102 J. A. Perez, F. Mateo and E. Mel6ndez-Hevia Electrophoresis 1982,3, 102- 106

Josi A- Fitima Mate0 and Nucleotide isotachophoretic assay: Method and Enrique MelCndez-Hevia

application for determination of ATP/ADP ratio in Departamento de Bioquimica, Facultad de Biologia,

- - several rat tissues

Universidad de La Laguna Separation of ATP and ADP was achieved in isotachophoresis by using malonate, inorganic phosphate (Pi), creatine phosphate and lactate as spacers, which isolate these nucleotides from other UV-absorbing products in rat tissues. By combining isotachophoresis with enzymatic end-point analysis of several rat tissues, calibra- tion for ATP and ADP was achieved obtaining an operative calibration curve that allows the use of isotachophoresis as the only analytical technique in further assays. ATP and ADP quantities of rat skeletal and smooth muscle, heart, liver, kidney and lung were determined by isotachophoresis. Individual variation was also calculated by analyzing 30 animals in the same control conditions, muscular tissues showing the greatest values for ATP and ADP, whereas kidney and lung show the smaller values. ATP/ADP ratio is discussed as a representative parameter for describing the energy content in biological tissues, there are also experimentel reasons which imply that other product concentrations in this parameter not be included. This ATP/ADP ratio is calculated in the rat tissues studied, as well as its individual variation, obtaining the larger values for muscular tissues and the smaller for kidney. A great individual variation is found in kidney, followed by heart and skeletal muscle, and the most constant values are found in liver and smooth muscle. The results obtained in this calibration method for isotachophoresis assay suggest the use of a similar procedure in order to apply this technique for other nucleotide analysis.

1 Introduction

Isotachophoresis is a very useful technique for analyzing biological samples because of its greater resolution power which allows analysis of small sample volume as well as that of products at low concentration in biological extracts. Nucleotide analysis, a very important aim in biological studies, has been resolved in part by isotachophoresis. Con- ventional techniques for nucleotide assays, i.e. enzymatic end-point, give good results but they not only take a long time, but they also need also specific reactions for each nucleotide and large sample volumes, conditions which are not always possible. Isotachophoresis provides an important experimental economy in the complete assay in only a short time using a very small volume. Several authors have devel- oped experimental methods for isotachophoretic nucleotide separation in commercial mixtures and biological samples [ 1-81; see [9] for a review. In these projects the quantifica- tion of each nucleotide band is as important as its resolution. This quantification cannot always be made by using stand- ard solutions because it is difficult to obtain and store pure samples of many nucleotides. Since isotachophoresis is adopted as an alternate method, it is necessary to compare the values obtained from the two analytical techniques, iso- tachophoresis and enzymatic end-point, in order to study the correlation between these two independent experimental data from the same sample.

Correspondence: Prof. Enrique Melendez-Hevia, Departamento de Bio- quimica, Facultad de Biologia, Universidad de La Laguna, Tenerife, Canary Islands, Spain.

Abbreviation: Pi: Inorganic phosphate

0 Verlag Chemie GmbH, D-6940 Weinheim 1982

Our interest in this work is the experimental determination of ATP/ADP ratio in several rat tissues. Isotachophoresis is an accurate method for this objective but there are two prob- lems here: resolution and calibration. Resolution involves a suitable separation which can be obtained by the addition of spacers to the sample, but afterwards, we must demonstrate that each peak in the isotachopherogram corresponds to only one nucleotide. Calibration with commercial nucleo- tides is possible but we cannot directly apply this calibration in routine procedure for biological sample analysis because it is necessary to prove that each nucleotide in the isotacho- phoresis running is not mixed with other UV-absorbing products. One way to prove this is by using another method of quantification and comparing the two kinds of data, which should give linearity. This method of calibration can thus show the resolution of each nucleotide and achieve its quan- tification. Therefore, there are four steps in the use of this technique: 1) peak identification in the isotachopherogram, which can be resolved by adding a certain quantity of each nucleotide to the sample; 2) separation and resolution, ob- tained by adding convenient spacers; 3) calibration, which involves knowing the quantity of nucleotide analyzed by another procedure; and 4) verification to the effect that all these conditions are met in the biological samples to be analyzed. Obviously, these four steps are not necessarily resolved in this order because, as discussed above, resolution and calibration, for example, must be resolved simultaneous- ly since only linearity in calibration demonstrates nucleotide resolution. In this work we have used commercial nucleo- tides and biological samples as mixtures in order to obtain an accurate technique useful for these analyses.

