UDC 579.64 Original scientific paper PROTEASE ACTIVITY OF HYPERICIN-INDUCED FUNGI Slavica SOLUJIC and Milorad MILOSEV Faculty of Science, Department of Chemistry, University of Kragujevac, Kragujevac, Serbia and Montenegro Solujié Slavica and Milorad MiloSev (2002): Protease activity of hypericin-induced fungi. - Mikrobiologija, Vol. 39, No 1-2, 9-18, Beograd. ‘The paper presents experimental results of testing the influence of hypericin as a redox and photosensitive compound on the protease activity in three generations of the hypericin-induced fungi Penicillium verrucosum, Penicillium cyclopium, and Penicillium chrysogenum. The ‘afluence of hypericin by itself was tested, as well as the joint effect of hypericin and light under conditions of exposure of the system to white light (4.5 k3). Prolone-4 development of the fungi on a modified nutrient base with hypericir. »1owed that the investigated fungi are not adaptable to the presence of hypericin, whose effect can be characterized as inhibition of enzymatic activity by about 70%. Key words: microorganisms, hypericin, protease activity, inhibition INTRODUCTION Hypericin is a naphthodianthron and a metabolic product of plants of the genus Hypericum such as Saint John's wort (Hypericum per;‘sratum). Interest in extract of this plant has been aroused by results indicating the possibility of stopping Tonesponding author: Slavica Solujié, Prirodno-matematitki fakultet, R. Domanoviéa 12, 34000 Kragujevac, Email:ssolujic@knez.uis.kg.ac.yu, Serbia and Montenegro.10 MIKROBIOLOGUA, Vol. 39, No. 1-2, 9-18, 2002. the development of certain viruses such as the herpes simplex virus, cytomegalovirus, and human immunodeficiency virus type 1 (CARPENTER et al., 1994). The antivirus activity of hypericin is a timely research problem about which there are distinct controversies. It is known that hypericin creates photodynamically induced cross links with membrane proteins, which leads to the conclusion that decrease of virus infectivity could arise from prevention of the bonding of viruses and formation of their capsids (WEINER et al.,1999). Other different biological properties of hypericin have been described, properties such as potent antidepressive activity (WHEATLEY, 1998), light- dependent inhibition of protein kinase C (JARVIS, 1994) and tyrosine kinase (DE Wirre et al., 1993) activity, and photosensitized inhibition of mitochondrial succinoxidase (THOMAS et al., 1992). Results indicating retrovirus inactivation in blood products (MISKOVSKY ef al., 1998) suggested the possibility of using hypericin as an anticancerogenic compound (VANDERWEREF ef al., 1996). Interaction of human serum albumin (HSA) with hypericin (KOHLER ef al., 1996) helps to overcome difficulties in solubilization and dispersion of hypericin in aqueous physiological solutions. The HSA-hypericin interaction results in (i) dissociation into the monomeric form of hypericin, which is aggregated in the aqueous phase, (ii) change in hydrophobicity of the tryptophan environment, (iii) formation of an H-bond between the carbonyl group of hypericin and the NI - H group of tryptophan, leading to appearance of a protonated-like carbonyl in hypericin and decrease in the strength of bonding at the NI -H site of tryptophan, and (iv) alteration of the tryptophan side-chain conformation. Hypericin is a photosensitive compound and under conditions of illumination produces singlet oxygen (PETRICH ef al., 1998)) and superoxide radicals or radicals of hypericin (SMIRNOV et al., 1999), which may be responsible for its biological activity. In keeping with what has been said above, the purpose of the present work was to determine the extent to which the tested fungal cultures are adaptable to the continuous presence of hypericin in the nutrient base. Attention is paid here to the level of protease activity under daylight conditions and conditions involving daily increase in the total energy level of the biological system by 4.5 kJ (Das et al., 1999). MATERIAL AND METHOD. Experimental part. -Microorganisms. Monosporic cultures of the fungi Penicillium verrucosum (Dierckx), Penicillium cyclopium (Westling), and Penicillium chrysogenum (Thom.) were obtained from the mycological collection of the Science Faculty of the University of Kragujevac. Fermentation. - Monosporic fungal cultures were obtained by means of exhaustion on pure agar (Merck) in petri dishes. Fungal cultures were raised on potato-glucose nutrient agar at 