Selenoamino Acids in Tissues of Rats Administered Inorganic Selenium Oscar E. Olson and Ivan S. Palmer There are conflicting reports in the literature concerning the synthesis of selenoamino acids from inorganic selenium in animals, and this work was undertaken to further investigate this. Pronose digests of acetone powders of liver and kidney tissue from mts administered “SeO; were subjected to fractionation by cation exchange chromatography using current methods for separating the various amino acids. Very little, if any, selenocystine was
found in the digests. However, good evidence was obtained for the occurrence of 2,7-diomino-4-thia-S-selenooctanedioic acid. It is suggested that the selenocysteine portion of this compound was formed by the reduction of the selenite to selenide with its subsequent incorporation into the amino acid by the action of serine hydrolase (E C 4.2.1.22). No selenomethionine was found under the conditions of this study.
T IS generally accepted that selenium replaces sulfur in many biological systems, and Shrift’ has summarized the literature reporting the occurrence of many selenium analogs of sulfur amino acids, including selenocystine and selenomethionine, in plants and microorganisms. There have also been reports of selenocystine and selenomethionine in animal proteins. Rosenfeld* concluded they were present in acid hydrolysates of wool from sheep administered %eO; orally. Godwin et al.3 reported selenomethionine in pronase digests of milk proteins from ewes orally administered 75SeO;. In both cases, the selenoamino acids might have been synthesized microbiologically in the rumen of the animals.4 McConnell and Wabni& found much of the radioactivity in the acid hydrolysates of proteins of dogs injected with 7SSeC14to appear on paper chromatograms in the areas where cystine and selenocystine and methionine and selenomethionine were found. Much of the work on animals, plants and microorganisms was done with paper chromatography, and Schwarz and Sweeney6 have pointed out the need for caution in using this method as the sole source of identification for selenium metabolites, since selenite was found to bind to and travel with disulfides during the paper chromatography. In addition, acid hydrolysis was sometimes used, and selenocystine has been reported to be unstable under these conditions.7 Jenkins’ could find no selenocystine or selenomethionine in serum from chicks administered 7SSeO; by intubation into the crop. Further, Cummins and Martin,g using ion exchange chromatography, found neither selenoamino acid present in pronase hydrolysates of liver from rabbits fed 75Se0,‘. Olson et al.”
I
From the Department of Chemistry, South Dakota Store University, Brookings, S. Dok. Received for publication July 1, 1975. Pubiished with the approval of the Director of the South Dakota Agricultural Experiment Station as Poper No. 1334 of the Journal series. Supported in part by PHS research grant number FD 00472-02 from the Food and Drug Administration. Reprint requests should be addressed to Ivan S. Palmer. Department of Chemistry, Experiment Station Biochemistry Section, South Dakota State University, Brookings. S. Dak. 57006. o 1976 by Grune & Stratton. Inc.
Metobohsm, Vol. 25, No. 3 (March), 1976
299
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have suggested that the likelihood of finding selenocystine is small, since in the presence of cysteine in comparatively high amounts most of any selenocysteine formed would probably react with cysteine and occur as the sulfide-selenide, 2,7-diamino-4-thia-5-selenaoctanedioic acid, which migrates differently from selenocystine. This work was undertaken to further investigate the occurrence of either amino acid in animals fed inorganic selenium. Ion exchange chromatography was used as an identification tool, taking into consideration the need for the use of buffers without added thiodiglycol in identifying selenocysteine because of the reaction in which the amino acid is in part converted to the 2-amino-4selena-5-thia-7-hydroxyheptanoic acid. ” Also considered were the possible presence of 2,7-diamino-4-thia-5-selenaoctanedioic acid and the relative instability of selenoamino acids. MATERIALS
AND METHODS
H2’%e03 was obtained from ICN isotope and Nuclear Division (Irvine, Calif.). Selenocystine and selenomethionine were obtained from Calbiochem (La Jolla, Calif.). A 50 x 0.9 cm column of Aminex A-4 cation exchange resin (Bio-Rad Laboratories, Richmond, Calif.) was used on a Beckman Model 120 amino acid analyzer modified for accelerated analysis for chromatographing the various solutions. The buffers for diluting the samples and for elution of the amino acids from the ion exchange column were those described by Moore et al.13 with some modifications. The pH 3.25 buffer had 4.0 ml of methanol added to each 100 ml just before its use. Further, thiodiglycol was not added to the pH 3.25 buffer for the development of some of the chromatograms nor to the pH 2.2 diluting buffer usedin preparing some solutions. Male Sprague-Dawley rats weighing about 60 g were placed on a Tori&r yeast diet t3containing 0.5 pg Se/g as selenite. They were maintained on this diet until they were killed. Pronase used for the hydrolysis of tissue preparations was obtained from Calbiochem. Its stated activity was 45,000 proteolytic units/g. Preparufion of tissues and hydrolysutes. Two 100 g rats were administered 0.8 ml of a water solution of H,75Se0, containing 80 pCi 75Se and 0.45 cg Se. One of the rats received the solution intraperitoneally, the other by stomach tube. The rats were then maintained on the Torufu yeast diet for 3 days, at which time they were killed in an ether chamber. The livers and kidneys were removed, washed with water, and blotted dry. After weighing, each was ground in about 20 volumes of cold acetone with a VirTis grinder. The mixtures were filtered through fiber glass and the insoluble material was washed well with acetone. It was then dried in vacua without added heat. The combined acetone extracts were evaporated in vacua to about I5 ml. Then 10 ml of hexane was added and the mixture was gently shaken for several minutes. The hexane and water layers were measured and counted. The hexane contained 0.071% and 0.048% of the total counts in the liver and kidney, respectively, while the water contained 0.88% and 0.29%. These data suggest the presence of some water-soluble but of very little lipid-related selenium in these tissues. About 80 mg of the acetone powder was suspended in 4.0 ml of 0.2 M phosphate buffer, pH 7.0, containing 8 mg of pronase. After mixing, the solution was placed in a water bath at 37”C, covered with a I mm layer of toluene, and gently shaken during the incubation period. At 24 hr, 2.0 ml of the buffer containing 4 mg of pronase was added, and the incubation was continued. At 48 hr, the mixture was removed, adjusted to pH 2.2 with 1N HCI and diluted to IO ml with pH 2.2 diluting buffer containing no thiodi lycol. It was then filtered through fiber glass with gentle suction. Between 66% and 72% of the A Se present in the various acetone powders was recovered in the filtrates, the remainder representing that retained on the filtration apparatus or in an insoluble residue present in the digests. A 3 ml portion of the filtrate was treated with 3 ml of unlabeled selenocystine solution (1.0 pmole/ml in pH 2.2 diluting buffer without thiodiglycol). Another 3 ml portion was treated with 3 ml of unlabeled selenomethionine solution (1.0 pmole/ml in pH 2.2 citrate buffer with thiodiglycol). The remainder was left untreated. The selenoamino acids were added as markers should confirmation of the location of peaks on the chromatograms be later needed. Each of the liver and the kidney preparations was treated in this manner.
