BIOINORGANICCHEMISTRY 8, 419-429 (1978)
419
Isolation, Purification, and Partial Chemical Characterization of Chromium(III) Fractions Existing in Brewer's Yeast and Sabouraud's Liquid Medium
JORMA KUMPULAINEN, PEKKA KOIVISTOINEN, and SEPPO LAHTINEN Department ofFood Chemistry and Technology, University of Helsinki, SF-00710, Helsinki 71, Finland
ABSTRACT
An ethanol extract of brewer's yeast which had been cultivated in a medium containing trivalent 51Cr was analyzed for 51Cr compounds by using petroleum ether extraction, gel filtration, cation and anion exchange chromatography and thin layer chromatography. Similar analytical procedures as for the above analysis were used for studying 51Cr compounds formed in the spent culture medium and in a sterile medium. Several 51Cr fractions were isolated from the three chromium sources, but one anionic 51Cr fraction present in the yeast and in the spent culture medium was not found in the sterile medium. Molecular weight estimations of the 51Cr fractions by gel filtration chromatography showed that the 51Cr ion exchange fractions contained several 51Cr compounds. The molecular weights of these compounds ranged from 150 to 1000 daltons and the molecular weights of 51Cr com· pounds separated from the yeast were markedly lower than those of the corresponding ion exchange fractions isolated from the culture medium. By using thin layer chromatography it was possible to isolate 51 Cr compounds from the main bulk of ninhydrin active impurities. The polarity of all 51Cr compounds was found to be greater than that of most amino acids. The 51Cr compounds isolated from the yeast were mixed with 125I_insulin and incubated, after which the solution was eluted through Sephadex G-50 gel to test if binding had occurred. Elution peaks of 51Cr and 125I_insulin showed that 51Cr compounds were not bound to the insulin.
INTRODUCTION The essential role of trivalent chromium in the maintenance of normal carbohydrate metabolism in man and animals has been extensively reviewed [1,2]. In addition to the carbohydrate metabolism, chromium may have other physiological functions [3,4,5,6,7] . Most trace elements are easily converted into a biologically active form after © Elsevier North-Holland, Inc., 1978
0006-3061/78/0008-0419$01.25
420
J. KUMPULAINEN ET AL.
absorption, but chromium, even when present in the trivalent state, does not readily convert into a biologically most active form in the mammalian organism [2, 3, 8] . This seems to be a unique feature of chromium. Biological material contains organic chromium (III) complex(es) whose chemical structure and biological activity have only partly been identified [2] . The availability for absorption and biological activity of at least one of these compounds is much greater than that of inorganic chromium or organic chromium (III) complexes prepared synthetically [2, 8]. Furthermore, brewer's yeast (Saccharomyces carlsbergensis) has been found to be the best source of this biologically most active form of chromium compound(s) [2,9]. Because trivalent chromium has, however, a very strong tendency to form octahedral complexes with biological ligands [10] , an attempt was made in the present study to resolve the following points: (1) The kinds of chromium (III) complexes that exist in the culture medium; (2) the capability of brewer's yeast to take up and metabolize chromium compounds existing in the medium; (3) and whether the chromium (III) complexes isolated from the yeast are capable of binding to the insulin molecule [14].
MATERIALS AND METHODS Yeast Cultivation The yeast strain (Saccharomyces uvarum ex S. carlsbergensis) was obtained from the Biotechnological Laboratory of the State Technological Research Center, Helsinki, and stored on Sabouraud's dextrose agar slants at +4°C in capped test tubes. Sabouraud's liquid medium containing 1% neopeptone (Difco) and 5% dextrose (Bacto dextrose, Difco) was used for the cultivations. Dextrose liquid was sterilized separately by autoclaving for 25 min at 116°C. Peptone liquid was autoclaved for 20 min at 121°C. Sterilized tracer chromium (CrCI 3 X 6H 20, Judex Chemicals, in 0.1 N HC1) , 51CrCl 3 X 6H 20 (The Radiochemical Centre added into a 2.0-liter Erlenmayer. Four cultivations were done under agitation and nonaerated conditions. Other cultivation characteristics are presented in Table 1. Three days after the inoculation, sterilized glucose solution was added to the medium to obtain a 5% concentration. The yeast obtained from the cultivation No.1 was used for the estimation of the molecular weight of the separated chromium (III) fractions. The yeast obtained from the cultivation No.4 was used to test possible bond formation between 125 1 insulin and partially purified 51 Cr labeled chromium (III) compounds.
