**6. References**

260 Gel Electrophoresis – Advanced Techniques

dependent *trans*-dihydrodiol dehydrogenases, almost all investigations have been done in mammalian tissues (Carbone et al., 2008; Chang et al, 2009; Chen et al., 2008) but only a few reports about these important enzymes have been done in fungi (Bezalel et al., 1997; Hammel, 1995; Sutherland et al., 1993) particularly in *Phanerochaete chrisosporium* (Bogan & Lamar, 1996; Muheim et al., 1991). At date, there is no any report about the detection of dihydrodiol dehydrogenase activities by means of electrophoretic zymograms in any organism. This methodology represents an interesting approach because in this way it is possible to detect, study and compare the different isoenzymes present in the cell-free extracts of the organism used as enzymatic source. In our own work, YR-1 strain possesses extraordinary metabolic machinery that premises it to survive in a very dangerous place

The results about the localization of DD activities in a differential centrifugation procedure from YR-1 grown in different carbon sources (Table 1), revealed that the activity measured with *cis*-naphthalene-diol as substrate and NADP+ as electron acceptor was only present in the supernatant fractions of each centrifugation speed, suggesting that all DD activity observed must be a soluble enzyme. At date, we cannot discard the possibility that the DD activity could be located in the lumen of some kind of microsomal bodies, because of the drastic ballistic treatment used to homogenize the cells. Actually, we are conducting different experiments employing density gradients and electron transmission microscopy to

Complementary analysis of DD activities by electrophoretic zymograms led us to detect eleven different activities and all of them were NADP+-dependent (Fig. 2) this represents the first report about the detection of DD activities by electrophoretic zymograms, a nondenaturing gel electrophoresis stained with a colored product of the enzymatic reaction.

Of the eleven bands detected, we described five different constitutive DD activities, DD 1-5, since them were observed when D-glucose was the carbon source and only DD-2 was solely induced by this sugar, since the others are induced at least for another carbon source. When *n*-decanol was used as a carbon source, we observed four out of five of the constitutive bands, lacking only the DD-2 band. Its noticeable that only when glycerol, ethanol, npentane and n-hexadecane were the carbon source to grow the fungus, not a single constitutive band was observed, may be due to the fact that these compounds only can be metabolized specifically by the induced enzymes. In glucose grown mycelium, all inducible dihydrodiol dehydrogenase activities were absent suggesting that they could be subject to

Surprisingly all the activities described here as DD are able to use *cis*-naphthalene-diol, since this substrate has been describes as bacterial specific (Cerniglia & Gibson 1977). The

Phenanthrene was the best inducer since when used as a carbon source four out of six inducible bands were observed, *n*-decanol and naphthalene were the second best inducers since each one led the induction of three different enzymes, sharing the bands denominated iDD1 and iDD2. Also in the case of the inducible enzymes glycerol, n-pentane and nhexadecane were unable to induce any activity. The specific induction of an activity must be

substrate reported for eukaryotic cells is the *trans*-naphthalene-diol (Cerniglia 1977).

how is a petroleum-contaminated soil.

carbon-catabolite repression (Fig. 2).

due to substrate specificity.

resolve this question.

Alvarado, C. Y., Gutiérrez-Corona, F. & Zazueta-Sandoval, R. (2002). Presence and physiologic regulation of alcohol oxydase activity in an indigenous fungus isolated from petroleum-contaminated soils. *Applied Biochemistry and Biotechnology*. Vol. 98- 100, No. 1-9 (March 2002), pp. 243-255. ISSN: 0273-2289.

Polyacrylamide Gel Electrophoresis an Important Tool for the

2002), pp. 348-356, ISSN 1347-4367.

1987), pp. 1017-1023. ISSN 0008-4166.

72, No. 7 (July 2006), pp. 426-434. ISSN 0099-2240.

(November 1951), pp. 265-275. ISSN 0021-9258.

(January 1991), pp. 369-375. ISSN 0014-2956.

221. ISBN 0 7167 1005 6.

1135-1144. ISSN 0045-6535.