301
SELENOAMINOACIDS
Chromatographic procedures. Before chromatographing the various hydrolysates, it was necessary to standardize the ion exchange column using known selenium compounds. Two different buffer systems were used, and both required standardization. This was done using ninhydrin color development by the procedure normally employed with the amino acid analyzer. Later, when the hydrolysates were chromatographed, ninhydrin color development was not used. Instead, the eluates were collected with a fraction collector directly off of the column and the radioactivity of the various fractions was measured. This resulted in a time difference between the two methods of measurement. Correction for this was made as previously described.” A source of 2,7-diamino-4-thia-5-selenaoctanedioic acid for use in the standardization was prepared as follows. A mixture of equimolar parts of cysteine and selenocystine was allowed to stand in pH 7.0 phosphate buffer for about 1 hr with occasional shaking. It was then adjusted to pH 2.2 with 1N HCI and diluted to volume with pH 2.2 diluting buffer without thiodiglycol. This also served as a source for cystine and selenocystine. In the search for selenocystine, only pH 3.25 buffer without added thiodiglycol was used. In looking for selenomethionine, both the pH 3.25 and pH 4.25 buffers, each containing thiodiglycol, were used, and the buffer change timer was set at 100 min. Chromatography of the hydrolysates. The hydrolysates were chromatographed using both buffer systems as further described in presenting the results. The eluates were collected in test tubes with a fraction collector and their radioactivity was measured by counting. In order to calculate recoveries from the column, a sample of the hydrolysate was counted at the same time. In verifying the positions of certain amino acids, some of the fractions were adjusted to pH 2.2 with IN HCI and chromatographed again, using ninhydrin color development. In addition, in some cases, the contents of a series of tubes were sampled and spotted on paper. After drying, spraying with ninhydrin and heating the papers, the positions of key amino acids could be established.
RESULTS AND DISCUSSION
The results of chromatographing hydrolysates in the absence of thiodiglycol are shown in Fig. 1. In addition, some of the results are summarized in Table 1. While both the liver and kidney hydrolysates had been prepared from two animals and all of them were chromatographed, only one set of data (that for the rat intraperitoneally injected) is shown since the findings for each set were so similar. Chromatogram A shows the positions of selenocystine and the 2,7-diamino4-thia-5-selenaoctanedioic acid. Aspartic acid and cystine peaks are included merely for comparison with normal amino acid chromatograms obtained with the amino acid analyzer. The peaks are illustrative of about 0.5 pmole of amino acid. Chromatogram B shows a peak in tubes 4-7 under which anionic substances, including selenite, should elute. Other peaks occur up to about tube 20, and these will be discussed to some extent later here. A very significant peak between tubes 51-57 suggests the presence of 2,7-diamino-4-thia-5_selenaoctanedioic acid. On the other hand, selenocystine itself, (tubes 70-78) appears to be present in very small amounts, if at all. When unlabeled selenocystine was added to the liver hydrolysate (Chromatogram C) the peak for “Se decreased in size where the 2,7-diamino-4ithia-5selenaoctanedioic acid elutes, while it increased where selenocystine elutes. This finding is in agreement with the presence of the former, suggesting an interchange between this compound and the unlabeled selenocystine to give labeled selenocystine. Chromatograph F supports this conclusion, since it also indicates this type of interchange. In view of this finding, only those hydrolysates of the second liver and the two kidney acetone powders that contained added selenocystine were chromatographed by the single buffer system.
t
_- _____ -_-_-
0,
ASP
-r
2
2, I
C
l-
2
I
C
2
I
C
2
I
C
2c
IC
0
TUBE
NO.
Fig. 1. Chromatogmms of ulenoamino acids elutod with pH 5.25 buffer without added thio diglycol. The olution mto was 1.0 ml/min. Each tuba contained 2.60 ml of lluate. The materials chromatogmphod wore as follows: (A) amino acids using ninhydrin color development; (B) liver hydrofysato; (C) liver hydrolysato plus 0.5 pmole selonocystine; (D) kidney hydrolysate plus 0.5 rmolo rlonocystino; (E) standard amino acid mixture plus selonocystine (0.5 fimole of mch amino acid) and “‘S&l; (F) lluate in tuba 54 from a previous chromatogmm rechromatogmphd with 0.5 Lmolo of selonocystino. The abbmviattons used in identifying the peaks an as follows: Asp = aspartic acid; CyS = cystine; CyS = 2,7diamino4-thia-5-selonaoctanedioc acid; I CySa = wlenocystine.