Cr(III) FRACTIONS IN BREWER'S YEAST
421
TABLE 1
Characteristics of the Yeast Cultivations
Number of cultivation
Cultivation time (d)
Medium volume (ml)
Cr conc. in medium (mg/l)
Specific activity of Cr (Ci/g)
I
10 8 7 8
2 X 640 2 X 640 1280 1280
0.63 0.78 0.78 0.0078
2.6 2.2 0.9 196.0
2 3 4
Preparation of Sterile Medium
A sterile medium for the study of chromium compounds fonned without influence of yeast or yeast metabolites was prepared in the same manner as the medium used for the cultivations. Olation of chromium in neutral conditions was prevented by adding 6 M HCl to the medium to obtain a pH value of 3.5, which was the same as that fonned as a result of yeast fennentation in the medium. Isolation and Purification of Chromium (III) Fractions
Yeast cells were harvested by centrifugation. The yeast was then washed with distilled water and the cells were disintegrated in a cold press (Biox). Ethanol was added to obtain a 50% ethanol concentration. The cell mass was extracted for 1-3 days under agitation. The ethanol was removed by centrifugation and the disintegrated cell mass was further washed three times with 50% ethanol. The ethanol solutions were subsequently combined and lipids were extracted with petroleum ether. In experiments dealing with chromium compounds in spent and sterile mediums, the petroleum ether extraction was the first step in the isolation procedure. After the lipid extraction the samples were concentrated to a small volume in a vacuum rotary evaporator at a temperature of +40°C and chromatographed on 2.5 X 45 cm Sephadex G-25 gel, which was then eluted with distilled water. Combined 2.0 ml fractions containing olCr were concentrated in a vacuum rotatory evaporator (Heidolph UV-I) and chromatographed on Dowex 50W X 8 (H+ form, 100-200 mesh) cation exchange resin using an increasing NH 4 0H concentration gradient elution (0.0-2. 0 M).
422
J. KUMPULAINEN ET Al
The 'Y active 2.0 ml fractions belonging to the same peaks were collected and combined. The 51Cr peak that had been eluted with water was chromatographed on Sephadex QAE A-25 anion exchange gel (C0 3 2- form) using an increasing (NH 4 hC0 3 concentration gradient elution (0.0-1.5 M), and 2.0 ml fractions were collected. Purification of isolated chromium (III) fractions was done on 20 X 20 cm thin layer chromatographic plates using a two dimensional n-butanol: acetic acid: water (2:1 :2) elution. The qualitative and quantitative analysis of chromium was based on 'Y activity measurements of 51Cr in a 'Y scintillation counter (Ultragamma 1280, LKB-Wallac) which incorporated an automatic sample changer and was coupled to a printer. Partial Chemical Characterization of Chromium (III) Fractions The average molecular weight of each isolated 51Cr fraction was estimated by gel filtration chromatography on 1.5 X 90 cm Sephadex G-25 columns. NaCI solution (0.1 %) was used for the elution. For preparation of the calibration line, 50% polyethyleneglycol solutions representing molecular weights from 500 to 1500 were used. The formula of the calibration line (Kav = 1.635 - 0.474 log MW; r2 = 99.99%) was calculated as a linear regression between logarithms of the molecular weights of the polyethyleneglycols and the corresponding Kav (= elution volume) values. The calibration line error was calculated by using Student's t-test and 95% confidence limits. Test for Binding of 51 Cr Compounds to the Insulin Molecule
The ability of partially purified 51 Cr compounds isolated from brewer's yeast to bind to the insulin molecule was tested as follows. The 51 Cr fractions were evaporated to dryness and dissolved in phosphate buffer, pH 7.4. Then 500 JLU of 125 1 labeled insulin (Insulin RIA-Kit, The Radiochemical Centre Ltd.) and 500 JLU of unlabeled insulin were added to the buffer and incubated for 2 hours at room temperature in capped test tubes. The solution was eluted through a 0.9 X 60 cm SephadexG-50 gel with phosphate buffer. Fractions of 2.0 ml were collected and the 'Y activity of both 51Cr and 1251 were measured simultaneously in a 'Y scintillation counter (Ultragamma 1280, LKB-Wallac) by the usual double labeling measurement technique.
RESULTS Incorporation of 51 Cr into Yeast Cells
Radioactivity measurements showed that 0.3-0.7% of the chromium added to the medium was taken up by the yeast cells. More chromium was
Cr(IlI) FRACTIONS IN BREWER'S YEAST CATION EXCHANGE
423 ANION EXCHANGE
STERILE MEOIUM
STERILE MEOIUM 16
120
12
~
!2
x
~
u
x
~
80
u
8
'0
H20
0.5 1.0 1.5 1.6
2.0
1.5
M NH,OH
CATION EXCHANGE
ANION EXCHANGE
MEOIUM AFTER YEAST CENTRIFUGATION
160
160
MEOIUM AFTER YEAST CENTRIFUGATION
120
x ~ 80
u
'0
\ H 20
A 0.5 1.0 1.5 1.6
2.0
M NH,OH
FIG. 1. 51 Cr peaks eluted in cation and anion exchanges of chromium fractions isolated by gel filtration from sterile medium and from medium after yeast cultivation (spent medium).