ISSN 0006-2960.

0836.

pp. 41-43.

Detection and Analysis of Enzymatic Activities by Electrophoretic Zymograms 263

Goto, S. & Takami, H. (1986). Classification of Ascoideaceous Yeasts Based on the Electrophoretic

Hammel, K. E. (1995). Mechanisms for Polycyclic Aromatic Hydrocarbon Degradation by

Hara, A., Taniguchi, H., Nakayama, T. & Sawada, H. (1990). Purification and Properties of

Higaki, Y., Kamiya, T., Usami, N., Shintani, S., Shiraishi, H., Ishikura, S., Yamamoto, I. &

Hunt, J. M. (1979). *Petroleum geochemistryand geology.* W. M. Freeman, San Francisco, CA. pp.

Hyötyläinen, T. & Olkari, A. (1999). The Toxicity and Concentrations of PAHs in Creosote-

Jeng, R. S., Shiyuan, Y. & Wayman M. (1987). Isoenzyme and Protein Patterns of Pentose-

Jouanneau, Y. & Meyer, C. (2006). Purification and Characterization of an Arene *Cis-*

Krauzova, V. I., Il'chenko, A. P., Sharyshev, A. A. & Lozinov, A. B. (1985). Possible Pathways

Laemmli, U. K. (1970). Cleavage of Structural Proteins During the Assembly of Head of

Lim, W. J., Park, S. R., Cho, S. J. Kim, M. K., Ryu, S. K., Hong, S. Y., Seo, W. T., Kim, H. &

Muheim, A., Waldner, R., Sanglard, D., Reiser, J., Schoemaker, H. E. & Leisola M. S. (1991).

Neidle, E., Hartnett, C., Ornston, L. N., Bairoch, A., Rekik, M. & Harayama, S. (1992). *Cis*-

*Biochemistry*. Vol. 108, No. 2 (August 1990), pp. 250-254. ISSN 0021-924X. Harayama, S. & Timmis, K. N. (1989). *In: Genetics of bacterial diversity*. Hopwood, D.A., and

Chater K.E.. eds. Academic Press, pp. 151-174. New York, USA.

*Microbiology*, Vol. 32, No. 4 (June 1986), pp. 271-282. ISSN 1349-8037.

Comparison of Enzymes and Coenzymes Q Systems. *The Journal of General and Applied* 

Ligninolytic Fungi. *Environmental Health Perspectives*, Vol. 103. Suppl. 5 (June 1995),

Multiple Forms of Dihydrodiol Dehydrogenases from Human Liver. *Journal of* 

Hara, A. (2002). Molecular Characterization of Two Monkey Dihydrodiol Dehydrogenases. *Drug Metabolism and Pharmacokinetics*. Vol. 17, No. 4 (September

Contaminated Lake Sediment. *Chemosphere.* Vol. 38, No. 5 (February 1999), pp.

Fermenting Yeasts. *Canadian Journal of Microbiology*. Vol. 33, No. 11 (November

Dihydrodiol Dehydrogenase Endowed with Broad Substrate Specificity Toward Polycyclic Hydrocarbon Hydrodiols. *Applied and Environmental Microbiology.* Vol.

of the Oxidation of Higher Alcohols by Membrane Fractions of Yeasts Cultured on Hexadecane and Hexadecanol. *Biochemistry.* Vol. 50, No. 5 (May 1985), pp. 609-615.

Bacteriophage T6. *Nature*. Vol. 227, No. 5259 (August 1970), pp. 680-685. ISSN 0028-

Yun, H. D. (2001). Cloning and characterization of an intracellular isoamylase gene from *Pectobcterium chrysantemi* PY35. *Biochemical and Biophysical Chemical Communications.* Vol. 287, No. 2 (September 2001), pp.348-354. ISSN 0006-921X. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein Measurement

with the Folin Phenol Reagent. *Journal of Biological Chemistry*, Vol. 193, No. 1

