Electrophoresis 1990, l l ?621-626

Arved Haas’ Marika Geldmacher-vonMallinckrodt2 ‘Childrens Hospital, University of Dusseldorf ’Institute of Legal Medicine, University of Erlangen

“Sandwich”-procedure for visualization of paraoxonases after isoelectric focusing

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Enzyme visualization with partially dehydrated agarose substrate layers: Detection of paraoxonase after thin-layer isoelectric focusing in agarose A new approach is described for studying the polymorphism of paraoxon hydrolyzing serum esterases after isoelectric focusing of native sera. Enzyme visualization is performed by a modified sandwich procedure which is faster and also affords higher resolution and considerably improved sensitivity. Up to seven paraoxon splitting isoenzymes can be visualized and clearly distinguished from arylesterases and phosphatases by using 3-naphtyl acetate, paraoxon, 5-bromo-4-chloro-3-indolyl acetate and 5-bromo-4-chloro-3-indolyl phosphate as substrates. The new technique is also able to differentiate between paraoxonase isoenzymes sensitive to EDTA and those which are EDTA-stable. Immunofixation with anti-human serum albuminantibodies revealed similar isoelectric points for these isoenzymes, although they are not assumed to be identical. The new technique may prove useful in other applications of enzyme visualization where diffusion of enzymes and/or cleavage products is the major problem.

1 Introduction

2 Materials and methods

Paraoxon, a potent inhibitor of cholinesterases (EC 3.1.1.7, EC 3.1.1 .8), is the active metabolite ofthe organo-phosphorus insecticide parathion (E 605). It is hydrolyzed and thus detoxified by an esterase activity (“paraoxonase”) which is found in the sera of man and various mammalian species [ 1-51. The natural substrate is unknown. Many attempts at isolating and characterizing this enzyme have been hampered by its pronounced lability [ I , 2,6-111. Several workers IS, 11-15] have obtained clear evidence that at least two paraoxon splitting activities exist, one inhibited by EDTA, the other EDTAstable and not distinguishable from serum albumin t 1 11. Serum paraoxonase as an entity has widely varying activity levels not only in different mammalian species [ 1,2,7,16,171 but also between human subjects 12, 13, 15, 18-23]. The statistical evaluation of these activity measurements favors a genetically determined polymorphism of EDTA-sensitive paraoxonase, but presently it is not clear how many enzymes are active with this substrate 1131. The goal of the present study was to determine the number of paraoxon splitting enzymes in serum after isoelectric focusing (IEF) under defined conditions and to check their identity with human serum arylesterase (EC 3.1.1.2), which also hydrolyzes phenyl acetate and P-naphthyl acetate (P-NA). Zymogram techniques known so far were inefficient, even in the more refined form described in [24-261; thus for enzyme visualization a new overlay-technique had to be developed. T o overcome the poor sensitivity and difficult handling of paraoxon as a substrate, the compatibility with other well-known esterase substrates 127-321 was studied.

2.1 Serum samples Human serum samples were obtained from healthy individuals, partially identified in a previous study [ 121 as having low, intermediate, or high serum paraoxonase activity. Samples were used fresh or kept frozen at -70 OC until use. To check the proper conditions for enzyme visualization, rabbit serum with high paraoxonase activity was always run in parallel as a control.

2.2 Chemicals Isogel Agarose-EF was obtained from LKB (Bromma, Sweden). Servalyts 3- 10, 4-6, P-NA, 5-bromo-4-chloro-3-indolyl acetate (BCI-A), 5-bromo-4-chloro-3-indolyl phosphate (BCI-P) potassium salt, Serva Blue R, Nitro Blue Tetrazolium (NBT) salt as well as Fast Blue Salt B were from Serva (Heidelberg, FRG). Paraoxon was supplied by Ehrenstorfer (Augsburg, FRG). Rabbit anti-human serum albuminIgG was obtained from D A K O (Copenhagen, Denmark), nitrocellulose (NC) membranes from Schleicher & Schull (Dassel, FRG), and India Ink from Pelikan (Hannover, FRG), Dowex 1 x 8 (100-200 mesh) and phenyl acetate (PA) were purchased from Sigma (Munich, FRG), all other chemicals from Merck (Darmstadt, FRG).