incorporated into cells when the cultivation time was increased from 7 to 10 days. The chromium content of the yeast cells correlated positively with the chromium content of the medium and was about 50-80% of the latter depending on the cultivation time. Depending on the extraction time 30-50% of the chromium in the yeast cells was extracted in 50% ethanol. Petroleum ether was used for the extraction of lipids from this solution and only a small amount « 0.1%) of the chromium in ethanol was lost in the petroleum ether phase. Gel Filtration and Ion Exchange Chromatography Chromium was eluted through the gel filtration column in a single peak and always at the same VeNt value (0.38-0.42). The results of the cation and anion exchanges of 51Cr fractions isolated by gel filtration from the sterile medium and the medium used for yeast cultivation are presented in Figure 1. The figure demonstrates that during cultivation the proportion of
424
J. KUMPULAINEN ET AL. ANION EXCHANGE
CATION EXCHANGE YEAST CULTIVATION
60
n
40
\"')~ 8
~ 20
~ 4
M
\2
YEAST CULTIVATION
10
n
x
x
1
1
\ H20
0.5 1.0 1.5
2.0
H
2
0
M NH 0H 4
0.2 0.4
0.6
0.8
1.5
1,0
1,5
M INH 4 )2C03
CATION EXCHANGE
ANION EXCHANGE
MEDIUM AF TER YEAST CENTRIFUGATION
160
120 o
x
x ~ 80 u
40
\ H 0
2
A 0.5 1.0 1.5 1.6 M NH 4 0H
2.0
H 2 0 0.2
0.4
0.6
0.8
M INH 4 ' 2 C0 3
FIG. 2. Cation and anion exchanges of 51Cr fraction isolated by ethanol extraction and gel fJItration from the yeast obtained from culture No.2. Ion exchanges of the 51Cr fraction isolated from the culture medium by gel fJItration are presented for comparison. cationic 51Cr fraction decreased from 57% to 8% of the total 51Cr of the medium. The anionic 51Cr peak eluted with 0.1 M (NH4 hCO a solution was drastically decreased as a result of cultivation. In addition, a new anionic 51Cr fraction, eluted with 0.4 M carbonate solution, was formed during the cultivation. Figure 2 shows the results of cation and anion exchanges of 51Cr fractions isolated by ethanol extraction and gel filtration from the yeast obtained from cultivation No: 2. Corresponding ion exchanges of 51Cr fraction isolated from the cultivation medium by gel filtration are included for comparison. The elution peaks of the 51Cr fractions isolated from the yeasts in cultivation No. 3 were exactly the same as those of No.2. Figure 2 shows that the yeast did not contain the chromium fractions of the medium eluted with water and 0.1 M carbonate solution. The 51Cr fraction eluted with 0.4 M carbonate solution seems to result from the metabolic activity of the yeast, as it is not found in the sterile medium. The cationic 51Cr fraction seems to be present in the yeast and in both the sterile and the spent culture mediums. The proportions of cationic and
425
Cr(III) FRACTIONS IN BREWER'S YEAST TABLE 2 Proportions of Cationic and Anionic 51 Cr Fractions (%) of the Total 51Cr Eluted from Different 51Cr Sources in Cation Exchanges Cr content of Cultivation time (d)
yeast (mg/kg)
medium (mgfl)
Proportion of cationic 51Cr fraction (%)
Proportion of anionic 51Cr fraction (%)
7
0.38
0.78
29.7
70.3
8
0.44
0.78
33.1
66.9
8
0.0051
0'.0078
49.6
50.4
0.48
0.63
41.5
58.5
10
Medium (10)
0.63
8.1
91.9
Sterile medium (10)
0.78
57.0
43.0
anionic 51Cr fractions of the total 51Cr eluted from different 51Cr sources in cation exchanges are summarized in Table 2.
Thin Layer Chromatography The cationic 51Cr fractions isolated from the yeast and from the yeast-free culture medium both contained ninhydrin active impurities, as also did the anionic 51Cr fraction isolated from the yeast-free culture medium. When using n-butanol; acetic acid: water (4:1:1) elution, which is commonly used for the separation of amino acids, several ninhydrin active impurities could be found in the cationic 51 Cr fraction isolated from yeast and spent medium. The anionic 51Cr fraction isolated from the yeast did not contain ninhydrin active compounds. Several elution systems were studied for the purification of 51Cr compounds on thin layer chromatographic plates. The best eluent proved to be butanol: acetic acid: water (2: 1 :2). By using this solvent and two-dimensional elution it was possible to separate the 51 Cr compounds from the main bulk ofninhydrin active impurities, which migrated faster than the 51 Cr compounds. The thin layer chromatographic experiments showed that the polarities of all the isolated 51Cr compounds were greater than those of most amino acids. 5lCr activity was found in spots that were slightly visible after ninhy-
J. KUMPULAINEN ET AL.
426 TABLE 3
Estimated Molecular Weights of Cationic 51Cr Fractions
M NH 4 0H Ve (ml) MW, daltons Estimate of error
Spent culture medium
Yeast
1.5 M
I.5M
71 763 674-864
67 979 864-1109
78 493 436-559
86 300 262-342
90 234
drin treatment, but ninhydrin activity of these 51Cr compounds could not be definitely ascertained. Estimation of Molecular Weight The results of the molecular weight estimations of the isolated 51Cr fractions are presented in Tables 3 and 4. Results of the molecular weight estimations show that neither cationic nor anionic 51Cr fractions are homogeneous. The cationic 51Cr fraction of yeast contains at least three 51Cr compounds and that of the medium two. The anionic 51Cr fraction of both the yeast and the medium contains two 51Cr compounds. Tables 2 and 3 show that both the cationic and the anionic 51Cr complexes isolated from the yeast were in general much lower in molecular weight than the corresponding compounds isolated from the medium. Cationic 51Cr complexes isolated from both yeast and medium contained a small olated, polymeric 51Cr fraction. TABLE 4 Estimated Molecular Weights of Anionic 51 Cr Fractions
M (NH 4 hCO a Ve (m)) MW, daltons Estimate of error
Spent culture medium
Yeast
0.4-0.5 M
0.4 M
76 559 489-638
90 234
90 234
98 142
Cr(III) FRACTIONS IN BREWER'S YEAST
427
200 r----.----..-........---.---.--., 16 M
M
O
120
12 S2
~ 80
8 [3
~
x
~
~ L.
