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Identification of biocytin in biotin proteins using high-voltage electrophoresis
470
SHORT
COMMUNICATIONS
J. B. LLOYD W. J. WHELAN Department of Biochemistry University of Miami School of Medicine Miami, Florida SSlb.2 Received May 6, 1969
Identification Using
of Biocytin High-Voltage
in Biotin
Proteins
Electrophoresisl
The first indication of the type of bond involved in the interaction of biotin with protein came from the isolation of biocytin (r-N-biotinyl-nlysine) from yeast by Wright et al. (1) and the determination of its structure (2). Using a crude preparation of propionyl holocarboxylase, Kosow and Lane (3) were able to show that in this protein biotin is covalently linked to the r-amino group of a lysine residue. Biocytin was identified using a combination of paper and ion-exchange chromatography. In this communication an alternative method for the identification of biocytin from proteins labeled in viva with d- [ carbonyl-14C] -biotin is presented. rats (4) were inIn vivo labeling of b&in proteins: Biotin-deficient jected intraperitoneally with 5 PC of d-[carbonyl-l*C]-biotin (57.4 mc/ mmole) in saline per 100 gm body weight 16 hr before sacrifice. The rats were killed by decapitation. The livers were quickly removed, rinsed in cold saline, and homogenized in 3 vol of medium (0.24 M sucrose, 0.025 M KCl, 0.001 M MgCl, in 0.05 M Tris HCl, pH 7.6). The crude homogenate was centrifuged at 600g for 10 min in a Sorvall RC-2B centrifuge, the pellet discarded, and the supernate centrifuged at 105,OOOg for 90 min in a Spinco model L ultracentrifuge. The supernate was fractionated with ammonium sulfate. The O-40% ammonium sulfate fraction was collected, dissolved, and dialyzed against water for 4 hr. This fraction containing 93% of the supernatant radioactivity was concentrated to a small volume. Proteins labeled in viva were subjected to digestion with Pronase (Calbiochem) . In a final volume of 2 ml the digestion mixt,ure contained: ‘This work was supported by a grant from the Medical Research Council of Canada. S. Litvak was the recipient of a Postdoctoral Fellowship from the University of Manitoba.
SHORT
471
COMMUNICATIONS
10-25 mg protein (6000-8000 cpm) ; 2 mg Pronase; 80 poles sodium phosphate, pH 7.4; 2 wales biocytin; and 50 pl absolute ethanol. Incubation was carried out at 37°C for 48 hr. The hydrolyaate was applied as a band on Whatman 3 MM paper and the chromatogram developed for 12-14 hr with n-butanol/acetic acid/water @O/20/20, v/v) solvent. An aliquot of the hydrolyzate and reference amino acids were applied on guide strips. The developed chromatograms were dried and one of the guide strips sprayed with ninhydrin. The other guide strip was cut in 2 X 2 cm pieces, placed in liquid scintillation vials with 15 ml of the scintillation fluid (5 gm 2,5-diphenyloxazone and 100 mg 1,4-bis-2-(5phenyloxazolyl) benzene per liter toluene) and counted in a Packard TriCarb liquid scintillation counter. Figure 1 shows that almost 75% of the total radioactivity of the chromatogram is associated with the band with an RI of 0.37 corresponding to biocytin, alanine, and tyrosine. The Rf of biotin in this system is 0.80. The band of Rf 0.35-0.40 was cut from the main chromatogram, eluted with 10 ml water, and concentrated to a small 90
80
70
,x ‘$c .t .-8 z
60
50
L z P y)
40
E al Y I4! 3c
2C
I(
0.1
0.2
0.3
0.4
0.6
0.7
0.8
0.9
1.0
Rf FIQ.
biotin
1. Distribution proteins.
of radioactivity
on chromatogram
of a Pronase
hydrolyzate
of
472
SHORT
COMMUNICATIONS
volume. The concentrated eluate was applied on a Whatman 3 MM paper and subjected to electrophoresis in a Savant high-voltage electrophoresis apparatus at 4000 V for 40 min at pH 1.9 (glacial acetic acid/9870 formic acid/water 87/25/888, v/v). On the same sheet standards (alanine, tyrosine, biocytin and [14C]-biotin) were applied to serve as guides. One of the guide strips was dried and sprayed with ninhydrin and the other was scanned for radioactivity. Figure 2 shows the electrophoretic separation of the three amino acids and biotin. The electrophoretic strip with the protein hydrolyzate was scanned for radioactivity by cutting it into
i
FIG.