0 173-0835/82/0204-0102S2.50/0

Electrophoresis 1982,3, 102-106 ATP/ADP ratio determination in rat tissues by isotachophoresis 103

2 Materials and methods of peaks in isotachopherograms was calculated according to Gower and Woledge [ 11.

2.1 Chemicals 2.4 Enzymatic assays of nucleotides

All chemicals used were of analytical reagent grade, pur- chased from Sigma Chem. Co. (St. Louis, Mo. USA) or E. Merck (Darmstadt, FRG). Nucleotides and enzymes were obtained from Sigma. In all isotachophoresis experiments, doubly distilled and deionized water was used to prepare all solutions, including electrolytes, spacers, and nucleotide standards, which were used within 72 h of preparation.

2.2 Biological samples

Male Wistar albino rats (200 g), fed on standard laboratory diet, were used for all experiments. Liver, lung, skeletal muscle, smooth muscle (diaphragm), heart and kidney were obtained under ether anesthesia, and immediately quick- frozen using large steel clamps previously frozen by liquid nitrogen, which press the tissue within 10 s after its extraction from the animals, forming a thin slice 101. Frozen tissue was then powdered in liquid nitrogen in a cold porcelain mortar, adding 7 ml of 24 % v/v ethanol in 0.78 M MC104 per gram of tissue, homogenized in an Omni Mixer (Sorvall Instru- ments, Newtown, Conn. USA) at 3000 rpm for 30 s and clarified by centrifugation in a Sorvall RC-SB centrifuge at 20 000 x g for 1 min in an SS-34 rotor at 4 "C. The pH ofthis supernatant was corrected to 7.1-7.3 by addition of K2C03. After neutralization, samples were again centrifuged under the same conditions to extract KC104 and the resulting supernatant was extracted and used immediately for iso- tachophoresis and enzymatic assays. All these operations were carried out at 3-4 "C in a cold chamber.

2.3 Isotachophoresis

Isotachophoretic assays were performed on the LKB 2 127 Tachophor (LKB Instruments, Bromma, Sweden) with a polytetrafluorethylene capillary column (6 10 x 0.5 mm) at 20 "C. 5 mM HCl corrected to pH 3.89 f 0.03 (critical value) with p-alanine was used as leading electrolyte; 5 mM caproic acid, pH 3.94, was used as terminating electrolyte and 0.1 % Triton X-100. (by volume) was added to the leading electrolyte to reduce electroendosmosis. Separations were achieved in 40 min at 75 pA initial constant current, which was reduced to 38 FA when the voltage reached 15 kV - the potential being 12-13 kV (at 38 pA) for the terminator pass- ing the detector. Running zones were detected by UV signal at 254 nm and registered on an LKB 2066 recorder at 500 mV signal range and 3 cm. min-' chart velocity. Integration

