[1] THIN-LAYER CHROMATOGRAPHY AND ELECTROPHORESIS 3

[1] T h i n - L a y e r C h r o m a t o g r a p h y a n d T h i n - L a y e r

E l ec t ropho re s i s o f P o l y a m i n e s a n d T h e i r D e r i v a t i v e s

By N I K O L A U S SELLER

Until a decade ago an impressive amount of work had been performed in polyamine biochemistry that was based on surface-chromatographic separations. In 1973 Bachrach I quoted 30 different solvents that had been suggested for paper chromatography, 11 solvents for thin-layer chroma- tography, and 12 different buffers for paper electrophoretic separations of the nonderivatized polyamines.

Most of the suggested methods were used for specific purposes and never gained widespread attention, but certain versions of paper electro- phoresis 2,3 found wide application in the quantitative assay of urinary polyamines until recently, when it was recognized that spermine concen- trations, as measured after paper electrophoretic separation, were higher than those found with other procedures owing to an interfering com- pound.

Little progress has been made since 1973 with regard to polyamine separations by surface chromatography. Not even the introduction of high-performance thin-layer silica gel plates by several companies has had an effect in this regard, because more sensitive and automated methods have been developed (see this volume [2,3]).

The main difficulty in the application to polyamine assay of paper- and thin-layer chromatography is the high polarity of the compounds, which makes their isolation from salt-containing aqueous solutions difficult, and amines and amino acids move together, so that extensive separations are required that are not easily obtained by unidimensional development of thin-layer chromatograms.

Thin-layer electrophoresis is slightly more advantageous in this re- spect. If the pH for the separations is appropriate, neutral amino acids stay near the origin, and only basic amino acids and peptides move to- ward the cathode together with the amines.

Thin-Layer Chromatography

The most complete study of thin-layer chromatographic determination of polyamines and their acetyl derivatives was reported by Hammond and

i U. Bachrach, "Funct ion of Naturally Occurring Polyamines." Academic Press, New

York, 1973. 2 A. Raina, Acta Physiol. Scand., Suppl. 218, 1 (1963). 3 D. H. Russell, C. C. Levy, S. C. Schimpf, and I. A. Hawk, CancerRes. 31, 1555 (1971).

Copyright © 1983 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 94 All rights of reproduction in any form reserved.

ISBN 0-12-181994-9

4 ANALYTICAL METHODS FOR AMINES [1]

Herbst. 4 Based on experience with paper chromatography, they chose two solvents and studied the chromatographic behavior of polyamines and basic amino acids, i.e., of those compounds that are eluted together in the same fraction of an ion-exchange chromatographic cleanup proce- dure .4

Using Whatman CC-41 cellulose thin-layer plates and diethylene gly- col monoethyl ether-propionic acid-water (14 : 3 : 3) saturated with NaCI, the following Rf values were obtained: spermine, 0.13; spermidine, 0.24; histidine, 0.29; ornithine, 0.34; histamine, 0.35; 1,3-diaminopropane, 0.36; putrescine, 0.40; arginine, 0.42; lysine, 0.43; cadaverine, 0.47; N j- acetylspermidine, 0.57; N8-acetylspermidine, 0.58; ethanolamine, 0.65; monoacetylspermine, 0.72; diacetylspermidine, 0.81; Nl,N~2-diacetyl - spermine, 0.83.

Regardless of the fact that it takes 5 hr to develop the chromatogram, the limitations of the method are obvious: it is not possible to determine putrescine in the presence of lysine and arginine. Unfortunately it is diffi- cult to eliminate these basic amino acids completely by a cleanup proce- dure. Even extraction of the polyamines with butanoF ,5 may leave traces in the polyamine fraction, and thus prohibit the determination of low concentrations of putrescine.

The system is suitable, however, for separating polyamines from ace- tylpolyamines, and it may therefore be considered in metabolic studies with labeled precursors if the study does not necessitate the determina- tion of both N j- and N8-acetylspermidine at the same time.

A better separation of these two biologically important spermidine conjugates 6,7 has been achieved by developing silica gel 60 G-24 plates (Brinkmann Instruments, Westbury, New York) using chloroform-meth- anol-ammonium hydroxide (2 : 2 : I), 8 but no data exist for this system for other polyamine derivatives.