C.I. I
CYS
I CYS
SELENOAMINO
303
ACIDS
Table 1. Summary of Some Data From the Various Chromatogmms 75Se Added to ChromatoMaterial
graph B
Liver
C
hydrolysate
liver hydrolysate Kidney
with
no
with
hydrolysate
75SeO;
Tubes
Tubes
Tuber
Tubes
Tubes
Tubes
(cpm)
(% of added)
4-7
4-20
35-40
45-49
51-57
52-53
70-78
23326
48.0
10.1
25.4
1.2
0.6
10.3
-
1.9~
23187
50.4
9.3
26.5
1.4
0.7
7.4
-
7.7
34015
52.2
9.3
20.4
1.2
0.5
7.9
-
6.0
69491
91.8
89.8
90.9
0.1
0.0
0.1
-
0.1
89.2
-
-
-
-
56.6
-
25.2
83.1
-
-
-
-
selenocystine
added E
Tubes
sslsnocystine
added D
Eluates
Chromatogrophed
added
Percent of Added 75Se in Various Fmctions’
75Se in
Column
with
selanocystine plus amino
standard
mixture
acid and
selenocystine F
lube
54 plus selenocystine**
11
75Se0;
plus amino
standard 12
%O;
M
liver
N
Kidney
*The **Tube
data
with
4.8
6.1
14380
-
84.1
-
-
-
-
21699
84.7
10.4
5.8
27.4
-
25.5
6.9
-
1.5
-
30713
05.7
8.9
26.3
32.1
5.6
-
1.3
-
-
with
selenomethionine
are not corrected
54 from
93.6
selenomethionine
hydrolysate
added
29032
mix
hydrolysate
added
acid
1840
a previous
for baselines
on the chromatograms.
chromotogram.
The kidney hydrolysate with added selenocystine also yielded peaks in the positions of the 2,7-diamino-4-thia-5selenaoctanedioic acid and selenocystine (Chromatogram D). A peak around tube 14 was present here as it was in Chromatogram C. This peak is not so obvious in Chromatogram B, and it was at first thought that this difference was in some way due to the added selenocystine. However, the other liver hydrolysate without added selenocystine did show the marked peak at tube 14. In view of this as well as of the similarity of the data for tubes 4-20 of Chromatograms B, C and D (see Table l), it appears that the difference was merely a matter of degree of resolution. In view of the report of Schwarz and Sweeney6 that selenite often cochromatographs with disulfides, it was felt that this possibility here should be studied. Chromatogram E shows that, in the presence of an amino acid mixture containing cystine and selenocystine, the selenite eluted early in the chromatogram, showing no interaction. It should be noted that small amounts of the radioactive selenium were retained on and bled slowly from the column during the entire elution period. A similar observation was made concerning the elution of the selenium in other chromatograms. Chromatogram K of Fig. 2 shows the locations of some selenium compounds when the two-buffer system with added thiodiglycol was used. The first of these, 2-amino-4-selena-S-thia-7-hydroxyheptanoic acid, is a mixed sulfurselenium compound formed as the results of a reaction of selenocystine with 2-mercaptoethanol, which occurs as a contaminant of the thiodiglycol in the buffer.” This reaction is incomplete as concerns the selenocystine, and the height of the peak and the raised baseline shown following it are intentionally somewhat exaggerated in this figure. The 2,7-diamino-4-thia-5-selenaoctanedioic
OLSON
304
f 4
IUbs 3712.6%
-
s 5 -
4
z
/)
I
!
ii
ii
ihi ii il\! i i
2-
u
:2yy~ 00
i
I
I4-
z
)
I 1 ii
“w 6-
2
PALMER
11
6_
I-
AND
I
IO
I
I
I
20
30
40
TUBE
I ’ Ii I
I
50
/
I
60
70
NO.
Fig. 2. Chmmotogmms of selonoomino acids eluted with pH 3.25 buffer followed by pH 4.25 buffer, both containing odded thiodiglycol. The elution mte was 1.0 ml/min. Each tube contained 2.86 ml of oluota. The moteriols chmmatogmphed were as followr: (K) seleneamino acids with omino acid standard solution (0.5 with ninhydrin color development; (1,) ‘%eO; selonomethionine; Imole of each amino acid); L1) ‘“GO;; (M) liver hydmlysato plus 0.5 #mole (N) kidney hydmlysate with 0.5 Imole selenomethionine. The obbreviations used in identifying acid; CyS = the peoks are as follows: CySeSEtOH = 2-amino4-selena-5-thio-7-hydroxyheptonoic CySe 2,7-diamino-4-thio-5-selenaoctonedioic
acid; CySe = selenocystine; SeMet = selenomethionine. I CySe
acid and the selenocystine elute in partially resolved peaks very soon after the buffer change, and selenomethionine is well resolved from them. Selenite (Chromatograms L, and L2) also reacts with something in the buffer containing the added thiodiglycol. Very probably, this is a reaction with the 2-mercaptoethanol to form 1,7-dihydroxy-3, 5-dithia-4-selenaheptane, according to the reaction of Painter. I4 Benesch and Benesch” have suggested that in
SELENOAMINO
ACIDS
305
strong acids protonation of disulfides followed by heterolytic cleavage forms a thiol and a sulfenium cation. If some similar reaction takes place in the case of the selenotrisulfide, this could account for the cationic nature of the selenium compound formed. At present, however, this is mere conjecture. Chromatograms M and N for the liver and kidney hydrolysates with added unlabeled selenomethionine are very similar. It is tempting to conclude that the large peak between tubes 35-40 is the result of the reaction of selenite that is present with 2-mercaptoethanol. However it consistently reaches a maximum one tube in advance of that obtained when radioactive selenite was added to the column, suggesting other possibilities. Quite possibly, the peak contains at least some selenite or half-selenocystine derivatives of 2-mercaptoethanol, but at this time other derivatives cannot be excluded. Whatever the case, comparison of the data in Table 1 for tubes 4-7 of chromatographs B, C, and D with tubes 4-7 of chromatographs M and N indicates that selenite is not a major constituent of this peak, since there is little change in its size as a result of using thiodiglycol in the buffer. Since tubes 51-57 plus tubes 70-78 contain twice the percentage of ‘%e added to the column that tubes 45-49 of chromatographs M and N contain, it appears that part of the half-selenocystine moiety has been converted to the 2-mercaptoethanol derivative in the buffers with the added thiodiglycol. However, since only about half of the ‘?Se in hydrolysates is eluted by the buffer without added thiodiglycol while about 85% is eluted when thiodiglycol is used, it appears that most of the ‘%e in tubes 35-40 derives from compounds eluting after the selenocystine in chromatograms B, C, and D. Such compounds