Table 1 . ATP and ADP values in rat wet tissues

Enzymatic assay of ATP was carried out by a continuous recording UV absorbance increase of NADPH to end-point at 340 nm, using a Hitachi 100-60 spectrophotometer (Hitachi, Ltd. Tokyo, Japan) with a Churchill thermocir- culator at 37 "C and a linear recorder, coupling glucose phosphorylation by hexokinase with glucose-6-phosphate dehydrogenase reaction 1 1 I. The assay mixture contained: 38 mM Tris-HC1 buffer, pH 7.4; 6.66 mM MgC12; 0.12 M KCl; 0.33 mM NADP', 50 mM glucose; 1.26 U glucose-6- phosphate dehydrogenase; 5 mU hexokinase and 10 pl of sample, in a total volume of 2.75 ml. The reaction was devel- oped until the increase of NADPH absorbance was ter- minated, the end-value being stoichiometric with the ATP content in the sample. Enzymatic ADP assay was realized by a continuous recording UV absorbance decay of NADH measured at 340 nm with the same apparatus, coupling pyruvate kinase and lactate dehydrogenase reactions [ 121. The assay mixture contained: Tris-HC1 buffer, MgC12 and KCl as in the ATP assay; 1 mM phosphoenolpyruvate; 0.32mM NADH; 57 U lactate dehydrogenase; 43 U pyruvate kinase, and 0.9 ml of sample in a total volume of 2.4 ml. The reaction was developed until absorbance decay of NADH was terminated, wich is stoichiometric with the ADP content in the sample.

3 Results and discussion

3.1 Nucleotide peak identification

Nucleotide peak identification in isotachopherograms was achieved by adding pure commercial standard nucleotides to tissue extracts, as shown in Fig. 1, where ATP, ADP and AMP, respectively, were added to rat liver extract for iso- tachophoresis. Use of this procedure, with the addition of spacers to samples, enabled the identification of seven nu- cleotides in a single assay. Fig. 2 shows an experimental isotachopherogram from rat liver representing our experi- ments concerning the use of spacers for nucleotide separa- tion developed in this work.

3.2 Resolution and quantification

The above experiments do not, however, assure us of the homogeneity of each nucleotide.band in the sample isotacho-

Percent individual

ATP/ADP ratio ATP, pmol/g ADP, pmol/g ATP/ADP ratio variation in

Skeletal muscle 3.70 f 0.48 0.84 f 0.002 4.40 f 0.60 13.63 Smooth muscle 3.95 20.20 0.90 * 0.040 4.36 f 0.06 1.37 Heart 3.71 f 1.03 1.05 f 0.950 3.46 f0.66 19.07 Liver 2.04 f 0.58 0.69 ? 0.195 2.95 f 0.02 0.85 Lung 1.73 2 0.24 0.60 f 0.120 2.95 f0.17 5.98 Kidney 1.10 2 0.15 0.71 f 0.190 1.73 f 0.68 39.48

104 J. A. Perez, F. Mateo and E. Melendez-Hevia

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Electrophoresis 1982,3, 102-106

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-

d

v\ Figure I . Identification of adenine nucleotides in the isotachopherogram from rat heart. 7 p1 of rat heart extract (see text) were injected without spacers in the Tachophor in all experiments, adding standard nucleotide solutions (2 p!, 1 gg) of (a) none; (b) ATP; (c) ADP and (d) AMP. This procedure allows the localization of each nucleotide in the original isotachopherogram (a). ATP and ADP peaks are resolved without spacer addition, since there is Pi in the heart extract. Other nucleotides or spurious UV-absorbing products may have contaminated these identified peaks, however, making necessary the use of spacers in further quanti- tative assays.

phoresis assay. In order to use isotachophoresis as an ana- lytical technique, it is necessary to demonstrate the propor- tionality between the peak area and the injected quantity of nucleotide. For the two nueleotides of interest in the present study (ATP and ADP), Fig. 3 and 4 show this proportionali- ty in corresponding calibration curves using standard solu- tions of commercial nucleotides. However, these graphs can- not be used as calibration curves for biological extract assays since the nucleotide bands may be contaminated by other UV-absorbing products in the sample with the same mobili- ty. Therefore, use of the enzymatic method is necessary for

T P

.I 2

iDP

(*lDH

\ 4

L 1 I I

1 2 3 4 5 6 7

ATPnmol

Figure 3. Linear relationship between ATP quantity and peak area in isotachopherograms, obtained with standard nucleotide solution (1 mM).

isotachophoretic calibration and, once precision is assured, isotachophoresis may be used thereafter as the only method for quantitative nucleotide analysis.

