Faculty of Medicine, Palackj University of Olomoz~c, Czechoslovakia. Preliminary Communication

Transcription

1 Faculty of Medicine, Palackj University of Olomoz~c, Czechoslovakia ELECTROPHORETIC SEPARATION OF SOLUBLE MUSHROOM PROTEINS IN ACRYLAMIDE GEL Preliminary Communication By M. Kalib and Z. Matlocha Some controversial views have appeared regarding the content of proteins inmushrooms(smotiacha, 1947, Sandev and Dushev, 1963, Pilit, 1965). As a part of this problem, to investigate the composition of protein fractions we carried out electrophoresis of mushroom extracts' in a 7.5% acrylamide gel. Freshly gathered mushrooms in amounts of about 100 g were homogenized in a turmix with equal weights of a 1% NaCl solution to maintain the proteins in solution. Centrifugation at g for 10 min yielded an opalescing supernate. This extract, like some plant extracts (S t e w a r d and B a r b e r, 1964), inhibited photopolymerization of acrylamide. We therefore used our own procedure for introducing the samples. We placed about pl of the fluid extract on the spacer gel, covered it with the buffer used and stoppered carefully in a glass tube (5 X 65 mm) with a specially prepared 3 mm thick acrylamide cylinder. This procedure proved suitable for all samples which inhibit polymerization, e. g. haemoglobin solutions. As differences occurred between the results obtained with lyophilized and fresh extracts, only the latter were used throughout this work. In the initial stages no quantitative protein determinations were made on the mushroom extracts, and therefore on account of the wide range of protein concentrations, series of extracts of increasing volume were analyzed electrophoretically. Electrophoresis was carried out in a usual manner (0 rns t ein and Davis 1962) with a tris-hydroxymethyl-aminomethane-glycine buffer of ph 8.6. The current was regulated to 4 mamp.lcolumn. In the course of electrophoresis the pigments present in the extracts moved toward the anode and separated into 2-4 fractions, blue, yellow or brown, red or black in. colour. The electrophoresis was stopped after 90 min when the fastest pigment fractions had approached to about 5 mm from the anode end of the gel. The proiein fractions were stained with Amido Black 10 B. In the course of staining and de-staining some pigments escaped the gel, but there remained, nevertheless, one or two zones, which were probably formed by pigmented proteins and which revealed the highest electrophoretic mobility. Evaluation of stained protein fractions was carried out directly in situ at 570 WL by means of a Unicam SP. 500 spectrophotometer equipped with a SP. 590 strip-scanning attachment (Bennett, 1962). Readings, made every 0.5 mm or less, served for the construction of curves. The electrophoretic separation itself, as well as evaluation which followed, was eminently reproducible, as we proved by duplicate analyses carried out in two different laboratories with different mushroom samples. We analyzed a total of 31 mushroom species and found some relations, the study of which will be extended in future. In repeated series of analyses indi-

2 PM Kalib u. Matlocha, Electrophoretic Separation of Mushroom Proteins 127 vidual mushroom species were characterized by the composition of individual protein fractions throughout the acrylamide gel. In this preliminary communication we wish to put on record only some of the correlations found. In many extracts we observed that the separation was reproducible only when carried out on the same day. Storage, even at +lo-+ 3" or in a frozen state, altered the ratio of the protein fractions within 24 h, but the behaviour of individual mushroom species was different. Very stable proteins occurred in Russula fellea. Amanita phalloides autolyzed under aseptic conditions in ligroine at room temperature, so that after a week only negligible protein amounts could be proved in the gel; practically only the pigmented zone was evident as much before as after the Amido Black staining procedure. In Phallus impudicus the primordium yielded only three very faint protein zones, whereas in samples from the stem of the mature fruit body many such zones have been demonstrated (Fig. 1); in the viscous extract from the velum universale almost no proteins could be detected. This indicated that proteosynthesis and protein differentiation occurred in the developing fruit body of P. impudicus. We have studied this questi.on in some other species, too, but here the differences that could be noted between young and old fruit bodies were not so evident. 0 I cm Fig. 1. Protein fractions in mature PIzallus irnfiudicus Ordinate: Length of acrylamide gel in cm. Arrow indicates the border between the spacer and the small pore gel. Abscissa: Optical density (0. D.) at 570 mp of stained proteins. In R. fellea electrophoresis was carried out simultaneously with extracts from lamellae, and in Xerocomus badius from the tubes and with extracts from the stems and caps. The results in Fig. 2 do not indicate essential differences in the