as peptides containing the half-selenocystine moiety linked to sulfhydryl groups or selenite in ether linkages are possibilities, but the matter requires further study for its resolution. Finally, the data in Fig. 2 confirm those in Fig. 1 in showing the presence of 2,7-diamino-4-thia-5-selenaoctanedioic acid. There seems little doubt that selenocysteine can be synthesized from selenite in the body of the rat. Surprisingly, the data do not confirm the presence of selenomethionine. Since only a very small part of the “Se administered to the rats actually appeared in the selenocysteine moiety, a very small amount of impurity in the ‘?SeO; might account for it. Chromatograms E, L,, and L2 give some indication of the purity of the H,75Se0a used. In chromatogram E, about 90% (see Table 1) of the 75Se placed on the column eluted with the anions in tubes 4-7. No other peak of significance was noted. In view of the method of preparation of the H,‘%eO,, organic forms of the element could not be present. Selenium halides or oxyhalides and hydrogen selenide would decompose to elemental selenium and/or selenite under the conditions existing in the solutions. The 10% not accounted for in the eluates from the column might be the insoluble elemental form. However, at least a part of this might be accounted for by the fact that past experience has indicated the fixation of very small amounts of inorganic selenium that bleed slowly from the column over extended elution with the buffers. The small peak at tube 4 in chromatograms L, and LZ suggests an incomplete reaction of the selenite with the thiodiglycol or the more likely presence of some ‘%e04. It seems, therefore, that the 75Se solution used contained largely ‘%eO; with possible small amounts of 75Se or 75SeO;. The elemental form, being highly insoluble should not have been involved in selenocysteine formation, and the well known similarity between selenate and selenite
306
OLSON
AND PLAMER
metabolism in the rat suggests that either form could be contributing to the formation of this compound. The data showing that the half-selenocystine moiety occurs in combination with the sulfur analog (and perhaps in combination with peptides containing sulfide groups) may explain why some have not found selenium present in tissues as selenocystine. However, the mechanism of formation of selenocysteine in the animal body is not clear. Intestinal microbial biosynthesis may occur, but the similarity in level of selenium found in the half-selenocystine moiety in the rats injected with or orally administered the selenite suggests otherwise. The failure to find selenomethionine also suggests another mode of formation, since microbial synthesis should probably yield both amino acids. Reduction of the selenite to selenide’(’ and subsequent substitution of this for sulfide in the reaction with serine that is catalyzed by L-serine hydrolase (cysteine synthase EC 4.2.1.22)” is a possible explanation and might also account for the failure to find selenomethionine. ACKNOWLEDGMENT The authors wish to acknowledge the assistance of E. I. Whitehead this study.
with certain phases of
REFERENCES I. Shrift A: Selenium compounds in nature and medicine. E. Metabolism of selenium by plants and microorganisms, in Klayman DL, Gunther WHH (ed): Organic selenium compounds: Their chemistry and biology. New York, Wiley, 1973, pp 763-814 2. Rosenfeld I: Biosynthesis of seleno-compounds from inorganic selenium by sheep. Proc Sot Exptl Biol Med 1Il:670-673, 1962 3. Godwin KO, Handreck KA, Fuss CN: Identification of 75Se selenomethionine in ewe milk protein following the intraruminal administration of Na7’Se03 as a single oral dose. Aust J Biol Sci 24:1251-1261, 1971 4. Hidiroglou M, Heaney DP, Jenkins KJ: Metabolism of inorganic selenium in rumen bacteria. Can J Physiol Pharmacol 46:229-232, 1968 5. McConnell KP, Wabnitz CH: Studies on the fixation of radioselenium in proteins. J Biol Chem 226:765-776, 1957 6. Schwarz K, Sweeney E: Selenite binding to sulfur amino acids. Federation Proc 23:421, 1964 7. Huber RE, Criddle RS: Comparison of the chemical properties of selenocysteine and selenocystine with their sulfur analogs. Arch Biochem Biophys 122:164173, 1967 8. Jenkins KJ: Evidence for the absence of selenocystine and selenomethionine in the serum proteins of chicks administered selenite. Can J Biochem 46:1417-1425, 1968 9. Cummins LM, Martin JL: Are selenocystine and selenomethionine synthesized in
vivo from sodium selenite in mammals? Biothem 6:3162-3168,1967 IO. Olson OE, Novacek El, Whitehead EI, Palmer IS: Investigations on selenium in wheat. Phytochemistry 9:1181-l 188, 1970 11. Walter R, Schlesinger DH, Schwartz IL: Chromatographic separation of isologous sulfur- and selenium-containing amino acids: Reductive scission of the selenium bond by mercaptans and selenols. Analytical Biochem 27:231-243, 1969 12. Moore S, Spackman CH, Stein WH: Chromatography of amino acids on sulfonated polystyrene resins. Analytical Chem 3O:l l851190, 1958 13. Tsay DT, Halverson AW, Palmer IS: Inactivity of dietary trimethylselenonium chloride against the necrogenic syndrome of the rat. Nutrition Rept Internatl2:203-207, 1970 14. Painter EP: The chemistry and toxicity of selenium compounds, with special reference to the selenium problem. Chem Rev 28:179213, 1941 15. Benesch RE, Benesch R: Mechanism of disulfide interchange in acid solution; role of sulfenium ions. J Am Chem Sot 80~1666-1669, 1958 16. Diplock AT, Caygill CPJ, Jeffery EH, Thomas C: The nature of the acid-volatile selenium in the liver of the male rat. Biochem J 134:283-293, 1973 17. Greenberg DH: Amino acid metabolism, in Snell EE, Luck JM, Boyer PD, Mackinney G: Ann Rev Biochemistry 33:633-666, 1964
found in the digests. However, good evidence was obtained for the occurrence of 2,7-diomino-4-thia-S-selenooctanedioic acid. It is suggested that the selenocysteine portion of this compound was formed by the reduction of the selenite to selenide with its subsequent incorporation into the amino acid by the action of serine hydrolase (E C 4.2.1.22). No selenomethionine was found under the conditions of this study.