There is little doubt that the application of high-performance thin-layer plates can improve polyamine separations and, especially, reduce the separation time. Moreover, staining with ninhydrin 4 can be substituted by a convenient and more sensitive staining method with fluorescamine (see below). This method has the additional advantage that the fluorescent spots can be extracted with organic solvents and the extracts used for

4 j . E. Hammond and E. Herbst , Anal. Biochem. 22, 474 (1968). 5 S. M. Rosenthal and C. W. Tabor, J. Pharmacol . Exp. Ther. 116, 131 (1956). 6 N. Seiler, J. Koch-Weser , B. KnOdgen, W. Richards, C. Tardif, F. N. Bolkenius, P. J.

Schechter, G. Tell, P. S. Mamont, J. R. Fozard, U. Bachrach, and E. Grosshans, Adv. , /vamine Res. 3, 197 (1981).

~ '~J. Bolkenius and N. Seiler, Int. J. Biochem. 13, 287 (1981). ~ ~,,kenship and T. Walle, Adv. Polyamine Res. 2, 97 (1978).

[1] THIN-LAYER CHROMATOGRAPHY AND ELECTROPHORESIS 5

determination of radioactivity. Nevertheless the limitations for free poly- amine separations are severe, as is clear even from these short consider- ations, so that applications will be restricted to specific analytical problems.

Thin-Layer Electrophoresis

The Separation System

Using the buffer of the first reported paper electrophoretic procedure for the determination of di- and polyamines, 9 rapid separations of the polyamines can be achieved on silica gel and cellulose thin-layer plates, usually with very regular spots. ~0 The method was therefore used by us to separate polyamines and their metabolites, mostly for the demonstration of the formation of radiolabeled polyamines from precursors. ~j,12

Thin-layer electrophoresis is carried out with a cooled plate apparatus (Camag, Muttenz, Switzerland). For the determination of the polyamines and related compounds (i.e., for compounds that are positively charged at pH 4.8), the samples are applied on the plates as streaks or small spots (diameter 3 mm) at a distance of 3 cm from one edge. Then the plates are evenly sprayed with pyridine-acetate buffer pH 4.809 (100 ml of pyridine, 75 ml of glacial acetic acid, and 30 g of citric acid monohydrate in 2375 ml of deionized water), and portions of 20 ml of the same buffer are poured into the troughs of the apparatus before each run. Plates and troughs are connected by buffer-soaked filter-paper strips.

Separations are usually carried out at 600 V (1-1.5 V/cm). If the appa- ratus is cooled to 0 °, a current of 90-100 mA is usually observed with 20 × 20 cm plates of 250 mm layer thickness.

Figure 1 shows separations of polyamines, acetylpolyamines, and ba- sic amino acids on (A) silica gel 1500 (Schleicher & Schuell, Dassell, Federal Republic of Germany) and (B) silica gel 60 (Merck, Darmstadt, Federal Republic of Germany) thin-layer plates.

Under these electrophoretic conditions, neutral amino acids remain near the origin and acidic amino acids move toward the anode. They do not interfere with polyamine separations.

The relative mobilities of the various compounds are similar on silica gel 1500, silica gel G (plaster of Paris as binder), and cellulose layers. However, the comparison of Fig. 1A with 1B shows that the mobility

9 F. G. Fischer and H. Bohn, Hoppe-Seyler's Z. Physiol. Chem. 308, 108 (1957). ~0 N. Seiler and B. Kn6dgen, J. Chromatogr. 131, 109 (1977). i1 H. A. Fischer, H. Korr, N. Seiler, and G. Werner, Brain Res. 39, 197 (1972). ~2 N. Seiler and M. J. A1-Therib, Biochem. J. 144, 29 (1974).