4°C. The fungi were incubated on a mineral nutrient base of the following composition (g/l): NaNOs, 1; K2HPOs, 1; MgSOs x8. SOLUSIG et al.: PROTEASE ACTIVITY OF HYPERICIN-INDUCED FUNGI 11 7 H20, 0.50; KCl, 0.50; FeSOs x 7 H20, 0.01; sucrose, 30; and distilled water, 1000 (at pH 7.3). The influence of hypericin and light on protease activity of the fungi was tested in 30 ml of Czapek’s nutrient medium with the addition of 30 mg (1 mg/m!) hypericin in the form of an ethanol solution and { ml of a spore suspension (2500 spores). One series of experimental fungi developed at room temperature without additional aeration under daylight conditions. Another series developed under identical conditions, but with daily exposure of the system to white light for aperiod of 15 min. Energy activation of the system by 4.5 kJ is achieved in this way. Samples, 3, 6, 9, 12, and 15 days old were separated from mycelium by means of straining and the clear fermentation solutions were used to measure changes of enzymatic activity. Obtainment of hypericin-induced fungi. -Fungal mycelium after one development period of 21 days on modified Czapek's medium with addition of hypericin was separated from the fermentation solution by means of straining, rinsed with distilled water, dried in an inert and pure air atmosphere, and reseeded on a fresh substrate of potato-glucose agar. Mycelium developed on the agar substrate for 7 days, after which it was reseeded on a new modified liquid substrate with hypericin. The fungi developed on this substrate for 21 days. They developed under alternating day-night conditions in one series and were exposed to white light for a period of 15 min in another. In differently aged samples (every third daiy relation to seeding), values of enzymatic activity were determined for fungi induced twice by hypericin under daylight conditions and conditions of illumination. The same procedure of separation and reseeding was repeated with the mycelia of cultures that had two development periods on a hypericin-modified substrate. In this way, two series of fungi induced three times were obtained that developed under daylight and in the presence of daily exposure to white light. Samples of the fermentation solutions were used to determine changes of extracellular protease activity (PE). The proteolytic activiry of fungi was measured in 1 ml of the nutrient base by Anson's method (DuDKA, cee on the basis of the amount of tyrosine or tryptophane, produced by hydrol From the curve obtained, the intensity of the activity was colorimet determined. Proteolytic activity of the fermentation liquids was determined by standard methods. RESULTS Figure 1 shows the inductive influence of hypericin (1 mg/ml) and white light (4.5 kJ) on values of proteolytic activity in three generations of the fungi P. verrucosum, P. cyclopium, and P. chrysogenum as determined over a test period of 18 days. The graphs on the left-hand side of the figure indicate hypericin influence on PE activity, while those on the right show influence of the joint effects of hypericin and light on changes of proteolytic a12 MIKROBIOLOGUA, Vol. 39, No. 1-2, 9-18, 2002. Peverrucosum P.verrucosum P.chrysogenum ea ~~ aye aoe P.cyclopium Pecyelopium t+ Fig. 1. - Influence of hypericin and light on changes of the proteolytic acitivity in three generations of the fungi P.verrucosum,P-cytopum and P. ch ysogemam Influence of hypericin in the substrate on activity of extracellular proteases in the fungus P. verrucosum is manifested in an increase of activity in relation to the standard culture during the period from the 9th to 12th day, with an activity maximum of 0.7 IU . In the second part of the development period, activity declines to 0,2 IU (30%), which is the level of activity until the end of the examined development period. The investigated sample with hypericin exhibits greater enzymatic activity, which can be induced by change in pH of the medium, since hypericin alters pH in the direction of acid values and is also an energy molecule that can be a donor of energy to the biological system. Positive proof of these assertions is seen in experimental results where the sample was daily exposed to white light, since the level of activity of pureS. SOLUJIC et al.: PROTEASE ACTIVITY OF HYPERICIN-INDUCED FUNG 13 SSOLUIC ¢ 2: PROTEASE ACT TT ——————eeere culture is enhanced by about 0.1 IU and is now on the level of hypericin-induced enhanced activity in the absence of