3 I28 KalAb u. Matlocha,Electrophoretic Separation of Mushroom Proteins 1966, Heft 2 Fig. 2. Protein fractions in Russ~rla fellea 1 - lamellae, 2 - stem Fig. 3. Protein fractions in Amanita phalloides 1 - bulb, 2 - stem, 3 -cap

4 PM Kalib u. Matlocha, Electrophoretic Separation of Mushroom Proteins 129 protein fractions from the parts of these mushrooms mentioned. A different picture was noted in A. phalloides from which the bulb, the stem and the cap were repeatedly analyzed. The most evident changes in the stained acrylamide gel occurred in the protein zones, marked by us A, B, C, and D. Fig. 3 gives the results for one fruit body. In some others peak A consisted of two clearly distinguished intense protein zones. This problem deserves further study. Although simiiar protein fractions occurred in related mushrooms, Coprinus atramentarius and C. comatus, within the Amanita genus were quite different (Fig. 4). I A. rubescens Fig. 4. Schematic comparison of protein fractions in some Amanila species 0n'the basis of our preliminary results we may consider electrophoresis of mushroom extracts in acrylamide gel to be more suitable than electrophoresis in some other media which we also examined. In this procedure the protein ' patterns of individual mushroom species are characteristic. Summary Electrophoresis of mi~shroom saline extracts was carried out in acrylamide gel and revealed characteristic protein fractions. In Phallus imprdicus proteosynthesis in the fruit body was proved in the course of its development. The cap of Amanita phalloides contained predominating amounts of a protein fraction occurring in minor concentrations in the bulb and the stem.

5 130 Kalib u. Matlocha, Electropheretic Separation of Mushroom Proteins 1966, Heft 2 Zusammenf msung Extrakte mit lziger NaC1-Losung aus 31 Pilzarten wurden auf Acrylamid - Gel der Elektrophorese unterworfen. Es wurden charakteristische Proteinfraktionen erhalten. Bei Phallus impudicus wurde die Proteinsynthese im Verlaufe der Entwicklung des Fruchtkorpers nachgewiesen. Im Hut von Amanita phalloicles wurden iiberwiegend Anteile einer Proteinfraktion gefunden, die in der Knolle und im Stil in geringeren Konzentrationen vorkommen. Acknowledgements Whe thank Prof. MUDr. V. Pel i k i n Dr. Sc., Director of the Third Dept. of Internal Med., Palackg University, for his kind permission to carry out our experiments in clinical laboratories. Messrs. L. Rychtera, B. Rihobek and B. KnofliEek, Members of the Mycol. Group at the Museum of Olomouc, advised us on the classification of some mushroom species. For linguistic assistance we are indebted to Dr. I. Sand e m an, Ruislip, England. References B en n e t t, J. P.: Biochem. Biophys. Acta 63,398 (1962) 0 r n s t e i n, L., and D a v i s, B. J.: Disc Electrophoresis, Distillation Product Industries, Preprinted 1962 P i 1 A t, A., and U S j. k, 0.: Mushrooms, p. 49, Spring Books, London 1965 S a n d e v, S., and D u s h e v, A.: Izv. Inst. Zelencukovi Kulturi "Marica", Plovdiv, Akad. Selskostopansk. Nauki Bulgar. 3, 265 (1963) (from Chem. Abstr. 61, c (1964)) S m o t 1 a c h a, F.: Atlas hub jedl9ch a nejedlgch, p. 55, Melantrich, Prague 1947 Steward, F. C., and Barber, J. T.: Ann. N. Y. Acad. Sci. 121, 525 (1964) Address: Doc. Ing. M. Kalib CSc., Faczclty of Medicine, Palackj University, I. P. Pavlova 6, Olomouc, Czechoslovakia