U
I. ;;;
40 10
20
30
40
FRACTION NUMBER
100r---'~--~-"""""--,--,, _
125 51
80
1 Cr
16
M
M
S2 x 60
S2 12 x
~
[3
....
~
8
20
4 ;;;
U
II>
~
[3
1.0
10
20
30
1.0
50
FRACTION NUMBER
FIG. 3. Results of elution through Sephadex G-SO column of 125 1 insulin and of cationic (above) and anionic (below) 51Cr fractions isolated from the yeast.
Test for Binding of 51Cr Compounds to the Insulin Molecule Figure 3 shows the results of elution on 0.9 X 60 cm Sephadex G-SO gel of cationic (above) and anionic (below) 51Cr fractions isolated from the yeast and that of 125 1 insulin. Figure 3 indicates that neither cationic nor anionic 51Cr compounds were bound to the insulin molecule. The 125 1 peak first eluted from the column is apparently dimeric or polymeric insulin, the second 1251 peak is due to monomeric native insulin, and the third peak is non-reacted 1251. The elution characteristics of the anionic nCr fraction used in the test differed from those of the anionic 51Cr fractions obtained in other ion exchanges in the respect that the fraction was elutable with 0.2 M (NH 4 hCO a solution.
428
J. KUMPULAINEN ET AL.
DISCUSSION The results of ion exchange chromatography show that the yeast was capable of taking up, metabolizing and excreting into the medium chromium complexes formed in the medium, and/or that the metabolism products excreted into the medium by the yeast reacted with chromium, forming new compounds which then were taken up by yeast cells. Furthermore, Table 2 shows that the increase in chromium content of the yeast between the 7th and 10th days of cultivation was the result of an increase in cationic chromium in the cells. This suggests that the cationic chromium cannot be transported out of the cells. The fact that the chromium content of yeast cells was always lower then that of the medium and correlated positively with it suggests that the chromium was not actively taken up by yeast cells but that transport rather was based on diffusion and the ionic concentration balance. These observations confirm the results of previous experiments in our laboratory in which a different strain of Saccharomyces carlsbergensis was used [11] . The results of the present study differ from those of Votava et aI. [12], who obtained only one anionic 51 Cr fraction from Saccharomyces cerevisies. Evans et al. [14] have reported that a partially purified chromium-containing fraction extracted from brewer's yeast was able to bind to the insulin molecule, so potentiating the glucose metabolic activity of insulin in vitro. The binding of chromium to insulin was not demonstrated unambiguously, however, since in their experiment only the insulin was labeled. The chromium compound, or a fraction resembling the chromium (III) complex, termed glucose tolerance factor (GTF) and isolated and purified by Mertz et aI. [2] from brewer's yeast, was not found in the present study. Apparently, not all yeast strains of S. carlsbergensis are able to synthesize GTF, or possibly specific cultivation conditions are necessary for the synthesis.
REFERENCES 1. Mertz, W., Phys. Rev. 49,162-239 (1969). 2. Mertz, W., Toepfer, E. W., Roginski, E. E. and Polansky, M. M., Fed. Proc. 33,32753280 (1974). 3. Trace elements in human nutrition, Report of a WHO Expert Committee. Wid. Hlth. Org. Rep. Ser., No. 532 (1973). 4. Roginski, E. E. and Mertz, W.,!. Nutr. 97,525-530 (1969). 5. Schroeder, H. A., Nason, A. P. and Tipton, 1. H., J. Chron. Dis. 23,123-142. (1970). 6. Wacker, W. E. C. and Vallee, B. E.,!. Bioi. Chern. 234,3257-3262 (1959). 7. Punsar, S., Eriimetsii, 0., Karvonen, M. 1., Ryhlinen, A. and Vornamo, J. Chron. Dis. 28,259-287 (1975). 8. Mertz, W., and Roginski, E. E., in Newer Trace Elements in Nutrition, W. Mertz and E. E. Cornatzer, ed., New York, 1971, p. 438.
Cr(III) FRACTIONS·IN BREWER'S YEAST
429
9. Toepfer, E. W., Mertz, W., Roginski, E. E. and Polansky, M. M., J. Agr. Food Chern. 21,69-73 (1973). 10. Rollinson, C. L., Rosenbloom, E. and Lindsay, J., Proc. 7th Intern. Congr. Nutr. 5, 692-697 (1967). 11. Kumpulainen, J. and Koivistoinen, P., to be published in Bioinorg. Chem (1978). 12. Votava, H. 1., Hahn, C. 1. and Evans, G. W., Biochern. Biophys. Res. Cornrnun. 55, 312-319 (1973). 13. Burkeholder, J. N. and Mertz, W., Proc. 7th Intern. Congr. Nutr. 5,701-705 (1966). 14. Evans, G. W., Roginski, E. E. and Mertz, W., Biophys. Res. Cornrnun. 50,718-722 (1973).
