NOTES
371
& TIPS
3. Daugherty, A., Becker, N. N., Scherrer, L., Sobel, B. E., Ackerman, J. J. H., Baynes, J. W., and Thorpe, S. R. (1989) Biochem. J. 264,829~835. 4. Daugherty, A. D., Kilbourn, M. R., Dence, C. S., Sobel, B. E., and Thorpe, S. R. (1992) Nucl. Med. Biol. 19,411-416. 5. Daugherty, A., Thorpe, S. R., Lange, L. G., Sobel, B. E., and Schonfeld, G. (1985) J. Biol. Chem. 260, 14,564-14,570. 6. Strobel, J. L., Cady, S. G., Borg, T. K., Terracio, L., Baynes, J. W., and Thorpe, S. R. (1986) J. Biol. Chem. 261, 7989-7994. 7. Moldoveanu, Z., Epps, J. M., Thorpe, (1988)J. Zmmunol. 140,208-213.
S. R., and
Mestecky,
J.
8. Shai, Y., Kirk, K. L., Channing, M. A., Dunn, B. B., Lesniak, M. A., Eastman, R. C., Finn, R. D., Roth, J., and Jacobson, K. A. (1989) Biochemistry 2&3,4801-4806. 9. Spadaro, A. C. C., Draghetta, W., de1 Lama, S. N., Camargo, A. C. M., and Greene, L. J. (1979) Anal. Biochem. 96,317-321. 10. Ahmed, M. U., Thorpe, S. R., and Baynes, J. W. (1986) J. Biol. Chem. 261,4889-4894. 11. Spiro,
TIME
(MIN)
FIG.
1. Structure of DLEDA and HPLC profile of an aliquot of DLEDA synthesis mixture after deblocking in HCl as described in the text. Peak at 49.7 min is DLEDA.
terized. The peaks at 49.7 and 70.7 min were eventually identified as DLEDA and monolactitol ethylenediamine, respectively; the peak at 66.9 min coeluted with ammonia. When the reaction mixture was fractionated by cation-exchange chromatography three major carbohydrate-containing peaks were recovered. The first peak contained little or no primary amine; the later two peaks contained sugar:amine in -2:l and 1:l ratios, consistent with their identification as dilactitol EDA and monolactitol EDA, respectively. The final purified DLEDA yielded one peak at 49.7 min on HPLC. DLEDA reacted readily with the ITC and HSE derivatives of several fluorophores. In all cases the ratio of sugar to fluorophore was 2.0 f 0.2:1 in the final purified glycoconjugate. Based on starting primary amine, 7080% of the theoretical DLEDA-fluorophore was recovered in each synthesis. Organic extraction was effective in separating the majority of excess fluorophores from DLEDA-Auorophore conjugates as previously reported (2). However, a final purification of DLEDA-fluorophore from any underivatized DLEDA and traces of unreacted fluorophore was still required. C-18 columns provided a rapid and convenient means of separating all the DLEDA conjugates in micromole amounts from both unreacted DLEDA and fluorophores. REFERENCES 1. Strobel, J. L., Baynes, J. W., and Thorpe, S. R. (1985) Arch. Biothem. Biophys. 240,635-645. 2. Baynes, J. W., Maxwell, J. L., Rahman, K. M., and Thorpe, S. R. (1988) Anal. Biochem. 170, 382-386.
R. (1966)
Methods
Enzymol.
8, 3-25.