1 I I 1 I I 0.5 1.0 1.5 2.0 2.5

Figure 4. Linear relationship between ADP quantity and peak area in isotachopherograms, obtained with standard nucleotide solution, as in Fig. 3. These curves, however, cannot be used as operative tools in tissue extract assays (see text).

Thus, the operative calibration curve was obtained using several dilutions of samples from different rat tissues, assay- ing ATP and ADP in each by enzymatic methods. Since en- zymatic methods are highy specific, each nucleotide quantity in the isotachophoretic assay may be precisely determined and its quantity correlated with its peak area. These results are shown in Fig. 5 , which is the operative calibration curve for further assays. In these results the linearity between the two values obtained by different procedures is clear, ac- counting for good resolution in the isotachophoresis meth- od.

Figure 2. Isotachopherogram from rat liver with addition of spacers, which shows the separation of seven nucleotides (UTP, GTP, ATP, ADP, NADH, AMP and NAD+). Spacers used were as follows: (1) malonate;(2) Pi; (3) creatine phosphate; (4) lactate; ( 5 ) aspartate; (6) glucose-6-phos- phate;(7) glutamateand(8)citrate. 7 klofratliverextract(seetext) wereap- plied in the Tachophor, adding 2 p1 of a solution containing 2 mM of each spacer. Note the peak with mobility between ATP and ADP that corres- ponds to GDP plus other nonidentified products.

ATP/ADP ratio determination in rat tissues by isotachophoresis 105 Electrophoresis 1982,3, 102-106

A D Pnmol

1 2 3 4 5

JJYOL/GRAM (ENZYMATIC ASSAY)

Figure 5. Calibration curve for ATP gnd ADP obtained from several rat tissue sample extracts analyzed by isotachophoresis with UV detector at 254 nm, and by spectrophotometric NADPH absorbance increase or NADH absorbance decay, respectively, in enzyme-coupled reactions (end-point) at 340 nm. These data show a linear relationship between the pnoles obtained by enzymatic methods and the area per tissue gram from the isotachophoresis technique. Thus, it allows us to use this ratio in fur- ther assays employing only isotachophoresis. The same calibration curve is valid for ATP and ADP since both nucleotides have the same molar ab- sorption coefficient at 254 nm and the conversion of percent transmission to extinction units has been carried out by the function used by Gower and Woledge [l]. 7 p1 of conveniently diluted samples were applied in iso- tachophoresis; 100 1.11 and 900 1.11 for ATP and ADP calculation, respec- tively, of the same samples were used in spectrophotometric enzymatic- coupled assay. Statistical data: r = 0.95; p = 0.001; 0 ATP; 0 ADP.

3.3 ATPIADP ratio

Several parameters have been used to express cellular energy content as well as phosphorylation capacity. Atkinson et al. [ 131 have defined the energy charge as the quotient (ATP+ 1/ 2ADP) / (ATP+ADP+AMP); a value that mainly relates to adenylate kinase activity, which involves these three nucleo- tides. The rest of the cellular processes involved with these nucleotides, however, are only related to two of these or to others such as GTP, UTP, etc., which are not considered in this parameter. This energy charge has been determined by Surholt [81 in wall musculature of the lugworm (Arenicola marina), analyzing ATP, ADP and AMP by isotachophore- sis, finding a significant decrease in the value of this parameter - which changes from 0.83 to 0.61 after 48 h of anaerobiosis, and to 0.56 after electrical stimulation, accor- ding to the metabolic changes involved in these physiological variations.

Another parameter frequently used to describe the energy capacity of the cell, and thermodynamically more signifi- cant, is the phosphorylation potential, defined as ATP/ADP x Pi. However, Pi is a product of an energy transfer chain in- volved mainly in many reactions where neither ATP nor ADP participate, such as those catalyzed by phosphorylases and many phosphatases, whereas in kinases, the principal limiting steps of metabolism, ATP and ADP are substrates or products but Pi is not. Thus, phosphorylation potential is a useful thermodynamic value for ATPase-free energy calcula-

tion, but in stoichiometric coupling, this value adds no more pertinent information about useful cell energy than do the ATP and ADP values. Furthermore, AMP variation is not significant in energetically altered biological systems such as hypoxia, whereas ATP and ADP variations are large, and Pi variations cannot be accounted for by ATP and ADP varia- tions [ 141. In light of these considerations we are inclined to acept the simple ATP/ADP ratio as the more significant parameter to describe the energetic content available in the cell.