light. Additivity of the activator effect of hypericin and light on proteases is not recorded. In this series of experiments, hypericin causes an increase of enzymatic activity by about 0.1 IU or around 16%. In the second generation of hypericin-induced fungi, both hypericin and hypericin plus light cause activation of enzymatic activity to the level of 0.6-0.7 1U in the first half of the development period, followed by a declining trend to a value of 0.2 TU, which is about 30% of total enzymatic activity, Hypericin in the absence of irradiation exerts more pronounced influence on enzymatic activity: the increase here is on the level of 0.3-0.5 IU or 16-50% during the first half of the development period, It is characteristic that after 12 days of development, protease activity decreases abruptly to a level of about 30% of the original level. ‘The third induction of the fungus P. verrucosum with hypericin is characterized by the absence of any increase in activity over the whole test period, since it is on the level of 0.2 IU, which is the constant minimal activity in the previous two experiments and reduced by about 70% in relation to the standard culture. In the fungus P. cyclopium, hypericin has a mote pronounced effect during the initial period of development, when enzymatic activity is on the level of 0.61U. After that, activity at first tends to be on the level observed in pure culture, then decreases by about 0.2 IU in the last quarter. The joint effect of hypericin and light is on the level of changes observed in the case of hypericin alone. Double induction of the fungus with photoinduced hypericin gives discernibly elevated enzymatic activity during the period from 3 to 6 days with an activity maximum at 9 days on the level of 0. 9 1U, which at the same time is the greatest activity measured. This value of activity is enhanced 100% in relation to the standard culture. However, activity after this is in permanent decline to the level of 0.2 TU, which represents a decrease of about 50%. In the absence of light, maximal activity occurs in the middle of the test period and is in the rangeof 0.6 IU. ‘Triple induction of the fungus P. cyclopium with hypericin gives a level of activity comprising only 30% of the total activity of pure culture. This scope of activity remains over the entire test period on the level of about 0.2 IU, both in the presence of hypericin and in the case of the joint effect of hypericin and light. In the fungus P. chrysogenum, we note a deviation from the behavior observed in the two previous fungi. To be specific, the first induction of proteases with hypericin in the substrate is without any visible influence. The second induction of the fungus significantly lowers enzymatic activity. The general trend of activity is similar, but activity values are lower by 0.1-0.4 IU. With activity of about 0.1 IU, inhibition is discemible on the 9th and 15th days, and maximal activity is about 22% lower. The third induction causes decrease of enzymatic activity to the level of 0.2 IU over the entire test period.14 MIKROBIOLOGUA, Vol. 39, No. 1-2, 9-18, 2002. ‘The joint effect of light and hypericin in the first induction only lowers the activity maximum by about 25% in samples 12 days old. The second induction by light and hypericin completely alters the picture of activity, since it declines markedly to the level of 0.1-0.15 TU only in the region of from 9 to 12 days, which is 12% of activity recorded in the standard culture. The third induction evoked by hypericin in combination with light is identical to that observed in all the other samples. Enzymatic activity over the entire test period is on the level of 0.2 IU, which is only about 30% of total enzymatic activity of the standard culture. Biological activity of hypericin has been characterized on the level of redox reactions and reactions of enzymes and nucleic acids. Hypericin is active even in the absence of light and preferentially lowers values of the redox potential, behaving here as an effective substrate for electron capture in physiological transfer reactions (LAVIE et al., 1989). In the presence of light, hypericin gives singlet oxygen (HADIUR et al., 1994), free radicals and in some cases semiquinones as well (WEINER ef al., 1992). In practical terms, this means that it is an effective substrate for reactions of electron transport and free radicals. Hypericin is also an irreversible inhibitor of certain protein