Iron Contamination in Adenosine Triphosphate: A Warning Richard E. McCarty Department Baltimore,
of Biology, Maryland
The Johns Hopkins
University,
21218
Patricia Soteropoulos noticed that when she mixed dithiothreitol and ATP solutions the mixture often became pink. Her observations prompted me to carry out preliminary experiments with different lots of dithiothreitol and ATP. The intensity of the pink color was independent of the source of the dithiothreitol, but varied with the source of the ATP. In 50 ITIM Tris-HCl, pH 8.0, the pigmented compound was found to have an absorption maximum at 492 nm. It seemed possible that a heavy metal contaminant in ATP formed a complex with dithiothreitol. In accordance with this idea, the addition of FeSO, to buffered solutions of dithiothreitol was found to cause the appearance of a pink color with a visible absorption spectrum very similar to that observed for the ATP-dithiothreitol mixtures. In this brief report, I show by a l,lO-phenanthroline assay, that commercial preparations of ATP are contaminated by varying amounts of iron. Materials and methods. ATP (disodium salt, 1.5 to 3 H,O) was purchased from Sigma Chemical Co. (St. Louis, MO), United States Biochemicals (Cleveland, OH), and Research Organics (Cleveland, OH). To avoid the accidental contamination of the samples, ATP was dissolved directly in the vials supplied by the vendors in water that had been purified by reverse osmosis and ion exchange. The pH of the samples was not adjusted. Aliquots of the 100 mM solutions were taken for iron determination. ANALYTICAL
BIOCHEMISTRY
205, 371-372 0003.2697192
(1992) $5.00
Copyright 8 1992 by Academic Press, Inc. A 11 -:-LL^ ^C _^-_^ ?I..^&:^- :.. ^_.. C,.“... --““....,,A
372
NOTES
l,lO-Phenanthroline forms a specific, tight complex with Fe2+ that can be conveniently quantitated by its absorption at 510 nm (1). FeSO, -7H,O, dissolved in 1 N acetic acid, was used as the standard. The l-ml assay mixture contained 5 to 8% sodium acetate, 0.1% NH,OH, and 0.01% l,lO-phenanthroline. A linear standard curve (r = 0.999) was obtained with FeSO, over the range of O-100 nmol Fe2+. The slope of this curve was 0.011 &mm /nmol Fe2+. The addition of ATP slowed complex formation, perhaps because of the low pH of the ATP solutions, but did not affect the final color yield. To ensure that maximum color development had occurred, 20 nmol of the FeSO, .7H,O standard was added together with ATP at the highest concentration used. One hour was found to suffice for full color development, even when the ATP concentration was 30 mM. Dithiothreitol was from Research Organics and l,lOphenanthroline from Fisher. Chelex 100 was from Bio-Rad. Results and discussion. Table 1 summarizes the results of the determination of iron in four different ATP solutions. There is a wide variation in iron contents, from 0.09 to over 2 pmol iron per mmole of ATP. Although a systematic study was not carried out, the rather high iron content of the USB ATP was confirmed in a different lot. l,lO-Phenanthroline forms a complex with Fe2+, but not Fe3+. NH,OH is added to reduce Fe3+ to Fe2+. Since no complex formation was observed in the absence of NH,OH with USB ATP, iron is likely present in the sample as Fe3+. The presence of iron in ATP has obvious consequences in metalloprotein research. The complications that the iron contamination in ATP may introduce into the study of other proteins and enzymes are less certain. Iron could inhibit enzyme activity or promote the oxidation of protein dithiols to disulfides. An indication that the latter possibility may hold true was obtained from a glimpse at the effect of the USB ATP on the oxidation of dithiothrietol in air. Dithiothrietol (1 mM in 50 mM Tris-HCl, pH 8.0) was incubated at room temperature in the presence and absence of 8 mM USB ATP (adjusted to pH 6.7). The iron concentration, contributed by the ATP was 18 pM. The SH content of aliquots was
TABLE
1
Determination of Iron in ATP Solutions
Source Sigma Sigma USB Research
Organics
Lot number 41H 7205 78F 7045 74108 987-65-5
Catalog number A-2383 A-6144 10585 1166A-1
Iron content (nmol/lOO pmol ATP) 41.5 9.3 229.2 17.6
& TIPS
determined spectrophotometrically (2). After 90 min, the sample without ATP lost 0.41 pmol SH, whereas that with ATP lost 0.77 pmol SH. The presence of iron in ATP could explain our observation that the rate of oxidation of the dithiol form of chloroplast coupling factor 1 varies. Chelex 100 effectively removes iron from ATP solutions. For example, the passage of 1.0 ml of an 80 InM ATP solution (pH 6.7) through a 0.7 X 3.4-cm column of Chelex 100 equilibrated with water decreased the iron content of the ATP from 221 to less than 8 nmol/lOO pmol ATP. For critical work, it is recommended that the iron content of ATP solutions be determined and that Chelex 100 be used to remove the iron contamination. AcknowEedgment. This National
Science
work
Foundation
was (DMB
supported 91-04742).
by a grant
from
the
REFERENCES 1. Day, R. A., and Underwood, pp. 605-606, Prentice-Hall, 2. Ellman, G. L. (1956) Arch.
