77

Kurze Mitteilungen Z. Anal. Chem. 276, 77-78 (1975) - 9 by Springer-Verlag 1975

Simple Screening-Test for the Detection of Lactoperoxidase Inhibitors F. Geike and C. D. Parasher Institnt f/it Pflanzenschutzmittelforschung, Biologische Bundesanstalt ftir Land- und Forstwirtschaft, Berlin

Table 1. Detection limits of some chemicals by inhibition of lactoperoxidase. (Average area of the inhibitory spots: 0.3 cm2) Inhibitor

Received April 18, 1975 Einjaches Verfahren zum Nachweis yon Lactoperoxidase-Inhibitoren

Nachw. yon Lactoperoxidase-Inhibitoren; Chromatographie, Dfinnschicht/Enzymatische Analyse; Screening-Test Peroxidases are widely distributed in higher plants, but also occur in animals and microorganisms. The peroxidases may be classified in three groups, the ferriprotoporphyrin peroxidases, the verdoperoxidases being found in myelocytes, milk and some miscellaneous tissues, and the flavoprotein peroxidases. Besides these three groups of peroxidases there is a group of metalloproteins, e.g. haemoglobin, which give an unspecific peroxidase reaction. Though the presence of a peroxidase in milk was first realized in 1881 [3] and the lactoperoxidase, as this enzyme is called, was obtained in a fairly pure form in 1932 [8] and the purification of the enzyme was still further improved in the early 40's [18-20] only little is known about the role of this enzyme in organisms. Since milk of disturbed udders shows raised leucocyte counts and a positive correlation between cell counts and lactoperoxidase activity [13] one might speculate that lactoperoxidase plays some part in the detoxification of certain toxins produced by bacteria, as was shown for the closely related verdoperoxidase from leucocytes by Agner [t, 2], and it has been suggested [9] that the presence of lactoperoxidase hastens the thermal destruction of milk bacteria. Only recently it has been demonstrated that lactoperoxidase catalyzes the iodination of tyrosine peptides [5] and proteins [6,15]. As little is known about inhibitors oflactoperoxidase and a lot of compounds like pesticides, drugs, and heavy metals which could be inhibitory to the enzyme might enter the milk, the development of a simple screening-test for the detection of lactoperoxidase inhibitors appears of utmost importance. So a previously developed method for the detection of peroxidase inhibitors [11] is modified for an application of this test to the detection of lactoperoxidase inhibitors. The substances under investigation were spotted onto handmade silica gel plates (20 • 20 cm). The plates then were sprayed each with 12 ml solution of 1.5 mg lactoperoxidase from bovine milk (EC 1.11.1.7- Boehringer, 160U/rag) in 0.1 M phosphate buffer pH 5.5 and subsequently were incubated for 30 rain at 25~C and 8 0 - 90 ~ relative humidity. After incubation 3 - 5 ml of substrate were applied. The following two solutions were used as substrates. For substrate 1 0.05 ml

Aniline o-Toluidine m-Toluidine p-Toluidine HgCI; p-Benzoquinone Anthraquinone 1,8-Dioxyanthraquinone 2,6-Dichloroqninone-4-chloroimide 2,3-Dichloro-5,6-dicyano1,4-benzoquinone Resorcinol Phloroglucinol

Detection limits in lag Substrate I

Substrate II

2.0 3.0 1.0 0.3 0.2 0.1 0.4 0.8

2.0 2.0 t.0 6.0 0.3 0.8 0.4 0.4

0.8

0.7

0.08 0.01 0.01

0.1 0.01 0.01

of guaiacol was mixed with 0.02 ml and 20 ml of perhydrol (Merck) and water, respectively. For substrate I I i ml ofa 1 methanolic solution of o-dianisidine was mixed with 0.04 ml and 10 ml of perhydrol and water, respectively. Both substrate solutions should be prepared immediately before use and were applied undiluted, The colour on the plates develops immediately after spraying the substrate. In the case of guaiacol the light reddish-brown colour is more or less stable whereas with o-dianisidine the turquoise colour turns to brown in the course of I h. The interpretation of the plates should take place in the first 3 min after application of the substrate. The results from Table 1 show that the aromatic amines tested (aniline and the toluidines) are inhibitory to lactoperoxidase. These findings are in good agreement with those of Rosenberg [16], but they seemed very surprising, as vegetable peroxidase systems are known to oxidize several of these amines very easily [17]. As the white inhibitory spots of aniline, and mand o-toluidine on the plates after some time turn to blue (specially when using substrate II) it may be supposed that these compounds have been taken as slow reacting alternative substrates by the enzyme, thus being competitive inhibitors with respect to the substrates used in this study. A reaction of aniline and m- and o-toluidine with H2Oz or with the complete substrate solution to form a coloured product m a y be excluded as without enzyme there is no colour development at all with these aromatic amines. Peroxidase has proved to be of relatively low sensitivity to HgCI2 [21] which is thought to be due to the absence of a thiol group in the active center. The