T IS generally accepted that selenium replaces sulfur in many biological systems, and Shrift’ has summarized the literature reporting the occurrence of many selenium analogs of sulfur amino acids, including selenocystine and selenomethionine, in plants and microorganisms. There have also been reports of selenocystine and selenomethionine in animal proteins. Rosenfeld* concluded they were present in acid hydrolysates of wool from sheep administered %eO; orally. Godwin et al.3 reported selenomethionine in pronase digests of milk proteins from ewes orally administered 75SeO;. In both cases, the selenoamino acids might have been synthesized microbiologically in the rumen of the animals.4 McConnell and Wabni& found much of the radioactivity in the acid hydrolysates of proteins of dogs injected with 7SSeC14to appear on paper chromatograms in the areas where cystine and selenocystine and methionine and selenomethionine were found. Much of the work on animals, plants and microorganisms was done with paper chromatography, and Schwarz and Sweeney6 have pointed out the need for caution in using this method as the sole source of identification for selenium metabolites, since selenite was found to bind to and travel with disulfides during the paper chromatography. In addition, acid hydrolysis was sometimes used, and selenocystine has been reported to be unstable under these conditions.7 Jenkins’ could find no selenocystine or selenomethionine in serum from chicks administered 7SSeO; by intubation into the crop. Further, Cummins and Martin,g using ion exchange chromatography, found neither selenoamino acid present in pronase hydrolysates of liver from rabbits fed 75Se0,‘. Olson et al.”
I
From the Department of Chemistry, South Dakota Store University, Brookings, S. Dok. Received for publication July 1, 1975. Pubiished with the approval of the Director of the South Dakota Agricultural Experiment Station as Poper No. 1334 of the Journal series. Supported in part by PHS research grant number FD 00472-02 from the Food and Drug Administration. Reprint requests should be addressed to Ivan S. Palmer. Department of Chemistry, Experiment Station Biochemistry Section, South Dakota State University, Brookings. S. Dak. 57006. o 1976 by Grune & Stratton. Inc.
Metobohsm, Vol. 25, No. 3 (March), 1976
299
300
OLSON
AND
PALMER
have suggested that the likelihood of finding selenocystine is small, since in the presence of cysteine in comparatively high amounts most of any selenocysteine formed would probably react with cysteine and occur as the sulfide-selenide, 2,7-diamino-4-thia-5-selenaoctanedioic acid, which migrates differently from selenocystine. This work was undertaken to further investigate the occurrence of either amino acid in animals fed inorganic selenium. Ion exchange chromatography was used as an identification tool, taking into consideration the need for the use of buffers without added thiodiglycol in identifying selenocysteine because of the reaction in which the amino acid is in part converted to the 2-amino-4selena-5-thia-7-hydroxyheptanoic acid. ” Also considered were the possible presence of 2,7-diamino-4-thia-5-selenaoctanedioic acid and the relative instability of selenoamino acids. MATERIALS
AND METHODS
H2’%e03 was obtained from ICN isotope and Nuclear Division (Irvine, Calif.). Selenocystine and selenomethionine were obtained from Calbiochem (La Jolla, Calif.). A 50 x 0.9 cm column of Aminex A-4 cation exchange resin (Bio-Rad Laboratories, Richmond, Calif.) was used on a Beckman Model 120 amino acid analyzer modified for accelerated analysis for chromatographing the various solutions. The buffers for diluting the samples and for elution of the amino acids from the ion exchange column were those described by Moore et al.13 with some modifications. The pH 3.25 buffer had 4.0 ml of methanol added to each 100 ml just before its use. Further, thiodiglycol was not added to the pH 3.25 buffer for the development of some of the chromatograms nor to the pH 2.2 diluting buffer usedin preparing some solutions. Male Sprague-Dawley rats weighing about 60 g were placed on a Tori&r yeast diet t3containing 0.5 pg Se/g as selenite. They were maintained on this diet until they were killed. Pronase used for the hydrolysis of tissue preparations was obtained from Calbiochem. Its stated activity was 45,000 proteolytic units/g. Preparufion of tissues and hydrolysutes. Two 100 g rats were administered 0.8 ml of a water solution of H,75Se0, containing 80 pCi 75Se and 0.45 cg Se. One of the rats received the solution intraperitoneally, the other by stomach tube. The rats were then maintained on the Torufu yeast diet for 3 days, at which time they were killed in an ether chamber. The livers and kidneys were removed, washed with water, and blotted dry. After weighing, each was ground in about 20 volumes of cold acetone with a VirTis grinder. The mixtures were filtered through fiber glass and the insoluble material was washed well with acetone. It was then dried in vacua without added heat. The combined acetone extracts were evaporated in vacua to about I5 ml. Then 10 ml of hexane was added and the mixture was gently shaken for several minutes. The hexane and water layers were measured and counted. The hexane contained 0.071% and 0.048% of the total counts in the liver and kidney, respectively, while the water contained 0.88% and 0.29%. These data suggest the presence of some water-soluble but of very little lipid-related selenium in these tissues. About 80 mg of the acetone powder was suspended in 4.0 ml of 0.2 M phosphate buffer, pH 7.0, containing 8 mg of pronase. After mixing, the solution was placed in a water bath at 37”C, covered with a I mm layer of toluene, and gently shaken during the incubation period. At 24 hr, 2.0 ml of the buffer containing 4 mg of pronase was added, and the incubation was continued. At 48 hr, the mixture was removed, adjusted to pH 2.2 with 1N HCI and diluted to IO ml with pH 2.2 diluting buffer containing no thiodi lycol. It was then filtered through fiber glass with gentle suction. Between 66% and 72% of the A Se present in the various acetone powders was recovered in the filtrates, the remainder representing that retained on the filtration apparatus or in an insoluble residue present in the digests. A 3 ml portion of the filtrate was treated with 3 ml of unlabeled selenocystine solution (1.0 pmole/ml in pH 2.2 diluting buffer without thiodiglycol). Another 3 ml portion was treated with 3 ml of unlabeled selenomethionine solution (1.0 pmole/ml in pH 2.2 citrate buffer with thiodiglycol). The remainder was left untreated. The selenoamino acids were added as markers should confirmation of the location of peaks on the chromatograms be later needed. Each of the liver and the kidney preparations was treated in this manner.