A. L. (1980) Quantitative Englewood Cliffs, NJ.
Analysis,
Biochem. Biophys. 82,70-81.
Measurement of the Activity of Individual Respiratory Chain Complexes in Isolated Fibroblast Mitochondria Shelagh A. Lowerson, Louise Taylor, Helen L. Briggs, and Douglass M. Turnbull Division of Clinical Neuroscience, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, England
Abnormalities of the mitochondrial respiratory chain are increasingly being recognized as important causes of human disease (1). In order to establish diagnoses and to understand the pathogenesis of these disorders it is essential to identify the site and severity of defects within the respiratory chain. Initially, the study of respiratory chain function used mitochondria prepared from solid tissues, such as biopsied skeletal muscle. Skin biopsy, however, provides a less invasive alternative for the patient, particularly for children, who form an important clinical group. We therefore wished to establish whether accurate diagnosis of respiratory chain disorders could be made using relatively small quantities of cultured skin fibroblasts. We have developed methods for preparing a mitochondrial fraction from six to eight 80-cm2 flasks and optimizing the measurement of the activity of individual respiratory chain complexes. We have demonstrated that these techniques are sufficiently sensitive to detect respiratory chain defects in patients. ANALYTICAL
All
BIOCHEMISTRY
Copyright 0 1992 riehts of remoduction
205,372-374
(19%)
0003-2697/92 $5.00 by Academic Press, Inc. in am form reserved.
371
& TIPS
3. Daugherty, A., Becker, N. N., Scherrer, L., Sobel, B. E., Ackerman, J. J. H., Baynes, J. W., and Thorpe, S. R. (1989) Biochem. J. 264,829~835. 4. Daugherty, A. D., Kilbourn, M. R., Dence, C. S., Sobel, B. E., and Thorpe, S. R. (1992) Nucl. Med. Biol. 19,411-416. 5. Daugherty, A., Thorpe, S. R., Lange, L. G., Sobel, B. E., and Schonfeld, G. (1985) J. Biol. Chem. 260, 14,564-14,570. 6. Strobel, J. L., Cady, S. G., Borg, T. K., Terracio, L., Baynes, J. W., and Thorpe, S. R. (1986) J. Biol. Chem. 261, 7989-7994. 7. Moldoveanu, Z., Epps, J. M., Thorpe, (1988)J. Zmmunol. 140,208-213.
S. R., and
Mestecky,
J.
8. Shai, Y., Kirk, K. L., Channing, M. A., Dunn, B. B., Lesniak, M. A., Eastman, R. C., Finn, R. D., Roth, J., and Jacobson, K. A. (1989) Biochemistry 2&3,4801-4806. 9. Spadaro, A. C. C., Draghetta, W., de1 Lama, S. N., Camargo, A. C. M., and Greene, L. J. (1979) Anal. Biochem. 96,317-321. 10. Ahmed, M. U., Thorpe, S. R., and Baynes, J. W. (1986) J. Biol. Chem. 261,4889-4894. 11. Spiro,
TIME
(MIN)
FIG.