78 possibility, however, of the existence of a thiol group at the active site of lactoperoxidase has not yet fully been ruled out. HgC1 a may be considered as a nonspecific inhibitor which may react with thiol groups from the active center as well as non-essential ones. So the observed inhibition of the enzyme by HgC12 may be due to a reaction with a thiol group of the active site or due to the high concentration of HgC12 at the area of application resulting in a precipitation of lactoperoxidase following reaction with nonessential thiol groups of the enzyme. F r o m results of screening-tests on urease [10] and ALA dehydratase [12] it is known that enzymes with thiol groups in the active center are about ten times more sensitive to Hg 2+ than enzymes without thiol groups at the active site. So the observed inhibition seems to be due to a reaction of HgC12 with non-essential thiol groups. The inhibition of lactoperoxidase by certain quinones is in good agreement with results of Klapper and Hackett [14] and from this laboratory [11] obtained with horse-radish peroxidase, which was more intensively inhibited by the quinone compounds studied. The strong inhibitory activity of resorcinol and phloroglucinol against lactoperoxidase found in this investigation is in good accordance with results showing strong inhibition of the enzyme by aromatic compounds possessing m-hydroxy groups [4, 9,16]. The results reported here present evidence for the applicability and reliability of this thin-layer chromatographic-enzymatic technique as a screening-test method for the detection of lactoperoxidase inhibitors. F r o m these results, however, an inhibition cannot be distinguished from an inactivation of the enzyme protein. Comparing the data with those from a recent paper [11] it is evident that there are some differences in the tolerance of lactoperoxidase and horse-radish peroxidase against the inhibitory effects of the quinones studied and HgC12.

Z. Anal. Chem., Band 276, Heft 1 (1975) This method seems to be very useful in finding new inhibitor-substances of lactoperoxidase for research as well as in toxicological screening-tests. It cannot, however, replace an intensive study of the inhibitors, detected by this method.

Acknowledgement.The authors wish to thank the Deutsche Forschungsgemeinschaft for the financial support of this work. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. tl. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21.

Agner, K. : Nature 159, 271 (1947) Agner, K. : Rec. Trav. Chim. Pays-Bas 74, 373 (1955) Arnold, C. : Arch. Pharm. 219, 41 (1881) Arnott, D. G., Doniach, I. : Biochem. J. 50, 473 (1952) Bayse, G. S., Michaels, A. W., Morrison, M.: Biochim. Biophys2 Acta 284, 30 (1972) David, G. S. : Biochem. Biophys. Res. Commun. 48, 464 (1972) Demeter, K.J., Steude, R.: Milchw. Forsch. 19, 122 (1937) Elliot, K. A. C. : Biochem. J. 26, 10 (1932) Elliot, K. A. C. : Biochem. J. 26, 1281 (1932) Geike, F. : Z. Anal. Chem. 258, 284 (1972) Geike, F., M61tgen, H., Parasher, C. D. : Z. Anal. Chem. 270, 367 (1974) Geike, F., Parasher, C. D., M61tgen, H. : Z. Anal. Chem. 270, 366 (1974) Kicrlneier, F., Kuhlmann, H.: Z. Lebensm.-Unters. Forsch. 150, 224 (1972) Klapper, M.H., Hackett, D.P.: J. Biol. Chem. 238, 3736 (1963) Marchalonis, J. J.: Biochem. J. 113, 299 (1969) Rosenberg, I. N. : Science 116, 503 (1952) Saunders, B. C., Holmes-Siedle, A. G., Stark, B. P. : Peroxidases. London: Butterworths 1964 Theorell, H., Akesson, A. : Arkiv Kemi Min. Geol. 16A, No. 8 (1942) Theorell, H., Akesson, A. : Arkiv Kemi Min. Geol. 17A, No. 7 (1943) Theorell, H., Paul, K. G. : Arkiv Kemi Min. Geol. 18A, No. 12 (1944) Webb, J. L. : Enzyme and metabolic inhibitors, Vol. II and III. New York: Acad. Press 1966

Dr. F. Geike, Institut ftir Pflanzenschutzmittelforschung, Biologische Bundesanstalt ftir Land- und Forstwirtschaft, D-1000 Berlin 33, K6nigin Luise-Str. 19