kinases, while it manifests a weaker effect in protein kinase A and casein kinase (TAKAHASHI et al., 1989). Activity of hypericin is manifested on the level of mechanisms governing biochemical reactions of proteins, as well as on the level of conformation of that part of the protein molecule where the catalytic center of enzymes is located. Also, hypericin reduces proliferation signals by interacting with protein in biological memberanes (DE WITTE et al., 1993) Our results (SOLUNE ef al., 1997))and results published elsewhere indicate that hypericin causes decrease in intracellular pH of the medium. Biochemical conditions are thereby created for intensification of redox reactions and highly charged interactions (FEHR ef al., 1994). Proteinases act precisely through mechanisms that seek charged substrates. It follows that this is not the decisive factor in cases where increase of enzymatic activity is not recorded. Its therefore possible to speak of structural influence of hypericin. The environment of the catalytic center is significantly altered by interaction of hypericin with charged amino acids, a situation that is quantified by the level of enzymatic activity. This accounts for gradual decline of enzymatic activity in the second or third series of experiments. The given thesis is supported by the level of activity in the third series, which is the same in all of the fungi, indicating lasting change in conditions of enzymatic activity as a consequence of creation of a chemical adduct with hypericin. Light activation of hypericin is without a visible effect, which confirms that the recorded changes are not based on change in the reaction mechanism. An exception is noted in the fungus P. cyclopium during the first half of the development period, but this can be ascribed to specificity of the fungus itself.$. SOLUJIC et al.: PROTEASE ACTIVITY OF HYPERICIN-INDUCED FUNGI 15 CONCLUSION On the ba can be drawn up: In the first and second generation of hypericin and hypericin plus light induced fungi P. verrucosum and P. cyclopium cause activation of enzymatic activity for about 15-30% in the first half of the development period followed by decrease activity for about 50% in the second part of the development period. The second induction of the fungus P. chrysogenum with hypericin and hypericin plus light lowers enzymatic activity for 30-70%. The third induction of the fungus P. verrucosum, P. cyclopium and P. chrysogenum with hypericin and bypericin plus light iz characterized by the absence of any increase in activity over the whole test period. The enzymatic activity are reduced by about 70% in relation to the standard culture. is of the results ofthe investigation, the following conclusions16 MIKROBIOLOGUA, Vol. 39, No. 1-2, 9-18, 2002, REFERENCES CanrenreR, S., M. J. Fen, G. A. Kraus, J. W. PETRICH (1994): Chemiluminescent activation of the antiviral activity of hypericin, Proc. Natl. Acad. Sci. USA, Vol. 91. 12273-12277. Ds Wirt, P., P. Acosmints, J. VANLINT, .W. MERLEVEDE, J. R. VANDENHEEDE (1993): Inkibition of epidermal growth factor receptor tyrosine kinase activity by hypeticin. Biochem, Pharmacol., 46, 929. Das, K., D. Asuby, J. Wan, W. Perrici (1999): Temperature Dependence of the Excited-state Intramolecular Proton Transfer Reaction in Hypericin and Hypocrellin A, J. Phys. Chem., 108, 1581-1585. Duka, A. (1982): Methods of experimental ecology (in Russian), Naukova Dumka, Kiev Fexin, M. J., S. L. Carenrer, J. W. Perici (1994): The role of oxygen in the photoinduced antiviral activity of hypericin. Bioorg. Med. Chem. Lett. 4, 1339-1344 Hapiur, C., A. Jeuner, P, JARDON (1994): Photosensitizatin by hypericin: ESR evidence for singlet ‘oxygen and superoxide anion radicals formation in an in vitro model, J. Photochem. Photobiol. B. Biol, 6, 67. Jarvis, D. W., A. J. Turser, L. F. Povink, R. S. TRAYLOR, S. Granr (1994): Induction of ‘Apoptotic DNA Fragmentation and Cell Death in HL-60 Human Promyelocytic Leukemia Celis by Pharmacological Inhibitors of Protein Kinase C-1. Cancer Research, 54, 1707- 1714. Kouer, M., J. Garert, J, FRiepRicn, H. FALK, J. Mever (1996): Hole-Burning spectroscopy of Proteins in External Fields: Human Serum Albumin Complexed with the Hypericinate Ion, J. Phys. Chem. 100, 8567-8572. Lavi, D., F. VALENTINE, B. Levin, Y. Mazur, G. Gato, D. Lavie D. WeRwer, D. MeRur.o (1989): Studies of the mechanisms of action of the antiretroviral agents hypericin and pseudohypericin, Proc. Natl. Acad. Sci 