1. Structure of DLEDA and HPLC profile of an aliquot of DLEDA synthesis mixture after deblocking in HCl as described in the text. Peak at 49.7 min is DLEDA.
terized. The peaks at 49.7 and 70.7 min were eventually identified as DLEDA and monolactitol ethylenediamine, respectively; the peak at 66.9 min coeluted with ammonia. When the reaction mixture was fractionated by cation-exchange chromatography three major carbohydrate-containing peaks were recovered. The first peak contained little or no primary amine; the later two peaks contained sugar:amine in -2:l and 1:l ratios, consistent with their identification as dilactitol EDA and monolactitol EDA, respectively. The final purified DLEDA yielded one peak at 49.7 min on HPLC. DLEDA reacted readily with the ITC and HSE derivatives of several fluorophores. In all cases the ratio of sugar to fluorophore was 2.0 f 0.2:1 in the final purified glycoconjugate. Based on starting primary amine, 7080% of the theoretical DLEDA-fluorophore was recovered in each synthesis. Organic extraction was effective in separating the majority of excess fluorophores from DLEDA-Auorophore conjugates as previously reported (2). However, a final purification of DLEDA-fluorophore from any underivatized DLEDA and traces of unreacted fluorophore was still required. C-18 columns provided a rapid and convenient means of separating all the DLEDA conjugates in micromole amounts from both unreacted DLEDA and fluorophores. REFERENCES 1. Strobel, J. L., Baynes, J. W., and Thorpe, S. R. (1985) Arch. Biothem. Biophys. 240,635-645. 2. Baynes, J. W., Maxwell, J. L., Rahman, K. M., and Thorpe, S. R. (1988) Anal. Biochem. 170, 382-386.
R. (1966)
Methods
Enzymol.
8, 3-25.
Iron Contamination in Adenosine Triphosphate: A Warning Richard E. McCarty Department Baltimore,
of Biology, Maryland
The Johns Hopkins
University,
21218
Patricia Soteropoulos noticed that when she mixed dithiothreitol and ATP solutions the mixture often became pink. Her observations prompted me to carry out preliminary experiments with different lots of dithiothreitol and ATP. The intensity of the pink color was independent of the source of the dithiothreitol, but varied with the source of the ATP. In 50 ITIM Tris-HCl, pH 8.0, the pigmented compound was found to have an absorption maximum at 492 nm. It seemed possible that a heavy metal contaminant in ATP formed a complex with dithiothreitol. In accordance with this idea, the addition of FeSO, to buffered solutions of dithiothreitol was found to cause the appearance of a pink color with a visible absorption spectrum very similar to that observed for the ATP-dithiothreitol mixtures. In this brief report, I show by a l,lO-phenanthroline assay, that commercial preparations of ATP are contaminated by varying amounts of iron. Materials and methods. ATP (disodium salt, 1.5 to 3 H,O) was purchased from Sigma Chemical Co. (St. Louis, MO), United States Biochemicals (Cleveland, OH), and Research Organics (Cleveland, OH). To avoid the accidental contamination of the samples, ATP was dissolved directly in the vials supplied by the vendors in water that had been purified by reverse osmosis and ion exchange. The pH of the samples was not adjusted. Aliquots of the 100 mM solutions were taken for iron determination. ANALYTICAL
BIOCHEMISTRY
205, 371-372 0003.2697192
(1992) $5.00
Copyright 8 1992 by Academic Press, Inc. A 11 -:-LL^ ^C _^-_^ ?I..^&:^- :.. ^_.. C,.“... --““....,,A
372
NOTES
l,lO-Phenanthroline forms a specific, tight complex with Fe2+ that can be conveniently quantitated by its absorption at 510 nm (1). FeSO, -7H,O, dissolved in 1 N acetic acid, was used as the standard. The l-ml assay mixture contained 5 to 8% sodium acetate, 0.1% NH,OH, and 0.01% l,lO-phenanthroline. A linear standard curve (r = 0.999) was obtained with FeSO, over the range of O-100 nmol Fe2+. The slope of this curve was 0.011 &mm /nmol Fe2+. The addition of ATP slowed complex formation, perhaps because of the low pH of the ATP solutions, but did not affect the final color yield. To ensure that maximum color development had occurred, 20 nmol of the FeSO, .7H,O standard was added together with ATP at the highest concentration