301
SELENOAMINOACIDS
Chromatographic procedures. Before chromatographing the various hydrolysates, it was necessary to standardize the ion exchange column using known selenium compounds. Two different buffer systems were used, and both required standardization. This was done using ninhydrin color development by the procedure normally employed with the amino acid analyzer. Later, when the hydrolysates were chromatographed, ninhydrin color development was not used. Instead, the eluates were collected with a fraction collector directly off of the column and the radioactivity of the various fractions was measured. This resulted in a time difference between the two methods of measurement. Correction for this was made as previously described.” A source of 2,7-diamino-4-thia-5-selenaoctanedioic acid for use in the standardization was prepared as follows. A mixture of equimolar parts of cysteine and selenocystine was allowed to stand in pH 7.0 phosphate buffer for about 1 hr with occasional shaking. It was then adjusted to pH 2.2 with 1N HCI and diluted to volume with pH 2.2 diluting buffer without thiodiglycol. This also served as a source for cystine and selenocystine. In the search for selenocystine, only pH 3.25 buffer without added thiodiglycol was used. In looking for selenomethionine, both the pH 3.25 and pH 4.25 buffers, each containing thiodiglycol, were used, and the buffer change timer was set at 100 min. Chromatography of the hydrolysates. The hydrolysates were chromatographed using both buffer systems as further described in presenting the results. The eluates were collected in test tubes with a fraction collector and their radioactivity was measured by counting. In order to calculate recoveries from the column, a sample of the hydrolysate was counted at the same time. In verifying the positions of certain amino acids, some of the fractions were adjusted to pH 2.2 with IN HCI and chromatographed again, using ninhydrin color development. In addition, in some cases, the contents of a series of tubes were sampled and spotted on paper. After drying, spraying with ninhydrin and heating the papers, the positions of key amino acids could be established.
RESULTS AND DISCUSSION
The results of chromatographing hydrolysates in the absence of thiodiglycol are shown in Fig. 1. In addition, some of the results are summarized in Table 1. While both the liver and kidney hydrolysates had been prepared from two animals and all of them were chromatographed, only one set of data (that for the rat intraperitoneally injected) is shown since the findings for each set were so similar. Chromatogram A shows the positions of selenocystine and the 2,7-diamino4-thia-5-selenaoctanedioic acid. Aspartic acid and cystine peaks are included merely for comparison with normal amino acid chromatograms obtained with the amino acid analyzer. The peaks are illustrative of about 0.5 pmole of amino acid. Chromatogram B shows a peak in tubes 4-7 under which anionic substances, including selenite, should elute. Other peaks occur up to about tube 20, and these will be discussed to some extent later here. A very significant peak between tubes 51-57 suggests the presence of 2,7-diamino-4-thia-5_selenaoctanedioic acid. On the other hand, selenocystine itself, (tubes 70-78) appears to be present in very small amounts, if at all. When unlabeled selenocystine was added to the liver hydrolysate (Chromatogram C) the peak for “Se decreased in size where the 2,7-diamino-4ithia-5selenaoctanedioic acid elutes, while it increased where selenocystine elutes. This finding is in agreement with the presence of the former, suggesting an interchange between this compound and the unlabeled selenocystine to give labeled selenocystine. Chromatograph F supports this conclusion, since it also indicates this type of interchange. In view of this finding, only those hydrolysates of the second liver and the two kidney acetone powders that contained added selenocystine were chromatographed by the single buffer system.
t
_- _____ -_-_-
0,
ASP
-r
2
2, I
C
l-
2
I
C
2
I
C
2
I
C
2c
IC
0
TUBE
NO.
Fig. 1. Chromatogmms of ulenoamino acids elutod with pH 5.25 buffer without added thio diglycol. The olution mto was 1.0 ml/min. Each tuba contained 2.60 ml of lluate. The materials chromatogmphod wore as follows: (A) amino acids using ninhydrin color development; (B) liver hydrofysato; (C) liver hydrolysato plus 0.5 pmole selonocystine; (D) kidney hydrolysate plus 0.5 rmolo rlonocystino; (E) standard amino acid mixture plus selonocystine (0.5 fimole of mch amino acid) and “‘S&l; (F) lluate in tuba 54 from a previous chromatogmm rechromatogmphd with 0.5 Lmolo of selonocystino. The abbmviattons used in identifying the peaks an as follows: Asp = aspartic acid; CyS = cystine; CyS = 2,7diamino4-thia-5-selonaoctanedioc acid; I CySa = wlenocystine.