Received 10 May 1977
419
Isolation, Purification, and Partial Chemical Characterization of Chromium(III) Fractions Existing in Brewer's Yeast and Sabouraud's Liquid Medium
JORMA KUMPULAINEN, PEKKA KOIVISTOINEN, and SEPPO LAHTINEN Department ofFood Chemistry and Technology, University of Helsinki, SF-00710, Helsinki 71, Finland
ABSTRACT
An ethanol extract of brewer's yeast which had been cultivated in a medium containing trivalent 51Cr was analyzed for 51Cr compounds by using petroleum ether extraction, gel filtration, cation and anion exchange chromatography and thin layer chromatography. Similar analytical procedures as for the above analysis were used for studying 51Cr compounds formed in the spent culture medium and in a sterile medium. Several 51Cr fractions were isolated from the three chromium sources, but one anionic 51Cr fraction present in the yeast and in the spent culture medium was not found in the sterile medium. Molecular weight estimations of the 51Cr fractions by gel filtration chromatography showed that the 51Cr ion exchange fractions contained several 51Cr compounds. The molecular weights of these compounds ranged from 150 to 1000 daltons and the molecular weights of 51Cr com· pounds separated from the yeast were markedly lower than those of the corresponding ion exchange fractions isolated from the culture medium. By using thin layer chromatography it was possible to isolate 51 Cr compounds from the main bulk of ninhydrin active impurities. The polarity of all 51Cr compounds was found to be greater than that of most amino acids. The 51Cr compounds isolated from the yeast were mixed with 125I_insulin and incubated, after which the solution was eluted through Sephadex G-50 gel to test if binding had occurred. Elution peaks of 51Cr and 125I_insulin showed that 51Cr compounds were not bound to the insulin.
INTRODUCTION The essential role of trivalent chromium in the maintenance of normal carbohydrate metabolism in man and animals has been extensively reviewed [1,2]. In addition to the carbohydrate metabolism, chromium may have other physiological functions [3,4,5,6,7] . Most trace elements are easily converted into a biologically active form after © Elsevier North-Holland, Inc., 1978
0006-3061/78/0008-0419$01.25
420
J. KUMPULAINEN ET AL.
absorption, but chromium, even when present in the trivalent state, does not readily convert into a biologically most active form in the mammalian organism [2, 3, 8] . This seems to be a unique feature of chromium. Biological material contains organic chromium (III) complex(es) whose chemical structure and biological activity have only partly been identified [2] . The availability for absorption and biological activity of at least one of these compounds is much greater than that of inorganic chromium or organic chromium (III) complexes prepared synthetically [2, 8]. Furthermore, brewer's yeast (Saccharomyces carlsbergensis) has been found to be the best source of this biologically most active form of chromium compound(s) [2,9]. Because trivalent chromium has, however, a very strong tendency to form octahedral complexes with biological ligands [10] , an attempt was made in the present study to resolve the following points: (1) The kinds of chromium (III) complexes that exist in the culture medium; (2) the capability of brewer's yeast to take up and metabolize chromium compounds existing in the medium; (3) and whether the chromium (III) complexes isolated from the yeast are capable of binding to the insulin molecule [14].
MATERIALS AND METHODS Yeast Cultivation The yeast strain (Saccharomyces uvarum ex S. carlsbergensis) was obtained from the Biotechnological Laboratory of the State Technological Research Center, Helsinki, and stored on Sabouraud's dextrose agar slants at +4°C in capped test tubes. Sabouraud's liquid medium containing 1% neopeptone (Difco) and 5% dextrose (Bacto dextrose, Difco) was used for the cultivations. Dextrose liquid was sterilized separately by autoclaving for 25 min at 116°C. Peptone liquid was autoclaved for 20 min at 121°C. Sterilized tracer chromium (CrCI 3 X 6H 20, Judex Chemicals, in 0.1 N HC1) , 51CrCl 3 X 6H 20 (The Radiochemical Centre added into a 2.0-liter Erlenmayer. Four cultivations were done under agitation and nonaerated conditions. Other cultivation characteristics are presented in Table 1. Three days after the inoculation, sterilized glucose solution was added to the medium to obtain a 5% concentration. The yeast obtained from the cultivation No.1 was used for the estimation of the molecular weight of the separated chromium (III) fractions. The yeast obtained from the cultivation No.4 was used to test possible bond formation between 125 1 insulin and partially purified 51 Cr labeled chromium (III) compounds.