6 A N A L Y T I C A L M E T H O D S F O R A M I N E S [ll

18 1 7 1

141516• 12 • ~ •

13 4 5 9 1 0 ~ •

23o• 1 •

14 0 16 1718

0 0 0 4 5 15

2 3 ~ ~ 9 7 • 1£}1~ 1 Q e

6 0 I g 12 1 • o Q

6

13

f

FIG. 1. Thin-layer electrophoretograms of polyamines, acetylpolyamines, and basic amino acids. Electrophoretic conditions: cooled plate apparatus (Camag, Muttenz, Switzerland), 600 V, 50 min; 0°; pyr idine-acet ic acid buffer, pH 4.8. 9 (A) Silica gel 1500 plate (Schleicher & Schuell, Dassell, Federal Republic of Germany). (B) Silica gel 60 plate (Merck, Darm- stadt, Federal Republic of Germany) (both 20 × 20 cm). 1 = 4-aminobutyric acid (GABA); 2 = arginine; 3 = histidine; 4 = lysine; 5 = ornithine; 6 = homocarnosine; 7 = putreanine; 8 = NI-acetylspermine; 9 = monoacetylputrescine; 10 = NS-acetylspermidine; 11 = Nt-acetyl - spermidine; 12 = spermidine; 13 = spermine; 14 = ethanolamine; 15 = histamine; 16 = putrescine; 17 = cadaverine; 18 = 1,3-diaminopropane. The figures were copied from origi- nal electrophoretograms. The distance from plate edge to origin is 40 mm; the spots (approx- imately 60 nmol) were visualized by reaction with ninhydrin.

pattern is totally different on Merck plates. The irregular shapes and low mobilities of the spermidine and spermine spots indicate a strong interac- tion with the thin-layer matrix, which obviously increases with the num- ber of positive charges and the length of the aliphatic chain. Spermine and monoacetylspermine interact especially strongly. Since the same obser- vations were made using high-performance thin-layer plates of the same company, ~° it is assumed that the polyamines interact with the polyvinyl- alcohol binder in these plates.

4-Aminobutyrate, the basic amino acids, and the monoamines (mono- acetylputrescine, ethanolamine) move faster on the Merck than on the Schleicher & Schuell plates. Neutral amino acids also tend to move some- what toward the cathode on the Merck plates. This might indicate a lower effective pH than is achieved on the Schleicher & Schuell silica gel plates (or cellulose layers) under the electrophoretic conditions.

Practical experience with electrophoretic separations of polyamines on thin layers is presently restricted to a few types of silica gel and cellulose layers. Other types of commercial plates may interact with the polyamines to a different degree. Since the interactions with the layer

[1] THIN-LAYER CHROMATOGRAPHY AND ELECTROPHORESIS 7

matrix are well reproducible, it is possible to use them for achieving certain separations. For instance, the separation of putrescine and etha- nolamine is more complete on Merck plates than on the other thin layers, and spermine interferes with N~-acetylspermidine only on Schleicher & Schuell plates. 1,3-Diaminopropane and cadaverine, on the other hand, are favorably separated on thin-layer plates of this type (Fig. 1).

It is self-evident that prolongation of the separation time will improve the separation of the slowly moving compounds, whereby putrescine and the other diamines may move into the paper strip and be lost. It is possible to decrease the separation time if only fast-moving components of a mix- ture (putrescine, spermidine, spermine) are to be separated.

The above separation system was chosen because of the regular spots obtainable and its applicability to polyamine assay and because of the determination of certain amino acids (4-aminobutyrate, glutamic acid, aspartic acid, glutathione)) 3 However, other buffers (with different pH and composition) may also turn out to be useful for the thin-layer electro- phoresis of polyamines. It may at least be worthwhile to test those buffer solutions that have previously been used wilh advantage in paper electro- phoretic separations. J

Staining with Ninhydrin and Fluorescamine

Staining of thin-layer chromatograms with ninhydrin, extraction of the colored spots, and colorimetric estimation have previously been used suc- cessfully? The same method is applicable to thin-layer electrophoreto- grams.

The plates are dried in an oven for 30 min at 90 °. They are then sprayed uniformly with ninhydrin reagent (1% ninhydrin and 1% 2,4,6- trimethylpyridine in absolute ethanol) and heated for 15 min at 60 °. The colored spots are scraped off, and silica gel is collected in small centrifuge tubes. (A method for scraping out spots with a simple homemade device is described by Seiler14.) The colored reaction product is eluted with 1 ml of 70% ethanol by mixing. After centrifugation, absorbance of the superna- rants is measured at 575 nm. With this method, 5-50 nmol of the amines can be measured (SD -+ 1% for the staining method and colorimetry). 4 Direct scanning of the plates is also feasible but has not been used for polyamine determinations.

More recently, spraying with fluorescamine or an o-phthalaldehyde reagent has been suggested for the detection and determination of amino acids and peptides, drugs, etc., on thin-layer chromatograms. These tech-

13 S. Sarhan, N. Seiler, J. Grove, and G. Bink, J. Chromatogr. 162, 561 (1979). 14 N. Seiler, Methods Biochem. Anal. 18, 259 (1970).

