Measurement and detection of peroxidase

P/ant Sc/e~e, 69 (1990)19 - 26 Elsevier Scientific Publishers Ireland Ltd.

MEASUREMENT

R. V A N

HUYSTEE

19

AND DETECTION OF PEROXIDASE

~*. C. B R E D A t. P. SESTO'. N. B E O P O U O L O S

b and R. E S N A U L T ~

"Dept. of Plant Sciences. University of Western Ontario. London, Ontar,o, N6A 5B7 ICanadoJ and "Unite Biologw motec~daire. lwtitut de PAysWloFie vegetale, CNRS, Gif.sur-Yvette 91198 tFrunce]

(ReceivedOctober 2nd, 19891 (Revision received January 24th. 1990~ (Accepted February 1st, 1990) The purity and the concentration based on the RZ value and the molar extinction coefficient was determined for the cationic peroxidase isolated from peanut (AmcAUAFpog~eaL.}suspension culture medium. The maintenance of a concentrated sample and. if necessary, suspension in slightly alakline buffer was found to be the best condition for storage. As little as 3 ng of perox. idase could be immuno-precipitated, subjected to electrophoresis and immunoprobed and still be detected. Key words: A rachi~ hypogaea L.; peroxidase; heine, immunoblot;concentration

Introduction Peroxidase, an e n z y m e f r e q u e n t l y used for plant physiological studies [1], has now been cloned from genes in tobacco [2] and potato [3~ N e v e r t h e l e s s , the methods used for quantitation of peroxidase are still ill-defined. In many cases the amounts are e i t h e r given as e n z y m e units or as enzymatic products of various intensities in z y m o g r a m s [4,5[ Yet, peroxidase may readily be b r o u g h t to high p u r i t y from the suspension medium of tissue cultures [6]. Once antibodies have been raised additional peroxidase may be purified by immuno-affinity c h r o m a t o g r a p h y [7]. The purity can be measured by the absorbance ratio, f r e q u e n t l y called the RZ, of the heine S o r e t band o v e r the protein value m e a s u r e d at 280 nm [8,9]. • To whom correspondence should be sent. Abbreviations: ABTS. 2~2'azin~di{3-ethylbenzthiazoline sulphonic acid]: 3AEC, 3-amino-9-ethylcarbazole;BSA, bovine serum albumin; PAGE, polyacrylamide gel electroph¢~ reals; PEG. polyethylene glycol; PMSF, phenyl methyl sulfonyl fluoride: RZ, absorbance ratio: 405 nmt280 nm; SDS. sodium dodecyl sulfate; TBS-T, 20 mM Tris (pH 7.5) containing 150 mM NaCI and 0.1°#oTween-20.

A high RZ value, indicating purity, has sometimes been difficult to maintain [6] particularly after prolonged storage of the peroxidase solution. Since one of the ways of measuring peroxidase concentration involves the use of the molar extinction coefficient this change in RZ has been studied in the p r e s e n t work. Also, detection of minimal amounts of peroxidase, as a protein, has been d e t e r m i n e d by immunoprecipitation, SDS-PAGE and immunoblots probed by means of the biotin-streptavidin amplification system. T h e s e p r o c e d u r e s may be valuable in future peroxidase assays. Materials and M e t h o d s Materials

Secondary biotylated donkey a n t i r a b b i t antibodies and s t r e p t a v i d i n linked horseradish peroxidase were purchased from A m e r s h a m International. The peroxidase s u b s t r a t e s 2~'azino-di~3-ethylbenzthiazoline sulphonic acid] (ABTS) and 3-amino-9-ethylcarbazole (3AEC) and phenyl m e t h y l sulfonyl fluoride (PMSF) were obtained from Sigma Chemical Company. Nitrocellulose m e m b r a n e s and Tween-20 w e r e

0168-9452tg0t$03.50 ~:" 1990Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

