buffer (pH 6 3 ) with 0.001M S-ethylmaleimide and a sulfhydryl concentration lrss t h a n 0.0009.11 and greater than A 0.001119 S-ethylmale0.0001N. imide solution in buffer is also prepared and the ahsorb:iiices of both solutions arc read at 300 mp. Solutions containing ercrything but I\'-ethylmaleimide serve as blanks. The difference in absorption lietween the reacted and unreacted .\~-cthyltiiaIeimide solutions is divided 11)- the molar extinction coefticient of t,he compound ; this quotient is equal to the niolar sulfhydryl concentration of the sample. The spectrophotonietric nieasiirenieiits are made in 1-em., matched silica cells in a Beckman D r spectrophotometer. The concentration of reactants may lie varied as long as the X-ethylmaleimide concentration is in 10% excess of the sulfhydryl concentration. The reaction can lie carried out, at neutral or acid pH ( 7 ) , although it proceeds soniewliat~faster u t neutrality ( 5 ) .
4
Table I.
Reaction of N-Ethylmaleirnide with Glutathione in Presence of NEthylmalearnic Acid
0 00lJ1 SEA1 aged ior 1 ~ e e k
$0 000531 GSH + O 0OlJl S-rtlil Ininlcwnic 'rcitl
:3
1 1
0 547
0 237 0 5i0
0 310
Determination of Sulfhydryl Content of Rat Liver Extract (Totd voliinir 0 1 c~:ic11rt.:ic+iori mixtiire, 1U nil.) Liver Calcd. Extract, GSR Absorbance pmoles XEM, 10 20 M g . / Added, at 3g0 A Sulfhydryl pmoles h1l.a kmoleu M p Absorbance Groups ... ... 0.625 0 2.53 1 ... 0.468 0: iSi 2 ... 0.315 0.310 5.00 ... 2.0 0,500 0.125 2.01 ... 4.0 0.378 0.247 3.98 1 2.0 0.346 0.279 4.50 1 4.0 0.220 0.405 6.53
Table II.
Flask So. 1 2 3 4 5 (i r
I
'1
++ ++ ++ +
Concr,ntratiori i n tcv-mn of rionvo1:itile orgaiiiv mntwid and determined ncrortling to illinc. I)ovine scwim ulljiiniin m stantlard. rmiiixtioii mnt:Liii(d : ~ l lcomponcmts except SI'.2Z :IF indica:itcd.
RESULTS
The molar extinction coefficient of S-ttliT-liiialeimide a t 300 n i p is 620. This agrccs n ith Grcgorv's rrsiilt (6) ohtained a t 302 nip. When varying eonccntrations of glutathione react with S-ethylmaleimide, the per cent decrease in absorbance of the latter a t 300 mp is exactly equal to the per cent of glutathione concentrntioii in relation to the S-ethylmaleimide concentration. Identical results, within euperinientsl error, \\ere obtained for homocysteine, mercaptoethanol, cysteine, and thioglycolic acid. The assav is unaffected by the presence of 18 other amino acids, Duponol (nu Pont), ascorbic acid, glucose, arid T'ersene. The reaction appears to be highly specific for sulfhydrvl g r o u p . Thiolhistidine and ergothioneine, lion ever, failed to react n ith S-ethylinnleirnide. S o positive nitroprusside test m s obtained with these preparations, in agreement n ith the work of other inveytigators (9). Honioc\rsteine, thiolactone, cysteine, and oxidized glutntliione do not react with S-ethylnialeimide under these conditions. .~-Ctliylnialeimidespontaneously decomposes in 0.1V phosphate (pH 6.8) a t a fairly slon- rate when stored in the refrigerator, as seen in experiment 3 of Table I . Thc decomposition product is S-ethylmaleamic acid, which does not ahsorb light a t 300 nip. If S-etli> 1nialeaniic acid could react with sulflivrlryl groups a t an appreciable rate. frmhly pre.pare.tl S-cthJ Imaleimide solutions n o d d Iiav(, to hc uscd. Tal)lc I