8 ANALYTICAL METHODS FOR AMINES [1]

niques were compared with the ninhydrin method, 15 but were not found to be advantageous, owing to high background fluorescence and low yields in fluorescent derivatives. However, commercial thin-layer plates can be submitted to a dipping technique with fluorescamine that gives low homo- geneous background fluorescence and reproducible yields of fluorescent derivatives. This method was originally designed for the estimation of certain amino acids after thin-layer electrophoretic separations, 13 but was also found to be suitable for the determination of polyamines and their derivatives on electrophoretograms. 7

The procedure is as follows: The dry thin-layer plates are heated to 50 ° before staining. Then they are dipped rapidly into a tank containing an alkaline solution (to ensure optimal reaction condition). For the prepara- tion of this solution, 100 ml of saturated NaOH in methanol are mixed with 100 ml of 1-butanol, and the mixture is gradually diluted with 600 ml of toluene. The plate is removed from the tank after 30 sec and dried in a horizontal position for a few minutes at room temperature and then com- pletely for 10 min at 110 °.

The staining solution contains 10 mg of fluorescamine in 100 ml of a mixture of acetone-l-butanol (1: 1). A stainless steel or glass tank (22 x 20 x 0.7 cm) containing approximately 200 ml of the fluorescamine solu- tion is advantageous for dipping. The plates are cooled to room tempera- ture, and then they are dipped twice into the fluorescamine solution with drying periods of 1-2 min. About 15 ml of the fluorescamine solution are needed per 20 x 20 cm plate. The solution should be prepared daily.

The fluorescamine derivatives are formed gradually. Before quantita- tive evaluation the plates are therefore stored for at least 2 hr at room temperature. If they are protected from dust and light, quantitative evalu- ation is, however, possible even 48 hr after staining.

Direct scanning of fluorescence is the most advantageous technique for quantitative evaluation. Fluorescence is activated at 320 nm, and total emitted light is measured (using a 420 nm cutoff filter in the path of the emitted light). It is also possible to extract the fluorescamine derivatives with methanol and to measure fluorescence intensity conventionally. Methanol extracts can also be used for the determination of radioactivity. In contrast to the colored reaction products with ninhydrin, the fluores- camine derivatives do not decrease counting efficiency by fluorescence quenching.

The relationship using in situ scanning of fluorescence is linear be- tween recorded curve areas and the amount of compound in the range

15 E. Schilz, K. D. Schnackerz , and R. W. Gracy, Anal. Biochem. 79, 33 (1977).

[1] THIN-LAYER CHROMATOGRAPHY AND ELECTROPHORESIS 9

from 200 to 2000 pmol per spot. In this range the reproducibility of the procedure (including tissue extraction and sample application) is ---8-10%.

Conclusion

The methods described in this chapter cannot compete in sensitivity, specificity, and precision with other existing methods of polyamine as- say 16'17 (see also this volume [2,4]). However, they are simple and rapid in the sense that they allow one to perform many separations in parallel and require only relatively unsophisticated, versatile equipment.

Surface chromatographic separations of nonderivatized polyamines should be especially useful for the identification of radiolabeled precur- sors of the polyamines and of the polyamines themselves. Although this possibility has been utilized in the past, ~1,~2,18 it has not been fully ex- ploited in polyamine metabolic studies.

Both methods, thin-layer chromatography and thin-layer electropho- resis, separate not only the amines and their conjugates, but in the same systems certain amino acids and metabolic intermediates. Thus, it is pos- sible to resolve, for instance, tissue or cell extracts to a considerable extent, especially if the two methods are used in two directions on the same thin-layer plate. Autoradiographs of the plates then give a good picture of the metabolic reactions that took place.

The specificity of some enzymatic assays can be considerably in- creased if these simple but powerful separation methods are employed. Examples are the demonstration of NS-acetylspermidine formation by nuclear acetyl-CoA:spermidine acetyltransferase 8 and the degradation of Nm-acetylspermidine to putrescine, in contrast with the deacetylation of NS-acetylspermidine by liver homogenates. 7,t8

It is therefore concluded that these more recent versions of the older thin-layer chromatography and electrophoretic methods for polyamine assays may still remain a valuable tool in future work.

16 N. Seiler, Clin. Chem. (Winston-Salem, N.C.) 23, 1519 (1977). ~7 N. Seiler, in "Polyamines in Biomedical Research" (J. M. Gaugas, ed.), p. 435. Wiley,

New York, 1980. ~8 N. Seiler, in "Polyamines in Biology and Medicine" (D. R. Morris and L. J. Marton,

eds.), p. 127. Dekker, New York, 1981.