2. Electrophoretic
sepa.ration
of alanine,
tyrosine,
biocytin,
and biotin
SHORT
473
COMMUNICATIONS
2 X 2 cm pieces and determining radioactivity with a Packard Tri-Carb liquid scintillation spectrometer. The radioactivity distribution (Fig. 3) shows that more than 90% of the radioactivity is a,ssociated with biocytin. SO
1
IO-
30
Torn Distance
FIG.
3. Distribution
of radioactivity
after
origin
electrophoresis
40
(cm)
of the eluate
from
paper
The method described here is less time consuming than the one reported by Kosow and Lane (3) and also provides better separation of tyrosine and biocytin. Even when carrier biocytin was omitted the radioactive spot was at a distance from tyrosine, which left little doubt that biocytin was the only labeled amino acid. This method has been applied successfully by us for the isolation and identification of labeled biocytin from various hiotin-containing proteins. REFERENCES 1. L. D. WRIGHT, E. AND K. FOLKERS, 2. R. L. PECK, D. E. 3. D. P. Kosow AND 4. K. DAICSHINAMURTI
L. CRESSON, H. R. SKEGC, T. R. WOOD, R. L. PECK, D. E. WOLF, J. Am. Chem. Sot. 74, 1996 (1952). WOLF, AND K. FOLRERS, J. Am. Chem. Sot. 74, 1999 (1952). M. D. LANE, Bioche~m. Biophus. Res. Commun. 7, 439 (1962). AND C. CHEAH-TAN, Can. J. Biochem. 46, 75 (1968).
474
SHORT COMMUNICATIONS
S. LITVAK R. L. 0. BOECKX K. DAKSHINAMURTI Department of Biochemistry University of Manitoba Winnipeg S, Canada Received April 14, 1969
A New Paper Chromatography Pyrimidine-Pyrimidine Deoxyriboside
Solvent System Resolving Riboside-Pyrimidine Mixtures
During studies on deoxyribonucleoside metabolism in tissue cultures, the need arose for a paper chromatography system that could quantitatively separate pyrimidine-pyrimidine riboside-pyrimidine deoxyriboside mixtures. A search of the literature and trial experiments showed that none of the systems usually employed for the analysis of nucleic acid constituents was entirely satisfactory for this purpose. The usual solvents were incapable of resolving such mixtures, produced salt-encrusted papers, or required development at elevated temperatures, It was found that the isobutyric acid-ammonia-water solvents of Magasanik et al. (1) and of Krebs and Hems (2) could be modified to separate these substances readily by decreasing the water and ammonia content and adding toluene. The composition of the most useful modification was: isobutyric acid, 160; water, 22; 0.1 M sodium EDTA, 3; concentrated ammonia, 2; and toluene, 20 parts by volume. This new solvent was used to assay the degradation of radioisotopically labeled thymidine to thymine by Ehrlich ascites carcinoma cells in tissue culture. The experimental procedure employed and the properties of the system are described below. Protein was precipitated from tissue culture supernates by addition of cold perchloric acid (final concentration 0.3 N) . After centrifugation, the supernates were neutralized with KOH and chilled to precipitate potassium perchlorate. Samples (volume < 0.5 ml) were applied as 1 X 3 cm bands along a horizontal line 20 cm below the upper edge of Whatman 3 MM paper (46 X 57 cm). The top of the paper was notched (Fig. 1) to reduce the wick area and thus decrease solvent flow rate. Otherwise flow rates were excessive and spots were diffuse and poorly separated.