gives data demonstrating that S-ethylmaleamic acid does not react with sulfhydryl groups in the conditions of the assay. The decrease in absorption of .\--ethvlmaleimide in the presriice of a constant amount of glutathione is the same with varying aniouiits of S-ethylmalenniic acid (experiments 3, 4, 5 , and 6) as in a freshly prepared .V-ethylmaleimide solution (experiments 1 and 2). Table I1 gives the rewlts obtained with this method in a cleproteinated rat liver extract. TIT^ milliliters of (.\.tract gave a sulfhydryl valve double that of 1 ml. of extract (experiments 1. 2, and 3) denionstrating compliance with Beer's Ian. Pretreatment of the extract for 10 minutes with sodium p-chloromercuribenzoate abolishes any rmction with ~Y-ctliylmaleimide, indicating that it is reacting only with sulfhydryl groups in the extract. Glutathione can be added to the extract (experiments 6 and i )and quantitatively tlrtermined oyer and above the amount of sulfhydryl groups initially prmcnt in the extract. Two milliliters of the filtrate from a 1 to 5 diluted, hemolyzed human blood saniple in 5y0metaphosphoric acid gave a reaction with S-ethylnialeimide equivalent to 0.75 pniole of sulfhydryl groups; 4 ml. of the same filtrate gave a value of 1.48 pmoles. -4s in the case with the liver extract. known amounts of slutathione added to the filtrate quantitatively rcacted with X-ethylmaleiniide. The reaction n ith blood filtratps was carried out in the presence of 4 ml. of 1J1 phosphate buffer (pH VOL. 30, NO.
7,JULY 1958
1293
0.8) to neutralize thc iiietapho’l)horit. acid. This did not affect the evtinction coefficient of S-ctliylmalcimide. Comm t’r c ia 1 r y st :t 11i i i ~q g :I 11)tiI 11ii i reacts with ,\’-eth\lmnleiniid~, only after being denatured with Lhqmiol. By reacting a 1% solution of crj.ht:tlliiic egg albumin with 0.001M A‘-ethylinaleimide in the prrsence of 0.5% Duponol for 15 minutes, 2.2 equivalents of sulfhydryl per mole of egg albumin (assuming mol. wt. = 46,000) react with ,V-ethylmaleimide. S o further reaction occurred after 1 hour. This same egg albumin sample when reacted with p-chloromercuribenzoate a t p H 4.6 for 15 minutes ( 3 ) gave ft value equal to about 3 sulfhydryl groups per mole of protein. (2
DISCUSSION
This procedure is fast, simple, and highly specific for sulfhydryl groups. The principal advantage is that no standard thiol solutions have to be assayed along with the unknown sample for comparison. As added glutathione was quantitatively recovered in a tissue
t>xtrnct and in a t h o d filtrate, the their results, thiol esters and ergothiomethod may he of use in determining neinc do not react with N-ethylmalethe thiol concentrations of other comimide in neutral, aqueous media. plc \ hiological $( ilutic m. l’tic mcthotl a1qicars to br liniited in ACKNOWLEDGMENT determining tlic total sulfhytlrvl rontcltit of protritii, 1)ocwze /~-c~hloromt~r- The technical assistance of Patricia Saples is gratefully acknowledged. c.uribenzo:ite rcwtcd with morc thiol groups in the same egg albumin sample. LITERATURE CITED Aforeover, Roberts and Rouser ( 7 ) found that .Y-rthylmaleiniide reacted ( 1 ) Alexander, S., -1.B i d . Chem. 227, 975 (195i). nith only 60% as ninny sulfhydryl (2) Benesch, R., Benesch, R. E., Cutcho, groups in bovine serum allmmin as did AI., Laufer, L., S c i e n c ~123, 981 p-chloromercuribenzoate. This assay (1956). would, however, obviate the need for (3) . . Bover. P. D., J . Ani. C h e m SOC.76, 4331 (1954). using nitroprusside as an evternal in(4) Friedmann, E., Biochem. et Biophys. dicator (8)to determine the extent to Acta 9, 65 (1952). which S-rthylmaleimide reacts with (5) Gregory, J. D., J . Ana. Chem. SOC. a protein. One per cent solutions with 77, 3922 (1955). ( 6 ) Johnson, M. J., J . Biol. Chem. 181, one reactive sulfhydryl group per 100,707 (1949). ‘ 000 molecular weight would be 0.0001M Roberts. E.. Rouser. G.. h A L . with respect to the sulfhydryl concenCHEM’.30,’1291 (1958). ’ tration, which is within the limits of Tsao, T. C., Bailey, K., Biochirn. et Biophys. Acta 1 1 , 102 (1953). sensitivity of the method. Williamson, S. R., Meldrum, N. E., Benesch et al. ( 2 )have advantageously Biochem. J . 