20 bought from Biorad laboratory and polyethylene glycol~aO00 from Prolabo. BSA fraction V was bought from Boehringer Mannheim (Canada Ltd~ and the Centricon concentrators from Amicon. Peanut cells grown in suspension culture were maintained routinely on a gyratory shaker for 14 days at which time the medium was replaced by fresh medium [10]. The spent medium was made to 70% (v/v) acetone and the proteins were precipitated from the resuspended pellet with 80% (w/v) ammonium sulfate precipitation [11]. The subsequent purification of the cationic isozyme was conducted as described previously [6]. Purity of the final protein was assessed by PAGE, and the molar absorption of the heine moiety was measured as before [12]. For weight determinations, an aliquot of peroxidase dissolved in water was filtered in a Centricon 10 micro-concentrator, lyophilized and finally weighed on an analytical Sartorius A 1205 balance. Protein dye-linking assays [6] were conducted using a standard graph obtained using dilutions of a stock BSA solution. Antiserum raised in rabbits against the purified peroxidase was passed over a peroxidaseSepharose column to obtain monospecific antibodies [7]. Then peroxidase at a concentration of 10-0.5 ng, calculated from dye binding [13] was incubated with monospecific antibodies in a final vlume of 6 ~1 for 1 h at ambient temperature; 1 ~1 of secondary antibodies was then added and allowed to react at 4°C overnight. Seven microliters of 20% (w/v) PEG 6000 was then added [14] to enhance precipitation [15]. After 1 h, a 100~1 sucrose (1 M) cushion containing 1% (v/v) Triton X-100 and 1% (w/v) sodium deoxycholate was inserted below the mixture prior to centrifugation at 15 000 × g for 30 rain. The pellet was carefully rinsed with detergent [16]. The rinsed pellet was mixed with 5 ~1 of electrophoresis buffer and 10 ~1 of sample buffer [16] and heated at 90°C for 3 rain. Electrophoresis on a 12% polyacrylamide gel (95 x 50 x 0.75 ram) containing 0.1% (w/v) SDS was conducted at 25 mA and 80 V for 2 h. Subsequent transblotting was done in a solution con

taining 25 mM Tris, 192 mM glycine and 20% (v/v) methanol at 40 mA and 30 V overnight. The nitrocellulose membrane was washed twice in 20 mM Tris (pH 7.5~ containing 150 mM NaCI and 0.1% Tween-20 (TBS-T) for 10 rain and finally in TBS-T for 1 h. The immunoprobe was conducted by exposure of the membrane to either preimmune serum or 1:300 (by volume) monospecific primary antibodies (2 rag" m l 4 in TBS-T at 4 °C overnight followed by 2 washings in TBS-T, each for 10 rain, and then exposure to biotylated secondary antirabbit antibodies (1: 300 dilution in TBS-T) for 1 h and an exposure to streptavidin peroxidase after a 15 min wash in TBS-T. After a final wash of the complex in TBS-T for 15 rain, the immunoblot was developed in 3 AEC [17]. The occurrence of secondary antibody-linked peroxidase on the blot was detected using AEC at 0.02% (w/v~ in 50 mM sodium acetate (p]! 5~ containing 0.03% (VN) H202. For spectral enzymatic assays the substrate ABTS at 0.55 rag. ml -t in 0.1 M potassium phosphate/citrate buffer (pH 4.3) containing 0.02% H202 was employed [17]. Appropriate dilutions of the enzyme in 10 ~1 were added to 1 ml of the substrate solution and these were allowed to incubate at 37 °C. Readings or scans were taken at 420 nm. All data were derived from at least 3 independent assays. Results and Discussion Purity of the protein was determined by PAGE [6] and this was reflected initially in a high RZ value. However, with storage of dilute solutions this value decreased (Table II. The readings for protein (at 280 nm) increased somewhat. This is probably not due to proteolysis since PMSF, the preserver of phytochrome {18], had no effect. Moreover, peroxidase has been proven to be quite resistant to trypsin treatment [19]. Also, the amount of protein determined by staining did not change significantly when samples were subjected to electrophoresis at the beginning and end of the storage period. However, the heme moiety has been known to dissociate from the protein particu-

21 Table I.

Variation in absorbance ratio 405/280 nm (RZ) with storage time and the effect of calcium.