C.I. I
CYS
I CYS
SELENOAMINO
303
ACIDS
Table 1. Summary of Some Data From the Various Chromatogmms 75Se Added to ChromatoMaterial
graph B
Liver
C
hydrolysate
liver hydrolysate Kidney
with
no
with
hydrolysate
75SeO;
Tubes
Tubes
Tuber
Tubes
Tubes
Tubes
(cpm)
(% of added)
4-7
4-20
35-40
45-49
51-57
52-53
70-78
23326
48.0
10.1
25.4
1.2
0.6
10.3
-
1.9~
23187
50.4
9.3
26.5
1.4
0.7
7.4
-
7.7
34015
52.2
9.3
20.4
1.2
0.5
7.9
-
6.0
69491
91.8
89.8
90.9
0.1
0.0
0.1
-
0.1
89.2
-
-
-
-
56.6
-
25.2
83.1
-
-
-
-
selenocystine
added E
Tubes
sslsnocystine
added D
Eluates
Chromatogrophed
added
Percent of Added 75Se in Various Fmctions’
75Se in
Column
with
selanocystine plus amino
standard
mixture
acid and
selenocystine F
lube
54 plus selenocystine**
11
75Se0;
plus amino
standard 12
%O;
M
liver
N
Kidney
*The **Tube
data
with
4.8
6.1
14380
-
84.1
-
-
-
-
21699
84.7
10.4
5.8
27.4
-
25.5
6.9
-
1.5
-
30713
05.7
8.9
26.3
32.1
5.6
-
1.3
-
-
with
selenomethionine
are not corrected
54 from
93.6
selenomethionine
hydrolysate
added
29032
mix
hydrolysate
added
acid
1840
a previous
for baselines
on the chromatograms.
chromotogram.
The kidney hydrolysate with added selenocystine also yielded peaks in the positions of the 2,7-diamino-4-thia-5selenaoctanedioic acid and selenocystine (Chromatogram D). A peak around tube 14 was present here as it was in Chromatogram C. This peak is not so obvious in Chromatogram B, and it was at first thought that this difference was in some way due to the added selenocystine. However, the other liver hydrolysate without added selenocystine did show the marked peak at tube 14. In view of this as well as of the similarity of the data for tubes 4-20 of Chromatograms B, C and D (see Table l), it appears that the difference was merely a matter of degree of resolution. In view of the report of Schwarz and Sweeney6 that selenite often cochromatographs with disulfides, it was felt that this possibility here should be studied. Chromatogram E shows that, in the presence of an amino acid mixture containing cystine and selenocystine, the selenite eluted early in the chromatogram, showing no interaction. It should be noted that small amounts of the radioactive selenium were retained on and bled slowly from the column during the entire elution period. A similar observation was made concerning the elution of the selenium in other chromatograms. Chromatogram K of Fig. 2 shows the locations of some selenium compounds when the two-buffer system with added thiodiglycol was used. The first of these, 2-amino-4-selena-S-thia-7-hydroxyheptanoic acid, is a mixed sulfurselenium compound formed as the results of a reaction of selenocystine with 2-mercaptoethanol, which occurs as a contaminant of the thiodiglycol in the buffer.” This reaction is incomplete as concerns the selenocystine, and the height of the peak and the raised baseline shown following it are intentionally somewhat exaggerated in this figure. The 2,7-diamino-4-thia-5-selenaoctanedioic
OLSON
304
f 4
IUbs 3712.6%
-
s 5 -
4
z
/)
I
!
ii
ii
ihi ii il\! i i
2-
u
:2yy~ 00
i
I
I4-
z
)
I 1 ii
“w 6-
2
PALMER
11
6_
I-
AND
I
IO
I
I
I
20
30
40
TUBE
I ’ Ii I
I
50
/
I
60
70
NO.
Fig. 2. Chmmotogmms of selonoomino acids eluted with pH 3.25 buffer followed by pH 4.25 buffer, both containing odded thiodiglycol. The elution mte was 1.0 ml/min. Each tube contained 2.86 ml of oluota. The moteriols chmmatogmphed were as followr: (K) seleneamino acids with omino acid standard solution (0.5 with ninhydrin color development; (1,) ‘%eO; selonomethionine; Imole of each amino acid); L1) ‘“GO;; (M) liver hydmlysato plus 0.5 #mole (N) kidney hydmlysate with 0.5 Imole selenomethionine. The obbreviations used in identifying acid; CyS = the peoks are as follows: CySeSEtOH = 2-amino4-selena-5-thio-7-hydroxyheptonoic CySe 2,7-diamino-4-thio-5-selenaoctonedioic
acid; CySe = selenocystine; SeMet = selenomethionine. I CySe
acid and the selenocystine elute in partially resolved peaks very soon after the buffer change, and selenomethionine is well resolved from them. Selenite (Chromatograms L, and L2) also reacts with something in the buffer containing the added thiodiglycol. Very probably, this is a reaction with the 2-mercaptoethanol to form 1,7-dihydroxy-3, 5-dithia-4-selenaheptane, according to the reaction of Painter. I4 Benesch and Benesch” have suggested that in
SELENOAMINO
ACIDS
305
strong acids protonation of disulfides followed by heterolytic cleavage forms a thiol and a sulfenium cation. If some similar reaction takes place in the case of the selenotrisulfide, this could account for the cationic nature of the selenium compound formed. At present, however, this is mere conjecture. Chromatograms M and N for the liver and kidney hydrolysates with added unlabeled selenomethionine are very similar. It is tempting to conclude that the large peak between tubes 35-40 is the result of the reaction of selenite that is present with 2-mercaptoethanol. However it consistently reaches a maximum one tube in advance of that obtained when radioactive selenite was added to the column, suggesting other possibilities. Quite possibly, the peak contains at least some selenite or half-selenocystine derivatives of 2-mercaptoethanol, but at this time other derivatives cannot be excluded. Whatever the case, comparison of the data in Table 1 for tubes 4-7 of chromatographs B, C, and D with tubes 4-7 of chromatographs M and N indicates that selenite is not a major constituent of this peak, since there is little change in its size as a result of using thiodiglycol in the buffer. Since tubes 51-57 plus tubes 70-78 contain twice the percentage of ‘%e added to the column that tubes 45-49 of chromatographs M and N contain, it appears that part of the half-selenocystine moiety has been converted to the 2-mercaptoethanol derivative in the buffers with the added thiodiglycol. However, since only about half of the ‘?Se in hydrolysates is eluted by the buffer without added