Cr(III) FRACTIONS IN BREWER'S YEAST
421
TABLE 1
Characteristics of the Yeast Cultivations
Number of cultivation
Cultivation time (d)
Medium volume (ml)
Cr conc. in medium (mg/l)
Specific activity of Cr (Ci/g)
I
10 8 7 8
2 X 640 2 X 640 1280 1280
0.63 0.78 0.78 0.0078
2.6 2.2 0.9 196.0
2 3 4
Preparation of Sterile Medium
A sterile medium for the study of chromium compounds fonned without influence of yeast or yeast metabolites was prepared in the same manner as the medium used for the cultivations. Olation of chromium in neutral conditions was prevented by adding 6 M HCl to the medium to obtain a pH value of 3.5, which was the same as that fonned as a result of yeast fennentation in the medium. Isolation and Purification of Chromium (III) Fractions
Yeast cells were harvested by centrifugation. The yeast was then washed with distilled water and the cells were disintegrated in a cold press (Biox). Ethanol was added to obtain a 50% ethanol concentration. The cell mass was extracted for 1-3 days under agitation. The ethanol was removed by centrifugation and the disintegrated cell mass was further washed three times with 50% ethanol. The ethanol solutions were subsequently combined and lipids were extracted with petroleum ether. In experiments dealing with chromium compounds in spent and sterile mediums, the petroleum ether extraction was the first step in the isolation procedure. After the lipid extraction the samples were concentrated to a small volume in a vacuum rotary evaporator at a temperature of +40°C and chromatographed on 2.5 X 45 cm Sephadex G-25 gel, which was then eluted with distilled water. Combined 2.0 ml fractions containing olCr were concentrated in a vacuum rotatory evaporator (Heidolph UV-I) and chromatographed on Dowex 50W X 8 (H+ form, 100-200 mesh) cation exchange resin using an increasing NH 4 0H concentration gradient elution (0.0-2. 0 M).
422
J. KUMPULAINEN ET Al
The 'Y active 2.0 ml fractions belonging to the same peaks were collected and combined. The 51Cr peak that had been eluted with water was chromatographed on Sephadex QAE A-25 anion exchange gel (C0 3 2- form) using an increasing (NH 4 hC0 3 concentration gradient elution (0.0-1.5 M), and 2.0 ml fractions were collected. Purification of isolated chromium (III) fractions was done on 20 X 20 cm thin layer chromatographic plates using a two dimensional n-butanol: acetic acid: water (2:1 :2) elution. The qualitative and quantitative analysis of chromium was based on 'Y activity measurements of 51Cr in a 'Y scintillation counter (Ultragamma 1280, LKB-Wallac) which incorporated an automatic sample changer and was coupled to a printer. Partial Chemical Characterization of Chromium (III) Fractions The average molecular weight of each isolated 51Cr fraction was estimated by gel filtration chromatography on 1.5 X 90 cm Sephadex G-25 columns. NaCI solution (0.1 %) was used for the elution. For preparation of the calibration line, 50% polyethyleneglycol solutions representing molecular weights from 500 to 1500 were used. The formula of the calibration line (Kav = 1.635 - 0.474 log MW; r2 = 99.99%) was calculated as a linear regression between logarithms of the molecular weights of the polyethyleneglycols and the corresponding Kav (= elution volume) values. The calibration line error was calculated by using Student's t-test and 95% confidence limits. Test for Binding of 51 Cr Compounds to the Insulin Molecule
The ability of partially purified 51 Cr compounds isolated from brewer's yeast to bind to the insulin molecule was tested as follows. The 51 Cr fractions were evaporated to dryness and dissolved in phosphate buffer, pH 7.4. Then 500 JLU of 125 1 labeled insulin (Insulin RIA-Kit, The Radiochemical Centre Ltd.) and 500 JLU of unlabeled insulin were added to the buffer and incubated for 2 hours at room temperature in capped test tubes. The solution was eluted through a 0.9 X 60 cm SephadexG-50 gel with phosphate buffer. Fractions of 2.0 ml were collected and the 'Y activity of both 51Cr and 1251 were measured simultaneously in a 'Y scintillation counter (Ultragamma 1280, LKB-Wallac) by the usual double labeling measurement technique.
RESULTS Incorporation of 51 Cr into Yeast Cells
Radioactivity measurements showed that 0.3-0.7% of the chromium added to the medium was taken up by the yeast cells. More chromium was
Cr(IlI) FRACTIONS IN BREWER'S YEAST CATION EXCHANGE
423 ANION EXCHANGE
STERILE MEOIUM
STERILE MEOIUM 16
120
12
~
!2
x
~
u
x
~
80
u
8
'0
H20
0.5 1.0 1.5 1.6
2.0
1.5
M NH,OH
CATION EXCHANGE
ANION EXCHANGE
MEOIUM AFTER YEAST CENTRIFUGATION
160
160
MEOIUM AFTER YEAST CENTRIFUGATION
120
x ~ 80
u
'0
\ H 20
A 0.5 1.0 1.5 1.6
2.0
M NH,OH
FIG. 1. 51 Cr peaks eluted in cation and anion exchanges of chromium fractions isolated by gel filtration from sterile medium and from medium after yeast cultivation (spent medium).