Sample peroxidase' in

Day

7%isopropanol

Absorbance at

RZ

Spec. act. (E.U. min" (mg protein}"}

405

280

1 5 10 19

0.57 0.30 0.26 0.25

0.23 0.28 0.31 0.32

2.5 1.1 0.8 0.8

1,336 771 661 508

7% isopropanol and 0.1 mM CaCl~

1 5 10 19

0.53 0.51 0.48 0.36

0.26 0.27 0.29 0.32

2.0 1.9 1.7 1.1

1333 863 8,~ 629

7% isopropanol and 10raM CaCI 7

1 5 10 19

0.47 0.46 0.45 0.42

0.23 0.23 0~5 0.30

2.0 2.0 1.8 1.4

1143 901 851 787

Lyophilizedb

19

0.55

0.24

2.3

1171

•210 ~g peroxidase in 3 ml 7% isopropanol stored at 6°C. 'Data for day 1 of the experiment are identical (see Fig. 1l

larly in dilute solutions [20]. The question then arises whether the heine alone is stable. The absorbance at 405 nm at the Soret band was seen to decrease more than the rise in absorbance at 280 nm (Table I). If commercial heine in solution was stored for the same time a similar decrease was observed (data not shown). The decrease of the absorbance could be diminished significantly when calcium, particularly at 10 raM, was added to the solution. Whether the calcium has a role in maintaining the heine moiety at the active site [21] was not further investigated. Treatments that tended to prevent the decrease of the RZ were an increase in pH from 5 to 7 and especially an increase in the concentration of the peroxidase in solution. The greatest concentration would occur in the iyophilized protein and values are then stabilized, as may be seen from Table I. What causes the RZ values to change. It is known that major pH changes will affect Soret band absorbance patterns of horseradish peroxidase [8]. A major shift from pH 5 to 10 causes the Soret band to move from 405 to 417 nm in

peanut peroxidase. But no shift in the pH of the storage solution of peanut peroxidase was detected. However, as is shown in Fig. 1 there is a shift of the Soret band of the cationic peroxidase on storage. Consequently, since the 405 nm absorbance occurs now on the shoulder of the new peak, the readings decrease. Oxidation of the heme will also give rise to a shift of the Soret band. The ideal condition for the maintenance of a high RZ was to store the peroxidase at high concentration. Therefore we stored the peroxidase in the lyophilized state. For quantitation of peroxidase, weight measurements can be made since peanut cells release substantial amounts of this peroxidase [6]. To obtain as accurate as possible an estimation, low molecular weight substances associated with the peroxidase were separated by Centricon 10 centrifugation. The drying of aliquots of filtrate and filter residue and the subtraction of the weight of the former from that of the latter gave a close approximation of the weight (Table II). This value agrees closely with that obtained

22

,'/5-

I:= 0~ ,5

--

L.

o

.<

I

I

280

I

350

405

4

s|o

nm

Fig. I. Absorption spectra of a peroxidase sample after 14 days of storage at 6 °C. The open square represents a subsample kept in 7% isopropanol containing 0.I mM CaCI,. The closed circle represents a subsample in 7% isopropanol containing I0 mM CaCIr The closed square represents a lyophilized subsample kept for 14 days and then resolubilized in 7% isopropanol.

using the molar extinction coefficient of the heme and therefore of the overall molecule. The Table il. Evaluation of peroxidase concentration u s i n g three independent approaches, Sample

Dye

Molar

mgprotein

binding*

absorption'

1

0.558

0.738

2 3 4 5 average

0.245 0261 0.610 0.572 0.45

0.612 0.266 o.854 0.645 0.623

Weight' 0.69

0,62 O.38 0.87 0.79 0.67

*Values derived from albumin standard curve and absorb, ance at 595 nm. 'Using absorbance at 405 nm and ( ' ~ , ~ ~[20~ 'Differences in dry weight of retentate and filtrate aliquots following passage through a Centricon I0 micro-

concentrator,

values obtained using the dye-binding technique with a BSA standard graph reached only 70% of those obtained using the extinction coefficient. Therefore a correction factor should be used when the dye-binding technique is used. The b e t t e r approach is to use the molar extinction coefficient for protein measuremerits of concentrated solutions. Finally, the lower limit of detection was determined that could be obtained when peroxidase is immunoprecipitated and detected by means of immunoblotting using secondary antib o d y linked to a biotin-streptavidin amplified enzyme localization technique. As a preliminary step the lowest amount of the antibody needed in the complete immunopreeipitation was determined. In some studies several