In addition, there are some practical and experimental reasons for discussing this topic. Inorganic phosphate as- say involves mitochondrial, cytoplasmic and extracellular evaluation; ATP and ADP assays involve only their mito- chondrial and cytoplasmic values since these nucleotides are not found in the extracellular space in significant quantities, and AMP assay involves preferentially the cytoplasmic value because its quantity in mitochondria is very low. ATPIADP ratio, presumably, is not thesame in cytoplasmic and mito- chondrial spaces, but from their whole assay the total energy content of the cell can be deduced. The inclusion of other product values in this parameter might unnecessarily cloud this data which is sufficiently significant by itself. It is clearly impossible to make an organ perfusion to wash the ex- tracellular space or to isolate mitochondria in order to assay their nucleotide quantities, since these procedures greatly alter the cellular steady state, whose situation is the object of our interest.

We have, therefore, assayed the ATP and ADP in six rat tissues from 30 rats under the same controlled conditions in order to compare their ATPlADP ratio. In Table 1 these results are presented, showing large values for muscle tissues and low ones for kidney. ATP/ADP ratio values are also shown in this table, as well as their individual variations. Kidney, heart and skeletal muscle are the tissues where this variation predominates, whereas liver and smooth muscle present the most fixed values. In all cases ATP/ADP ratio is always greater than 1 and its variation possibly represents the maximum allowed variation in their normal steady state.

4 Concluding remarks

The results described in this work show the necessity of calibrating the isotachophoresis method using other meth- ods, such as the enzymatic technique. At the same time this procedure allows the calibration of the technique and, on having a linear relationship between values obtained by two independent methods in the analysis of the same sample, insures the resolution. Calibration is, thus, a good approach for resolution and separation of products by isotachophore- sis and a similar method should be employed in order to achieve other nucleotide separations for their assay by iso- tachophoresis. Since isotachophoresis is a more accurate method, there being fewer steps subject to error in its exper- imental development as well as other practical advantages discussed above, this technique may be presumed to be of great use in many biological assays. ATP and ADP values, as well as ATP/ADP ratio and its individual variation in assayed rat tissues, suggest the different normal steady states in these organs and their permissible variability, which is specific for each tissue and very distinct among all analyzed

106 J. C. Rajan and L. Klein Electrophoresis 1982,3, 106-109

tissues. This ATP/ADP ratio seems to be the most con- venient value for expressing the energy content in the whole cell.

This work was supported by a research grant from the Comi- sidn Asesora de Investigacidn Cienti3ca y Tknica, reference number 4119-79, for the study of “in vitro” metabolic models of glycolysis. The authors wish to thank Prof. An- tonio Bethencourt, Rector of this university, and #rof. Fran- cisco Sa’nchez, Vice-Rector for ScientiJic Research in this university, for their interest in this work and their eflorts to provide this department with the analytical apparatus neces- sary for this line of research.

Received November 16. 1981

5

1 2 3

References

Gower, D. C. and Woledge, R. C., Sci. Tools 1977, 24, 17-21. Eriksson, G., Anal. Biochem. 1980,109, 239-246. Wielders, J. P. M. and Muller, J. L. M., Anal. Biochem. 1980, f03, 386-393.

4 Woledge, R. C. and Reilly, P., in: Adam, A. and Schots, C. (Eds.), Biochemical and biological applications of isotachophoresis, Elsevier, Amsterdam and New York 1980, pp. 103-108.

5 Holloway, C. J., Husmann-Holloway, S. and Brunner, G., Electro- phoresis, 1981, 2, 25-31.

6 Husmann-Holloway, S., in: Allen, R. C. and Amaud, P. (Eds.), Elec- trophoresis ’81, Walter de Gruyter, Berlin 1981, pp. 781-795.