thiodiglycol while about 85% is eluted when thiodiglycol is used, it appears that most of the ‘%e in tubes 35-40 derives from compounds eluting after the selenocystine in chromatograms B, C, and D. Such compounds as peptides containing the half-selenocystine moiety linked to sulfhydryl groups or selenite in ether linkages are possibilities, but the matter requires further study for its resolution. Finally, the data in Fig. 2 confirm those in Fig. 1 in showing the presence of 2,7-diamino-4-thia-5-selenaoctanedioic acid. There seems little doubt that selenocysteine can be synthesized from selenite in the body of the rat. Surprisingly, the data do not confirm the presence of selenomethionine. Since only a very small part of the “Se administered to the rats actually appeared in the selenocysteine moiety, a very small amount of impurity in the ‘?SeO; might account for it. Chromatograms E, L,, and L2 give some indication of the purity of the H,75Se0a used. In chromatogram E, about 90% (see Table 1) of the 75Se placed on the column eluted with the anions in tubes 4-7. No other peak of significance was noted. In view of the method of preparation of the H,‘%eO,, organic forms of the element could not be present. Selenium halides or oxyhalides and hydrogen selenide would decompose to elemental selenium and/or selenite under the conditions existing in the solutions. The 10% not accounted for in the eluates from the column might be the insoluble elemental form. However, at least a part of this might be accounted for by the fact that past experience has indicated the fixation of very small amounts of inorganic selenium that bleed slowly from the column over extended elution with the buffers. The small peak at tube 4 in chromatograms L, and LZ suggests an incomplete reaction of the selenite with the thiodiglycol or the more likely presence of some ‘%e04. It seems, therefore, that the 75Se solution used contained largely ‘%eO; with possible small amounts of 75Se or 75SeO;. The elemental form, being highly insoluble should not have been involved in selenocysteine formation, and the well known similarity between selenate and selenite
306
OLSON
AND PLAMER
metabolism in the rat suggests that either form could be contributing to the formation of this compound. The data showing that the half-selenocystine moiety occurs in combination with the sulfur analog (and perhaps in combination with peptides containing sulfide groups) may explain why some have not found selenium present in tissues as selenocystine. However, the mechanism of formation of selenocysteine in the animal body is not clear. Intestinal microbial biosynthesis may occur, but the similarity in level of selenium found in the half-selenocystine moiety in the rats injected with or orally administered the selenite suggests otherwise. The failure to find selenomethionine also suggests another mode of formation, since microbial synthesis should probably yield both amino acids. Reduction of the selenite to selenide’(’ and subsequent substitution of this for sulfide in the reaction with serine that is catalyzed by L-serine hydrolase (cysteine synthase EC 4.2.1.22)” is a possible explanation and might also account for the failure to find selenomethionine. ACKNOWLEDGMENT The authors wish to acknowledge the assistance of E. I. Whitehead this study.
with certain phases of
REFERENCES I. Shrift A: Selenium compounds in nature and medicine. E. Metabolism of selenium by plants and microorganisms, in Klayman DL, Gunther WHH (ed): Organic selenium compounds: Their chemistry and biology. New York, Wiley, 1973, pp 763-814 2. Rosenfeld I: Biosynthesis of seleno-compounds from inorganic selenium by sheep. Proc Sot Exptl Biol Med 1Il:670-673, 1962 3. Godwin KO, Handreck KA, Fuss CN: Identification of 75Se selenomethionine in ewe milk protein following the intraruminal administration of Na7’Se03 as a single oral dose. Aust J Biol Sci 24:1251-1261, 1971 4. Hidiroglou M, Heaney DP, Jenkins KJ: Metabolism of inorganic selenium in rumen bacteria. Can J Physiol Pharmacol 46:229-232, 1968 5. McConnell KP, Wabnitz CH: Studies on the fixation of radioselenium in proteins. J Biol Chem 226:765-776, 1957 6. Schwarz K, Sweeney E: Selenite binding to sulfur amino acids. Federation Proc 23:421, 1964 7. Huber RE, Criddle RS: Comparison of the chemical properties of selenocysteine and selenocystine with their sulfur analogs. Arch Biochem Biophys 122:164173, 1967 8. Jenkins KJ: Evidence for the absence of selenocystine and selenomethionine in the serum proteins of chicks administered selenite. Can J Biochem 46:1417-1425, 1968 9. Cummins LM, Martin JL: Are selenocystine and selenomethionine synthesized in
vivo from sodium selenite in mammals? Biothem 6:3162-3168,1967 IO. Olson OE, Novacek El, Whitehead EI, Palmer IS: Investigations on selenium in wheat. Phytochemistry 9:1181-l 188, 1970 11. Walter R, Schlesinger DH, Schwartz IL: Chromatographic separation of isologous sulfur- and selenium-containing amino acids: Reductive scission of the selenium bond by mercaptans and selenols. Analytical Biochem 27:231-243, 1969 12. Moore S, Spackman CH, Stein WH: Chromatography of amino acids on sulfonated polystyrene resins. Analytical Chem 3O:l l851190, 1958 13. Tsay DT, Halverson AW, Palmer IS: Inactivity of dietary trimethylselenonium chloride against the necrogenic syndrome of the rat. Nutrition Rept Internatl2:203-207, 1970 14. Painter EP: The chemistry and toxicity of selenium compounds, with special reference to the selenium problem. Chem Rev 28:179213, 1941 15. Benesch RE, Benesch R: Mechanism of disulfide interchange in acid solution; role of sulfenium ions. J Am Chem Sot 80~1666-1669, 1958 16. Diplock AT, Caygill CPJ, Jeffery EH, Thomas C: The nature of the acid-volatile selenium in the liver of the male rat. Biochem J 134:283-293, 1973 17. Greenberg DH: Amino acid metabolism, in Snell EE, Luck JM, Boyer PD, Mackinney G: Ann Rev Biochemistry 33:633-666, 1964