used. One hour was found to suffice for full color development, even when the ATP concentration was 30 mM. Dithiothreitol was from Research Organics and l,lOphenanthroline from Fisher. Chelex 100 was from Bio-Rad. Results and discussion. Table 1 summarizes the results of the determination of iron in four different ATP solutions. There is a wide variation in iron contents, from 0.09 to over 2 pmol iron per mmole of ATP. Although a systematic study was not carried out, the rather high iron content of the USB ATP was confirmed in a different lot. l,lO-Phenanthroline forms a complex with Fe2+, but not Fe3+. NH,OH is added to reduce Fe3+ to Fe2+. Since no complex formation was observed in the absence of NH,OH with USB ATP, iron is likely present in the sample as Fe3+. The presence of iron in ATP has obvious consequences in metalloprotein research. The complications that the iron contamination in ATP may introduce into the study of other proteins and enzymes are less certain. Iron could inhibit enzyme activity or promote the oxidation of protein dithiols to disulfides. An indication that the latter possibility may hold true was obtained from a glimpse at the effect of the USB ATP on the oxidation of dithiothrietol in air. Dithiothrietol (1 mM in 50 mM Tris-HCl, pH 8.0) was incubated at room temperature in the presence and absence of 8 mM USB ATP (adjusted to pH 6.7). The iron concentration, contributed by the ATP was 18 pM. The SH content of aliquots was
TABLE
1
Determination of Iron in ATP Solutions
Source Sigma Sigma USB Research
Organics
Lot number 41H 7205 78F 7045 74108 987-65-5
Catalog number A-2383 A-6144 10585 1166A-1
Iron content (nmol/lOO pmol ATP) 41.5 9.3 229.2 17.6
& TIPS
determined spectrophotometrically (2). After 90 min, the sample without ATP lost 0.41 pmol SH, whereas that with ATP lost 0.77 pmol SH. The presence of iron in ATP could explain our observation that the rate of oxidation of the dithiol form of chloroplast coupling factor 1 varies. Chelex 100 effectively removes iron from ATP solutions. For example, the passage of 1.0 ml of an 80 InM ATP solution (pH 6.7) through a 0.7 X 3.4-cm column of Chelex 100 equilibrated with water decreased the iron content of the ATP from 221 to less than 8 nmol/lOO pmol ATP. For critical work, it is recommended that the iron content of ATP solutions be determined and that Chelex 100 be used to remove the iron contamination. AcknowEedgment. This National
Science
work
Foundation
was (DMB
supported 91-04742).
by a grant
from
the
REFERENCES 1. Day, R. A., and Underwood, pp. 605-606, Prentice-Hall, 2. Ellman, G. L. (1956) Arch.
A. L. (1980) Quantitative Englewood Cliffs, NJ.
Analysis,
Biochem. Biophys. 82,70-81.
Measurement of the Activity of Individual Respiratory Chain Complexes in Isolated Fibroblast Mitochondria Shelagh A. Lowerson, Louise Taylor, Helen L. Briggs, and Douglass M. Turnbull Division of Clinical Neuroscience, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, England
Abnormalities of the mitochondrial respiratory chain are increasingly being recognized as important causes of human disease (1). In order to establish diagnoses and to understand the pathogenesis of these disorders it is essential to identify the site and severity of defects within the respiratory chain. Initially, the study of respiratory chain function used mitochondria prepared from solid tissues, such as biopsied skeletal muscle. Skin biopsy, however, provides a less invasive alternative for the patient, particularly for children, who form an important clinical group. We therefore wished to establish whether accurate diagnosis of respiratory chain disorders could be made using relatively small quantities of cultured skin fibroblasts. We have developed methods for preparing a mitochondrial fraction from six to eight 80-cm2 flasks and optimizing the measurement of the activity of individual respiratory chain complexes. We have demonstrated that these techniques are sufficiently sensitive to detect respiratory chain defects in patients. ANALYTICAL
All
BIOCHEMISTRY
Copyright 0 1992 riehts of remoduction
205,372-374
(19%)
0003-2697/92 $5.00 by Academic Press, Inc. in am form reserved.