7 Lustorff, J. and Holloway, C. J., in: Allen, R. C. and Arnaud, P. (Eds.), Electrophoresis ’81, Walter de Gruyter, Berlin 1981, pp.

8 Surholt,B.,HoppeSeyler’sZ.Physiol. Chem. 1977,348,1455-1461. 9 Holloway, C. J. and Liistorff, J., Electrophoresis 1980, I , 129-136.

10 Wollenberger, A., Ristau, 0. and Schoffa, G., PJugers Archiv. ges. Physiol. Menschen, Tiere, 1960, 270, 399.

1 1 Lamprecht, W. and Trautschold, I., in: Bergmeyer, H. U. (Ed.), Methods of enzymatic analysis, Vol. 4, Verlag Chemie, Academic Press, New York and London 1974, pp. 2101-2110.

12 Jaworek, D., Gruber, W. and Bergmeyer, H. U., in: Bergmeyer, H. U. (Ed.), Methods of enzymatic analysis, Vol. 4, Verlag Chemie, Academic Press, New York and London 1974, pp. 2127-2131.

13 Atkinson, D. E., Biochemistry, 1968, 7, 4030-4035. 14 Lowry, 0. H., Passonneau, J. V., Hasselberger, F. X. and Schulz, D.

797-808.

W., J. Biol. Chem. 1964,239, 18-30.

Jess C. Rajan and LeRoy’ Klein

Departments of Orthopaedics and

Periodic acid-Schiff staining of type I1 collagen and its cyanogen bromide peptides after poly acrylamide gel

Biochemistry, Case Western Reserve electrophoresis University School of Medicine, Cleveland, OH

Pepsin-solubilized type I1 collagen and some of its CNBr-derived peptides could be stained for carbohydrate with periodic acid-Schiff (PAS) after polyacrylamide gel electrophoresis. PAS intensely stained an aldehyde-containing peptide (M, > 10 000) which was not detected with Coomassie Brilliant Blue R-250 stain. Types1 and I11 collagens and their CNBr peptides stained poorly, if at all, with PAS.

1 Introduction

Cartilage contains a genetically distinct collagen (type 11) with three identical a1 (11) chains [ 11. Type I1 collagen differs from a1 (I) and a2 chains of type I collagen and type 111 collagen in its primary structure, hydroxylysine content and the extent of glycosylation of the hydroxylysine residues. The functional role of the hexoses linked to hydroxylysine residues in a1 (11) either as Gal-Hyl or as Glc-Gal-Hyl re- mains unclear. For comparative studies of the different genetically determined collagen types from several tissues as well as from several species, the technique of CNBr cleavage has been used. The CNBr peptides are analyzed either by CM-cellulose chromatography [ll, or by sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis 12-41. During chromatographic elution the peptides are monitored by measuring their ultraviolet absorbance below 230 nm, while after polyacrylamide gel electrophoresis peptides are

Correspondence: Dr. J. C. Rajan, 5 11 Wearn Building, University Hos- pitals, Cleveland, Ohio 44 106, USA

Abbreviations: SDS: Sodium dodecyl sulfate; PAS: Periodic acid-Schiff

visualized by staining with Coomassie Brilliant Blue R-250. The present report describes the carbohydrate staining of a1 chains of type I1 collagen and some of its CNBr peptides with PAS reagent after polyacrylamide gel electrophoresis.

2 Materials and methods

2.1 Type I and type I11 collagens

From 2-week-old rat skins types I and I11 collagens were solubilized by pepsin treatment and purified by agarose column chromatography as described earlier [51.

2.2 Type I1 collagen

Sternal cartilage obtained from 18-day-old chicks were cleaned of adhering tissues, cut into small pieces and ex- tracted overnight with chloroform-methanol (2 : 1 v/v) and then with methanol for 6 h at 4 “C. The defatted tissue was freeze-dried and powdered in a Wiley mill with dry ice. The

0 173-083 5/82/0204-0106 $2.50/0 0 Verlag Chemie GmbH, D-6940 Weinheim 1982