86, 5963-6967. MiSkovsky, P., D. JANCURA, S. SaNcHEZ-CorTEs, E. KociSova, L. CHINsky (1998): Antiretrovirally ‘Active Drug Hypericin Binds the ITA Subdomain of Human Serum Albumin, J. Am. Chem., Sov: 120, 6374-6379 Peraici, J. W., M. S. GorDox, M. CaGLé (1998): Structure and Energetics of Ground-State Hypericin - Comparison of Experiment and Theory, J. Phys. Chem. A, 102, 1647-16 Sminnov, A.V., K. Das, D. S. English, Z. Wan, D. A. Kraus, J. W. Petrich (1999): Excited - State Intramolecular H Atom Transfer of Hypericin and Hypocrellin A Investigated by Fluorescence Upconversion, J. Phys. Chem. A, 103, 7949-7957. Sowund, S., S. SUKOLAK. Ls. Comic, Ls. Kestié (1997): Biochemical reaction in vitro of certain fungi to the presence of hypericin, Acta veterinaria Yugoslav. 47, 331-344, Tuomas, C., R. S. Mac Gitt, G. C, Miter, R. S. PARDIN! (1992): Photoactivation of hypericin generates singlet oxygen in mitochondria and inhibits succinoxidase, Photochem. Photobiol, 55, 4 TTakAHAstt, I, S. NAKANISHI, E, HOBAYASHT, N. NAKANO, K. SuZuKI, T. TaMAoxt, (1989): Hypericin and pseudohypericin specifically inhibit protein kinase C: possiblereaction to their antiretroviral activity. Bichem, Biophys. Res. Commun., 165, 1207-1212. ‘Vanper Werr, Q. M., R. E. SAXTON, A. CHANG, D. HORTON, M. B. PAIVvA, J. ANDERSON, C. FOOTE, J. Soupant, A. Matuey, D. J. Casto (1996): Hypericin: A New Laser Phototargeting Agent for Human Cancer Cells, Laryngoscope 106, 479-483, Wenner, L., E. RoTH, Y. Mazur, I. SiLMAN (1999): Targeted Cross-linking f a Molten Globule From of Acetylcholinesterase by the Virucidal Agent Hypericin, Biochemistry, 38, 11401-11405. WueaTtey, D. (1998): Hypericum Extract - Potential in the Treatment of Depression, CNS Drugs, Vol 6, p. 431-440. Weiner, L., Y. MAZUR (1992): EPR studies of hypericin. Photogeneration of free radicals and superoxide, J. Chem. Soc. Perkin Trans. 2, 1439. Recieved November 15, 2004 ‘Accepted January 16, 20058. SOLUJIC et al.: PROTEASE ACTIVITY OF HYPERICIN-INDUCED FUNGI 7 See eee ee AKTIVNOST PROTEAZA HIPERICIN-INDUKOVANIH GLJIVA Slavica SOLUJIC i Milorad MILOSEV Prirodno-matematitki fakultet, Kragujevac, Srbija i Cma Gora Izvod U radu su prikazani rezultati uticaja 8istog hipericina i zdruzenog efekta hipericina i bele svetlosti na ukupnu aktivnost proteolititkih enzima kod tri generacije gljiva Penicillium verrucosum, Penicillium cyclopium i Penicillium chrysogenum koje su se razvijale na izmenjenoj Czapek-ovoj hranljivoj podlozi uz dodatak 30 mg etanolskog rastvora hipericina . Eksperimentalni uzorci kultura su svakodnevno ozrativani belom svetloSéu u trajanju od 15 minuta pri emu je bioloSki sistem dodatno aktiviran sa 4,5 kJ energije. Hipericin u uslovima svoje fotosenzibilizacije favorizuje produkcijy slobodnih, visoko energetskih radikala, Sto je uslov promene obima i mehanizma dejstva proteaza ispitivanih gljiva. Proces indukcije enzimske aktivnosti ispitivanih gljiva obavljen je kori8éenjem gljiva koje su se razvijale nekoliko razvojnih perioda u trajanju od 21 dan na podlozi obogacenoj sa Img/ml etanolskog rastvora hipericina. Nakon toga, kulture su presejane na istu krompir-glukoznu podlogu i posle razvoja od 7 dana korigcene 2a ponovijeni eksperiment, uz dodatak hipericina. U ogledu je okarakterisan kvantitavni udeo istog hipericina i fotosenzi- bilisanog hipericina pri produZenom delovanju, na ukupnu aktivnost proteaza ispitivanih gljiva iz roda Penicillium. Eksperimentalni rezultati uticaja cistog hipericina , kao i hipericina i svetlosti na aktivnost proteaza kod tri generacije gljiva iz roda Penicillium daju povoda sledecim zakljuécima: U prvoj i drugoj generaciji ghjiva gajenih na izmenjenoj hranljivoj podiozi u prisustvu hipericina, odnosno hipericina i svetlosti, primeceno je povedanje aktivnosti proteaza kod gliiva P. verrucosum i P. cyclopium za oko 15- 30% u prvoj polovini razvojnog perioda, odnosno , smanjene aktivnosti od oko 50% u drugom delu razvojnog perioda. Dvostruka indukcija metabolizma gljive P. chrysogenum sa hipericinom, odnosno fotosenzibilisanim hipericinom, utite na umanjenu aktivnost proteaza u obimu od 30-70%. Treéa indukcija metabolizma gliiva P. verrucosum, P. cyclopium i P. chrysogenum sa hipericinom, odnosno fotosenzibilisanim hipericinom, pokazuje intenzivno umanjenu aktivnost proteaza u obimu od 70% u odnosu na aktivnost ovih enzima kod kulture koja se razvijala u odsustvu hipericina. Primljeno 15. novembra 2004. Odobreno 16. januara 2005.