SHORT
COMMUNICATIONS
J. B. LLOYD W. J. WHELAN Department of Biochemistry University of Miami School of Medicine Miami, Florida SSlb.2 Received May 6, 1969
Identification Using
of Biocytin High-Voltage
in Biotin
Proteins
Electrophoresisl
The first indication of the type of bond involved in the interaction of biotin with protein came from the isolation of biocytin (r-N-biotinyl-nlysine) from yeast by Wright et al. (1) and the determination of its structure (2). Using a crude preparation of propionyl holocarboxylase, Kosow and Lane (3) were able to show that in this protein biotin is covalently linked to the r-amino group of a lysine residue. Biocytin was identified using a combination of paper and ion-exchange chromatography. In this communication an alternative method for the identification of biocytin from proteins labeled in viva with d- [ carbonyl-14C] -biotin is presented. rats (4) were inIn vivo labeling of b&in proteins: Biotin-deficient jected intraperitoneally with 5 PC of d-[carbonyl-l*C]-biotin (57.4 mc/ mmole) in saline per 100 gm body weight 16 hr before sacrifice. The rats were killed by decapitation. The livers were quickly removed, rinsed in cold saline, and homogenized in 3 vol of medium (0.24 M sucrose, 0.025 M KCl, 0.001 M MgCl, in 0.05 M Tris HCl, pH 7.6). The crude homogenate was centrifuged at 600g for 10 min in a Sorvall RC-2B centrifuge, the pellet discarded, and the supernate centrifuged at 105,OOOg for 90 min in a Spinco model L ultracentrifuge. The supernate was fractionated with ammonium sulfate. The O-40% ammonium sulfate fraction was collected, dissolved, and dialyzed against water for 4 hr. This fraction containing 93% of the supernatant radioactivity was concentrated to a small volume. Proteins labeled in viva were subjected to digestion with Pronase (Calbiochem) . In a final volume of 2 ml the digestion mixt,ure contained: ‘This work was supported by a grant from the Medical Research Council of Canada. S. Litvak was the recipient of a Postdoctoral Fellowship from the University of Manitoba.
SHORT
471
COMMUNICATIONS
10-25 mg protein (6000-8000 cpm) ; 2 mg Pronase; 80 poles sodium phosphate, pH 7.4; 2 wales biocytin; and 50 pl absolute ethanol. Incubation was carried out at 37°C for 48 hr. The hydrolyaate was applied as a band on Whatman 3 MM paper and the chromatogram developed for 12-14 hr with n-butanol/acetic acid/water @O/20/20, v/v) solvent. An aliquot of the hydrolyzate and reference amino acids were applied on guide strips. The developed chromatograms were dried and one of the guide strips sprayed with ninhydrin. The other guide strip was cut in 2 X 2 cm pieces, placed in liquid scintillation vials with 15 ml of the scintillation fluid (5 gm 2,5-diphenyloxazone and 100 mg 1,4-bis-2-(5phenyloxazolyl) benzene per liter toluene) and counted in a Packard TriCarb liquid scintillation counter. Figure 1 shows that almost 75% of the total radioactivity of the chromatogram is associated with the band with an RI of 0.37 corresponding to biocytin, alanine, and tyrosine. The Rf of biotin in this system is 0.80. The band of Rf 0.35-0.40 was cut from the main chromatogram, eluted with 10 ml water, and concentrated to a small 90
80
70
,x ‘$c .t .-8 z
60
50
L z P y)
40
E al Y I4! 3c
2C
I(
0.1
0.2
0.3
0.4
0.6
0.7
0.8
0.9
1.0
Rf FIQ.
biotin
1. Distribution proteins.
of radioactivity
on chromatogram
of a Pronase
hydrolyzate
of
472
SHORT
COMMUNICATIONS
volume. The concentrated eluate was applied on a Whatman 3 MM paper and subjected to electrophoresis in a Savant high-voltage electrophoresis apparatus at 4000 V for 40 min at pH 1.9 (glacial acetic acid/9870 formic acid/water 87/25/888, v/v). On the same sheet standards (alanine, tyrosine, biocytin and [14C]-biotin) were applied to serve as guides. One of the guide strips was dried and sprayed with ninhydrin and the other was scanned for radioactivity. Figure 2 shows the electrophoretic separation of the three amino acids and biotin. The electrophoretic strip with the protein hydrolyzate was scanned for radioactivity by cutting it into
i
FIG.