incorporated into cells when the cultivation time was increased from 7 to 10 days. The chromium content of the yeast cells correlated positively with the chromium content of the medium and was about 50-80% of the latter depending on the cultivation time. Depending on the extraction time 30-50% of the chromium in the yeast cells was extracted in 50% ethanol. Petroleum ether was used for the extraction of lipids from this solution and only a small amount « 0.1%) of the chromium in ethanol was lost in the petroleum ether phase. Gel Filtration and Ion Exchange Chromatography Chromium was eluted through the gel filtration column in a single peak and always at the same VeNt value (0.38-0.42). The results of the cation and anion exchanges of 51Cr fractions isolated by gel filtration from the sterile medium and the medium used for yeast cultivation are presented in Figure 1. The figure demonstrates that during cultivation the proportion of
424
J. KUMPULAINEN ET AL. ANION EXCHANGE
CATION EXCHANGE YEAST CULTIVATION
60
n
40
\"')~ 8
~ 20
~ 4
M
\2
YEAST CULTIVATION
10
n
x
x
1
1
\ H20
0.5 1.0 1.5
2.0
H
2
0
M NH 0H 4
0.2 0.4
0.6
0.8
1.5
1,0
1,5
M INH 4 )2C03
CATION EXCHANGE
ANION EXCHANGE
MEDIUM AF TER YEAST CENTRIFUGATION
160
120 o
x
x ~ 80 u
40
\ H 0
2
A 0.5 1.0 1.5 1.6 M NH 4 0H
2.0
H 2 0 0.2
0.4
0.6
0.8
M INH 4 ' 2 C0 3
FIG. 2. Cation and anion exchanges of 51Cr fraction isolated by ethanol extraction and gel fJItration from the yeast obtained from culture No.2. Ion exchanges of the 51Cr fraction isolated from the culture medium by gel fJItration are presented for comparison. cationic 51Cr fraction decreased from 57% to 8% of the total 51Cr of the medium. The anionic 51Cr peak eluted with 0.1 M (NH4 hCO a solution was drastically decreased as a result of cultivation. In addition, a new anionic 51Cr fraction, eluted with 0.4 M carbonate solution, was formed during the cultivation. Figure 2 shows the results of cation and anion exchanges of 51Cr fractions isolated by ethanol extraction and gel filtration from the yeast obtained from cultivation No: 2. Corresponding ion exchanges of 51Cr fraction isolated from the cultivation medium by gel filtration are included for comparison. The elution peaks of the 51Cr fractions isolated from the yeasts in cultivation No. 3 were exactly the same as those of No.2. Figure 2 shows that the yeast did not contain the chromium fractions of the medium eluted with water and 0.1 M carbonate solution. The 51Cr fraction eluted with 0.4 M carbonate solution seems to result from the metabolic activity of the yeast, as it is not found in the sterile medium. The cationic 51Cr fraction seems to be present in the yeast and in both the sterile and the spent culture mediums. The proportions of cationic and
425
Cr(III) FRACTIONS IN BREWER'S YEAST TABLE 2 Proportions of Cationic and Anionic 51 Cr Fractions (%) of the Total 51Cr Eluted from Different 51Cr Sources in Cation Exchanges Cr content of Cultivation time (d)
yeast (mg/kg)
medium (mgfl)
Proportion of cationic 51Cr fraction (%)
Proportion of anionic 51Cr fraction (%)
7
0.38
0.78
29.7
70.3
8
0.44
0.78
33.1
66.9
8
0.0051
0'.0078
49.6
50.4
0.48
0.63
41.5
58.5
10
Medium (10)
0.63
8.1
91.9
Sterile medium (10)
0.78
57.0
43.0
anionic 51Cr fractions of the total 51Cr eluted from different 51Cr sources in cation exchanges are summarized in Table 2.
Thin Layer Chromatography The cationic 51Cr fractions isolated from the yeast and from the yeast-free culture medium both contained ninhydrin active impurities, as also did the anionic 51Cr fraction isolated from the yeast-free culture medium. When using n-butanol; acetic acid: water (4:1:1) elution, which is commonly used for the separation of amino acids, several ninhydrin active impurities could be found in the cationic 51 Cr fraction isolated from yeast and spent medium. The anionic 51Cr fraction isolated from the yeast did not contain ninhydrin active compounds. Several elution systems were studied for the purification of 51Cr compounds on thin layer chromatographic plates. The best eluent proved to be butanol: acetic acid: water (2: 1 :2). By using this solvent and two-dimensional elution it was possible to separate the 51 Cr compounds from the main bulk ofninhydrin active impurities, which migrated faster than the 51 Cr compounds. The thin layer chromatographic experiments showed that the polarities of all the isolated 51Cr compounds were greater than those of most amino acids. 5lCr activity was found in spots that were slightly visible after ninhy-
J. KUMPULAINEN ET AL.
426 TABLE 3
Estimated Molecular Weights of Cationic 51Cr Fractions
M NH 4 0H Ve (ml) MW, daltons Estimate of error
Spent culture medium
Yeast
1.5 M
I.5M
71 763 674-864
67 979 864-1109
78 493 436-559
86 300 262-342
90 234
drin treatment, but ninhydrin activity of these 51Cr compounds could not be definitely ascertained. Estimation of Molecular Weight The results of the molecular weight estimations of the isolated 51Cr fractions are presented in Tables 3 and 4. Results of the molecular weight estimations show that neither cationic nor anionic 51Cr fractions are homogeneous. The cationic 51Cr fraction of yeast contains at least three 51Cr compounds and that of the medium two. The anionic 51Cr fraction of both the yeast and the medium contains two 51Cr compounds. Tables 2 and 3 show that both the cationic and the anionic 51Cr complexes isolated from the yeast were in general much lower in molecular weight than the corresponding compounds isolated from the medium. Cationic 51Cr complexes isolated from both yeast and medium contained a small olated, polymeric 51Cr fraction. TABLE 4 Estimated Molecular Weights of Anionic 51 Cr Fractions
M (NH 4 hCO a Ve (m)) MW, daltons Estimate of error
Spent culture medium
Yeast
0.4-0.5 M
0.4 M
76 559 489-638
90 234
90 234
98 142
Cr(III) FRACTIONS IN BREWER'S YEAST
427
200 r----.----..-........---.---.--., 16 M
M
O
120
12 S2
~ 80
8 [3
~
x
~
~ L.