Purification and Properties of an Aryl-Alcohol Dehydrogenase from the White-Rot Fungus *Phanerochaete chrysosporium*. *European Journal of Biochemistry*. Vol. 195, No. 2

Diol Dehydrogenases Encoded by the TOL pWWO Plasmid *xylL G*ene and the *Acinetobacter calcoaceticus* Chromosomal *benD* Gene Are Members of the Short –


Atlas, R. M. (1995). Bioremediation. *Chemical and Engineering News*. Vol. 73, No. 14 (April

Auret, B. J., Boyd, D. R., Robinson, P. M. & Watson, C.G. (1971). The NIH Shift During the

Bergmeyer, H. U. (1983). Alkoholdehydrogenase. *In*: Bergmeyer, H.U. (ed) *Methods of enzymatic analysis*, Verlag Chemie, Weinheim, Vol. 11, 3rd edn. pp-139-145. Bezalel, L., Hadar, Y. & Cerniglia, C.E. (1997). Enzymatic Mechanisms Involved in

Camacho, M. R. L., Durón, C. A. & Zazueta-Sandoval, R. (2010). Analysis of Glycerol

*Leeuwenhoek*. Vol. 98, No. 4, (November 2010), pp. 437-445, ISSN 0003-6072. Carbone, V., Hara, A. & El-Kabbani, O. (2008). Structural and Functional Features of Dimeric

Cerniglia, C. E. (1997). Fungal Metabolism of Polycyclic Aromatic Hydrocarbons: Past,

Chang, C. H., Chen, L. Y., Chan, P. C., Yeh, T. K., Kuo, J. S., Ko, J. C. & Fang, Y. H. (2009).

Chen, J., Adikari, M., Pallai, R., Parckh, K. H. & Simpkins, H. (2008). Dihydrodiol

Durón-Castellanos, A., Zazueta-Novoa, V., Silva-Jiménez, H., Alvarado-Caudillo, Y., Peña

Hydroxylation of Aromatic Substrates by Fungi. *Journal of the Chemical Society D: Chemical Communications* No. 24, (December 1971), pp. 1585-1587. ISSN 1364-548X. Bartnicki-García, S. & Nickerson, W. J. (1962). Nutrition, Growth and Morphogenesis of

*Mucor rouxii. Journal of Bacteriology*. Vol. 84, No. 4 (October, 1962), pp. 841-858.

Phenanthrene Degradation by the White-Rot Fungus *Pleurotus ostreatus*. *Applied and Environmental Microbiology*. Vol. 63, No. 7, (July 1997) pp. 2495-2501. ISSN 0099-2240. Bogan, B. W. & Lamar, R. T. (1996). Polycyclic Aromatic Hydrocarbon-Degrading

Capabilities of *Phanerochaete laevis* HHB-1625 and Its Extracellular Ligninolytic enzymes. *Applied and Environmental Microbiol*ogy. Vol. 62, No. 5, (May 1996), pp.

Dehydrogenase Activities Present in *Mucor circinelloides* YR-1. *Antonie Van* 

Dihydrodiol Dehydrogenases. *Cellular and Molecular Life Science*. Vol. 65, No. 10,

Present and Future Applications in Bioremediation. *Journal of Industrial Microbiology and Biotechnology*. Vol. 19, No. 5-6 (November 1997), pp. 324-333. ISSN 1367-5435. Cerniglia, C. E. & Gibson, D. T. (1977). Metabolism of Naphthalene by *Cunninghamella* 

*elegans*. *Applied and Environmental Microbiology*. Vol. 34, No. 4, (October 1977), pp.