23

hundred fold excess antibodies have been used [14~ The data in Fig. 2 shows that an ~fold excess of antibody over antigen sufficed; thereby confirming earlierdata [7]. Dilutions were made from stock peroxidase solution of which the protein content had been determined by dye-binding. To enhance the immunoprecipitation of the primary antibody complex P E G 6000 was added [15].The washed immunoprecipitated pellet was subjected to electrophoresis,blotted and probed with fresh primary antibody followed by secondary. The biotin-linkedantibody was exposed to streptavidin linked to indicator peroxidase. Following washing and incubation with the peroxidase

s u b s t r a t e the p r e s e n c e of the original p e a n u t peroxidase subjected to e l e c t r o p h o r e s i s was examined. T h a t the original peroxidase did not react with the s u b s t r a t e offered at the end of the p r o c e d u r e [22] was d e m o n s t r a t e d by the channels A - C in Fig. 3. T h e s e results also show that p r e i m m u n e s e r u m did not p e r m i t recognition of the m a r k e r peroxidase (A) or the immunoprecipitated p e a n u t peroxidase (B,C). (The p r i m a r y and secondary antibodies used in the immunoprecipitation are visible a f t e r probing). H o w e v e r , when the blotted m a r k e r peroxidase was immunoprobed with specific antibodies t h e r e was recognition as shown (F). Immunoprecipitation without the use of sec-

/e

0.1

/

I m

O

0.01

I 1

5 ndn.

Fig. 2. Peroxidase activity remaining in the incubation mixture following immunoprecipitat/on with various amounts of antibody. Purified peroxidase (7.5 ng} was immunopre~ipitated with none {@I, 15 ng (O}.30 ng {f-l}and 60 ng (ms)of primary antibody. The peroxidase activity remaining in the incubation medium was assayed with ABTS in a scanning speetrophoto meter (3 min for the control and 5 min for all other samples}.

24

A

B

C

D

E

F

G

H

)

I

/ r

Fig. 3. Detection of peroxidase following immunoprecipitation, electrophoresis and immunoblotting. Initially samples A - F contained 6 ng of peroxidase and G and H contained 3 ng. Samples for channels B - E received 300 ng primary antibody: G and H only 100 ng. The sample for channels C and I) were not incubated with secondary antibody for immunoprecipitation and were incubated for 4 h; all others were incubated overnight. Channel H did not receive PEG treatment for immunoprecipitation. Electrophoresic migration proceeded from the top downwards. Channels A - C were immunoblotted with preimmune serum. After electrophoresis the peroxidase streptavidin complex was developed with 3AEC. (The solid line indicates the position of peanut peroxidase on the gel and the broken line the position of the primary antibody).

ondary antibody will occur but with reduced efficiency (D, faint band). A clear indication of the presence of peroxidase at 3 ng can only be obtained with the aid of secondary antibody (3G,H). With 0.5 ng even this amplification was insufficient {data similar to B and C). This is in good agreement with the assays of horseradish peroxidase, where spot tests were use [23]. This detection is nearly a 1000-fold more sensitive then that normally found [24].

When PEG was added to peroxidase, it enhanced the enzymatic activity slightly (Fig. 4}. In enzymatic assays with decreasing amounts of peroxidase it was noted that 1.5 ng elicited an absorbance value that was indistinguishable from the background values. The same amount of peroxidase in the presence of 10% PEG elicited a value of 0.05 A 420 nm. The activity shown was obtained during a 30-rain incubation at 37 °C and is stable for at least 2 h.

25

0.4 e~

o

"

I

/. tO

US

• ********************************* q

................ "

2.5

5.0110

Fig. 4. Measurement of activity of peroxide held at 6°C in the presence (11} and the absence I e ) of 10% PEG overnight and allowed to react subsequently at 37°C for I h.

Acknowledgement

4

This research was supported by a grant from Natural Sciences and Engineering Research Council of Canada. The authors acknowledge the help of A. Hollmann and S. Singh and in particular of T. Joseph in part of this study.

5

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