U
I. ;;;
40 10
20
30
40
FRACTION NUMBER
100r---'~--~-"""""--,--,, _
125 51
80
1 Cr
16
M
M
S2 x 60
S2 12 x
~
[3
....
~
8
20
4 ;;;
U
II>
~
[3
1.0
10
20
30
1.0
50
FRACTION NUMBER
FIG. 3. Results of elution through Sephadex G-SO column of 125 1 insulin and of cationic (above) and anionic (below) 51Cr fractions isolated from the yeast.
Test for Binding of 51Cr Compounds to the Insulin Molecule Figure 3 shows the results of elution on 0.9 X 60 cm Sephadex G-SO gel of cationic (above) and anionic (below) 51Cr fractions isolated from the yeast and that of 125 1 insulin. Figure 3 indicates that neither cationic nor anionic 51Cr compounds were bound to the insulin molecule. The 125 1 peak first eluted from the column is apparently dimeric or polymeric insulin, the second 1251 peak is due to monomeric native insulin, and the third peak is non-reacted 1251. The elution characteristics of the anionic nCr fraction used in the test differed from those of the anionic 51Cr fractions obtained in other ion exchanges in the respect that the fraction was elutable with 0.2 M (NH 4 hCO a solution.
428
J. KUMPULAINEN ET AL.
DISCUSSION The results of ion exchange chromatography show that the yeast was capable of taking up, metabolizing and excreting into the medium chromium complexes formed in the medium, and/or that the metabolism products excreted into the medium by the yeast reacted with chromium, forming new compounds which then were taken up by yeast cells. Furthermore, Table 2 shows that the increase in chromium content of the yeast between the 7th and 10th days of cultivation was the result of an increase in cationic chromium in the cells. This suggests that the cationic chromium cannot be transported out of the cells. The fact that the chromium content of yeast cells was always lower then that of the medium and correlated positively with it suggests that the chromium was not actively taken up by yeast cells but that transport rather was based on diffusion and the ionic concentration balance. These observations confirm the results of previous experiments in our laboratory in which a different strain of Saccharomyces carlsbergensis was used [11] . The results of the present study differ from those of Votava et aI. [12], who obtained only one anionic 51 Cr fraction from Saccharomyces cerevisies. Evans et al. [14] have reported that a partially purified chromium-containing fraction extracted from brewer's yeast was able to bind to the insulin molecule, so potentiating the glucose metabolic activity of insulin in vitro. The binding of chromium to insulin was not demonstrated unambiguously, however, since in their experiment only the insulin was labeled. The chromium compound, or a fraction resembling the chromium (III) complex, termed glucose tolerance factor (GTF) and isolated and purified by Mertz et aI. [2] from brewer's yeast, was not found in the present study. Apparently, not all yeast strains of S. carlsbergensis are able to synthesize GTF, or possibly specific cultivation conditions are necessary for the synthesis.
REFERENCES 1. Mertz, W., Phys. Rev. 49,162-239 (1969). 2. Mertz, W., Toepfer, E. W., Roginski, E. E. and Polansky, M. M., Fed. Proc. 33,32753280 (1974). 3. Trace elements in human nutrition, Report of a WHO Expert Committee. Wid. Hlth. Org. Rep. Ser., No. 532 (1973). 4. Roginski, E. E. and Mertz, W.,!. Nutr. 97,525-530 (1969). 5. Schroeder, H. A., Nason, A. P. and Tipton, 1. H., J. Chron. Dis. 23,123-142. (1970). 6. Wacker, W. E. C. and Vallee, B. E.,!. Bioi. Chern. 234,3257-3262 (1959). 7. Punsar, S., Eriimetsii, 0., Karvonen, M. 1., Ryhlinen, A. and Vornamo, J. Chron. Dis. 28,259-287 (1975). 8. Mertz, W., and Roginski, E. E., in Newer Trace Elements in Nutrition, W. Mertz and E. E. Cornatzer, ed., New York, 1971, p. 438.
Cr(III) FRACTIONS·IN BREWER'S YEAST
429
9. Toepfer, E. W., Mertz, W., Roginski, E. E. and Polansky, M. M., J. Agr. Food Chern. 21,69-73 (1973). 10. Rollinson, C. L., Rosenbloom, E. and Lindsay, J., Proc. 7th Intern. Congr. Nutr. 5, 692-697 (1967). 11. Kumpulainen, J. and Koivistoinen, P., to be published in Bioinorg. Chem (1978). 12. Votava, H. 1., Hahn, C. 1. and Evans, G. W., Biochern. Biophys. Res. Cornrnun. 55, 312-319 (1973). 13. Burkeholder, J. N. and Mertz, W., Proc. 7th Intern. Congr. Nutr. 5,701-705 (1966). 14. Evans, G. W., Roginski, E. E. and Mertz, W., Biophys. Res. Cornrnun. 50,718-722 (1973).
Received 10 May 1977