2.3 Preparation of agarose thin-layer plates

Correspondence: Prof. Dr. Marika Geldmacher-von Mallinckrodt. Institute of Legal Medicine, University of Erlangen, Universitatsstr. 22, D-8520 Erlangen, Federal Republic of Germany Abbreviations: AP-Tween, alkaline phosphate buffer/Tween 20: BCI-A, S-bromo-4-chloro-3-indolyl acetate; BCI-P, 5-brorno-4-chloro-3-indolyl phosphate. potassium salt; p-NA, 0-naphthyl acetate; E 600, paraoxon: EDTA, ethylenediaminetetraacetate; IEF, isoelectric focusing; NBT, Nitro Blue Tetrazolium salt; NC, nitrocellulose; PA, phenyl acetate; PBS. phosphate buffered saline

0VCH Verlagsgesellschaft mbH, D-6940 Weinheim. 1990

The Gel Casting Kit from LKB was used to prepare agarose thin-layer gels according to the LKB Instruction Manual 18 1 8 A. The gels contained 0.8 % w/v agarose, 3 % w/v Servalyt carrier ampholytes p H 2-1 1 and 1 % w/v carrier ampholytes p H 4-6. The maximum storage time of gels was 6 days.

2.4 IEF

The electrode solutions were 1 N NaOH and 0.05 N H , S 0 4 at the cathode or anode, respectively. Serum samples were ap0 I73-0835/90/0808-0621 $3.50+.25/0

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Eleclrophoresis 1990. 11. 621-626

A . Haar and M . Geldmacher von Mallinckrodt

plied at a distance of 2.5 cm from the cathodic site (10 pL/cm) using applicator strips (Serva) or applicator troughs (20 x 0.10 cm), cut out from silicon rubber :;beets. IEF was per formed in an LKB Multiphor 21 17 chamber in combination with an LKB 2 103 Power Supply, at a coolant temperature of 0 'C. current limited to 20 mA, voltage to 2000 V, and power to 35 W. Electrophoresis was terminated after attaining 1200 VH.

2.6 Protein staining The gels were stained according to LKB Instruction Manual 18 18 A with Coomassie Brilliant Blue R-250, on NC membranes by using India ink [361. The N C membranes were incubated overnight in India ink solution, diluted 1:lOOO in phosphate buffered saline, containing 0.05 % Tween 20 (Tween-PBS). Subsequently, they were briefly washed with PBS-Tween.

2.5 Enzyme visualization 2.7 Immunofixation The following substrates were used: 'BCI-A, BCI P, both amplified by the redoxing reagent NBT. P-NA, PA and para oxon. All solutions were prepared directly prior to use. Enzyme visualization was achieved in three ways: (i) With BCI A as a substrate, proteins were transferred after electrophoresis from the gel to NC membranes, using the print technique [24].Three mg BCI A were dissolved in I mL N,Ndimethylformamide and this solution was added to 12 mLO. I M phosphate buffer, pH 7.3, containing 2 M NaCl I 3 3 I and 4 mM MgS04.About 3 min after placing the NC membraneinto this mixture, 5 mg NBT in an aqueous solution and 2 mg phenazine methosulfate were added with gentle shaking. The incubation was continued for at least 2 h at 37 "C in the dark. With BCI-P as a substrate, enzyme visualization was carried out according to [ 341 after protein transfer to N C membranes: 10 mg NBT were dissolved at 37 "C in 30 mL Tris buffer (0.1 MTris-HC1,O.l MNaCI, 5 m~MgCI,,pl

Enzyme visualization with partially dehydrated agarose substrate layers: detection of paraoxonase after thin-layer isoelectric focusing in agarose.

A new approach is described for studying the polymorphism of paraoxon hydrolyzing serum esterases after isoelectric focusing of native sera. Enzyme vi...
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