2. Electrophoretic
sepa.ration
of alanine,
tyrosine,
biocytin,
and biotin
SHORT
473
COMMUNICATIONS
2 X 2 cm pieces and determining radioactivity with a Packard Tri-Carb liquid scintillation spectrometer. The radioactivity distribution (Fig. 3) shows that more than 90% of the radioactivity is a,ssociated with biocytin. SO
1
IO-
30
Torn Distance
FIG.
3. Distribution
of radioactivity
after
origin
electrophoresis
40
(cm)
of the eluate
from
paper
The method described here is less time consuming than the one reported by Kosow and Lane (3) and also provides better separation of tyrosine and biocytin. Even when carrier biocytin was omitted the radioactive spot was at a distance from tyrosine, which left little doubt that biocytin was the only labeled amino acid. This method has been applied successfully by us for the isolation and identification of labeled biocytin from various hiotin-containing proteins. REFERENCES 1. L. D. WRIGHT, E. AND K. FOLKERS, 2. R. L. PECK, D. E. 3. D. P. Kosow AND 4. K. DAICSHINAMURTI
L. CRESSON, H. R. SKEGC, T. R. WOOD, R. L. PECK, D. E. WOLF, J. Am. Chem. Sot. 74, 1996 (1952). WOLF, AND K. FOLRERS, J. Am. Chem. Sot. 74, 1999 (1952). M. D. LANE, Bioche~m. Biophus. Res. Commun. 7, 439 (1962). AND C. CHEAH-TAN, Can. J. Biochem. 46, 75 (1968).
474
SHORT COMMUNICATIONS
S. LITVAK R. L. 0. BOECKX K. DAKSHINAMURTI Department of Biochemistry University of Manitoba Winnipeg S, Canada Received April 14, 1969
A New Paper Chromatography Pyrimidine-Pyrimidine Deoxyriboside
Solvent System Resolving Riboside-Pyrimidine Mixtures
During studies on deoxyribonucleoside metabolism in tissue cultures, the need arose for a paper chromatography system that could quantitatively separate pyrimidine-pyrimidine riboside-pyrimidine deoxyriboside mixtures. A search of the literature and trial experiments showed that none of the systems usually employed for the analysis of nucleic acid constituents was entirely satisfactory for this purpose. The usual solvents were incapable of resolving such mixtures, produced salt-encrusted papers, or required development at elevated temperatures, It was found that the isobutyric acid-ammonia-water solvents of Magasanik et al. (1) and of Krebs and Hems (2) could be modified to separate these substances readily by decreasing the water and ammonia content and adding toluene. The composition of the most useful modification was: isobutyric acid, 160; water, 22; 0.1 M sodium EDTA, 3; concentrated ammonia, 2; and toluene, 20 parts by volume. This new solvent was used to assay the degradation of radioisotopically labeled thymidine to thymine by Ehrlich ascites carcinoma cells in tissue culture. The experimental procedure employed and the properties of the system are described below. Protein was precipitated from tissue culture supernates by addition of cold perchloric acid (final concentration 0.3 N) . After centrifugation, the supernates were neutralized with KOH and chilled to precipitate potassium perchlorate. Samples (volume < 0.5 ml) were applied as 1 X 3 cm bands along a horizontal line 20 cm below the upper edge of Whatman 3 MM paper (46 X 57 cm). The top of the paper was notched (Fig. 1) to reduce the wick area and thus decrease solvent flow rate. Otherwise flow rates were excessive and spots were diffuse and poorly separated.