Overexpression of Dihydrodiol Dehydrogenase as a Prognostic Marker in Resected Gastric Cancer Patients. *Digestive Diseases and Sciences*. Vol. 54, No. 2 (February

Dehydrogenases Regulate the Generation of Reactive Oxygen Species and the Development of Cisplatin Resistance in Human Ovarian Carcinoma Cells*. Cancer Chemotherapy and Pharmacology*. Vol. 61, No. 6 (May 2008), pp. 979-987. ISSN 0344-5704. Cook, F. D., Jobson, A., Phillippe, R. & Westlake, D. W. S. (1974). Biodegradability and crude

oil composition. *Canadian Journal of Microbiology.* Vol. 20, No. 7 (July 1974), pp. 915-

Cabrera, E. & Zazueta-Sandoval, R. (2005). Detection of NAD+-Dependent Alcohol Dehydrogenase Activities in YR-1 Strain of *Mucor circinelloides,* a Potential Bioremediator of Petroleum-Contaminated Soils. *Applied Biochemistry and Biotechnology.* Vol. 121-124, No. 1-3 (March 2005), pp. 279-288. ISSN 0273-2289. Ferris, J. P., MacDonald, L. H., Patrie, M. A. & Martin, M. A. (1976). Aryl Hydrocarbon

Hydroxylase Activity in the Fungus *Cunninghamella bainieri*: Evidence for the Presence of Cytochrome P-450. *Archives of Biochemistry and Biophysics*. Vol. 175, No.

1995), pp. 32-42. ISSN 0009-2347. DOI: 10.1021/cen-v073n014.p032

ISSN 0021-9193.

1597-1603. ISSN 0099-2240.

363-370. ISSN 0099-2240.

928. ISSN 0008-4166.

2009), pp. 342-347. ISSN 0163-2116.

2 (August 1976), pp. 443-452. ISSN 0003-9861.

(May 2008), pp. 1464-1474, ISSN 1420-682X.


**15** 

*Brazil* 

**Applications of Zymography** 

**(Substrate-SDS-PAGE) for Peptidase** 

Claudia M. d'Avila-Levy1, André L. S. Santos2, Patrícia Cuervo1,

*2Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes,* 

Peptidases are enzymes that catalyze the hydrolysis of peptide bonds in proteins or peptides. The hydrolysis can be specific or unspecific, leading to highly regulated cleavage of specific peptide bonds, or to complete degradation of proteins to oligopeptides and/or amino acids. Peptidases can be classified as endo- or exopeptidases, the latter only act near the ends of the polypeptide chain. Endopeptidases are divided into six major families by virtue of the specific chemistry of their active site: aspartic, serine, metallo-, cysteine,

Zymography is an electrophoretic technique, based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and a substrate (e.g. gelatin, casein, albumin, hemoglobin, etc.) co-polymerized with the polyacrylamide matrix. Proteins are prepared by the standard SDS-PAGE buffer under non-reducing conditions (no boiling and no reducing agent), and are separated by molecular mass in the standard denaturing SDS-PAGE co-polymerized with a protein substrate. After electrophoresis, peptidases are renatured by the removal of the denaturing SDS by a non-ionic detergent, such as Triton X-100, followed by incubation in conditions specific for each peptidase activity (time, temperature, ions, ionic strength), when the enzymes hydrolyze the embedded substrate, then proteolytic activity can be visualized as cleared bands on a Coomassie stained background (Heussen and Dowdle, 1980). Therefore, only endopeptidases can be detected by substrate-SDS-PAGE, which requires a considerable degradation of the substrate for visualization of the degradation haloes. Alternatively, an overlay with specific chromogenic or fluorogenic peptide substrate can be done after SDS-PAGE separation of the proteins and renaturation with Triton X-100, which allows the detection of specific peptidases in complex

This technique has many benets: (1) it is relatively inexpensive, requires short assaying times, and peptidases with distinct molecular masses can be detected on a single gel; (2)

glutamic and threonine peptidases (Rawlings et al. 2010).

**1. Introduction** 

biological samples.

**Screening in a Post-Genomic Era** 

José Batista de Jesus1,3 and Marta H. Branquinha2

*Universidade Federal do Rio de Janeiro, Rio de Janeiro 3Universidade Federal de São João Del Rei, São João Del Rei* 

*1Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro* 

Chain Alcohol Dehydrogenase Superfamily. *European Journal of Biochemistry*. Vol. 204, No. 1, (February 1992), pp. 113-120. ISSN 0014-2956.