26, 815 (1932). used X-ethylnialeimide to detect thiols Woodward, G. E., Fry, E. G., and thiol esters as pink spots on paper J . B i d . Chem. 97, 465 (1932). chromatograms in a strongly alkaline, RECEIVEDfor review October 2, 1957. nonaqueous medium. In contrast to Accepted Fehruary 20, 1958.
Gradient Elution of Disaccharides on a Stearic Acid-Treated Charcoal Column NANCY HOBAN and JONATHAN W. WHITE, Jr. Eastern Regional Research Laboratory, Philadelphia 1 8, Pa.
b During investigations on the minor sugars of honey, a technique for the isolation of disaccharides, not separable by paper chromatography, was needed. This paper describes the separation by gradient elution of four such pairs of sugars: turanosesucrose, isom a Itose-gentiobiose, ma Itulose-nigerose, and melibiose-lactose. Four other pairs of sugars (maltulosesucrose, maltulose-maltose, sucrosemaltose, and turanose-isomaltose), although separable on paper, were also separated by this method.
P
APCR chromatography
has been valuable for separating many carbohydrate mixtures. However, in some cases, occurrence of several sugars having similar R, values makes isolation by this technique difficult or impossible. This method presents many esperimental problems, such as uneven solvent fronts, sensitivities to temperature change, considerable losses of the original material, and, in the case of oligosaccharides, the time necessary for
1294
ANALYTICAL CHEMISTRY
adequate separation. Impurities from the cellulose contaminate the sugars during the extraction process ( I S ) and interfere with the determination of physical properties. Because of these limitations, many workers have modified the paper method or used other chromatographic procedures. Bayly and Bourne shortened the separation time by converting the disaccharides into the S-benzylglycosylamine derivatives directly on paper. This accelerated the spot travel by decreasing its affinity t o the cellulosewater phase ( 5 ) . T u and Ward obtained excellent separation of several disaccharides having similar R, values by using a thermocolumn (17). Foster used ionophoresis successfully for separating such pairs as maltose and cellobiose ( 9 ) . Gradient elution, where the concentration of eluent is increased continuously, has been applied to various mistures with success (4, 8, 10, 14, 15, 20). Improved separation over stepwisr elution is achieved by reducing the tailing of zones (3). Alni ( 2 ) used
gradient elution on a charcoal column treated with stearic acid for separation of carbohydrates, demonstrating that adjacent members of oligosaccharide series may be clearly separated. He did not attempt separations of sugars of the same molecular weight. This paper describes an application of the gradient elution method where, by continuously increasing the concentration of ethanol in a system, disaccharides having similar R, values are separated. I n some cases (Figure 2), separation is complete while in others slight overlapping of the zones occurs. EXPERIMENTAL
The eight pairs of disaccharides studied are shown in Table I with their R, values. All samples were commercial sugars, except isomaltose, which was obtained from the enzymic hydrolyzate of NRRL B-512 dextran; maltulose, which was prepared by the isomerization of maltose by lime water (19); and nigerose, which was obtained by the hydrolysis of nigeran ( 2 1 ) .
