82
E/rctrophorr\i.\ 1992. 13. 82--86
M . A. Ficldrs
Mary Ann Fieldes Department of Biology, Wilfrid Laurier University, Waterloo, Ontario
An explanation of the achromatic bands produced by peroxidase isozymes in polyacrylamide electrophoresis gels stained for malate dehydrogenase When plant tissue extracts are electrophoresed on polyacrylamide gels 2nd the gels are stained for malate dehydrogenase by the standard NAD-dependent dehydrogenase reaction, terminating in the formation of reduced Nitroblue Tetrazolium (NBT), achromatic bands, in addition to the expected chromatic bands, are observed. The achromatic bands are seen when the staining conditions favor a generalized background staining of the gel and have been shown, in a previous study, to be caused by peroxidase isozymes [l].The present study examined the mechanism by which peroxidase produced the achromatic bands using horseradish peroxidase (HRP). The generalized background staining resulted from the phenazine methosulfate (PMS)-mediated reduction of NBT. This reduction was enhanced by H 2 0 2and suppressed by HRP. Peroxidase apparently catalyzes the peroxidative oxidation of reduced PMS, which suppresses the generalized reduction of NBT in gel regions containing peroxidase isozymes producing the achromatic bands. In contrast, however, H R P also appears to catalyze the peroxidative oxidation of reduced NAD, but this reaction increases the reduction of NBT. The results are discussed in the context of the mechanisms proposed by others for the PMS-mediated reduction of NBT and for the peroxidase-catalyzed NADHdependent formation of H,O,. This peroxidase-catalyzed reaction has been proposed for the plant peroxidases involved in lignification.
1 Introduction In this laboratory, we use polyacrylamide gel electrophoresis to separate the malate dehydrogenase ( M D H ) isozymes in plant tissue extracts and stain the gels using the standard NAD-dependent, MDH-catalyzed conversion of malate to oxaloacetate. The staining procedure involves the malate/ NAD/phenazine methosulfate (PMS) electron transfer reaction (l), which terminates with the reduction of yellow Nitroblue Tetrazolium (NBT) into its purple-blue in soluble formazan (NBTH,), and the M D H isozymes are detected as purple-blue bands.
ir
MDH+ I oxaloacetate
NAD
PMSH,
2
ir
NBT
3
!L NADH 11PMS
+
JLBTH,
(1) However, PMS-NBT mixtures tend to be unstable and considerable background coloration can occur under certain staining conditions, e.g. bright light, alkaline pH, or elevated temperature. When we stain our electrophoresed plant extracts under conditions which favor background staining, achromatic bands are observed in addition to the chromatic M D H bands. By comparing the banding patterns, relative band intensities, and relative mobilities of the peroxidase isozymes in specific tissue extracts with Correspondence: Dr. Mary Ann Fieldes, Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, Canada, N2L 3C5 Abbreviations: HRP, horseradish peroxidase: MDH, malate dehydrogenase; NBT, Nitroblue Tetrazolium; NBTH2, reduced Nitroblue ‘Ietrazolium; PER. peroxidase; PMS, phenarine methosulfate; PMSHZ,reduced phenazine methosulfate
0VCH Verlag~gesells~liaftm h H , D-6Y40 Weinheim,
1992
those of the achromatic bands,we have shown [l]that, with one exception, these achromatic bands are caused by peroxidase (PER; EC 1.11.1.7).The exception is a band detected only in seedlings, which is possibly caused by catalase or by a superoxide dismutase with high specificity. In practical terms, there are two consequences ofthe achromatic bands. First, the banding patterns for the two enzyme systems in some types of tissue extract overlap and, at some gel concentrations, the achromatic bands disrupt the M D H banding pattern. This occurs even when the staining conditions d o not generate background staining and could result in misinterpretation of the M D H banding pattern. Secondly, we sometimes find it useful to obtain data for PER isozymes from the achromatic bands. Our studies involve screening large numbers of tissue extracts from individual plants at different developmental stages and, particularly in the case of leaf and seedling studies, the extract volumes are relatively small. We normally use the guaiacol/H,O, staining procedure [I] to examine PER isozymes. However, if the volumes of the extracts are limited we can stain for M D H , generating a moderately stained background, and obtain relative mobility data for the PER and M D H isozymes, simultaneously, from the achromatic and the chromatic bands. The present study was conducted for two reasons. The first was that there has been considerable discussion of the mechanisms involved in the PMSH,-mediated reduction of NBT under aerobic conditions. The mechanism by which peroxidase suppresses this reduction is, therefore, of general interest.The second was because we are also interested in the physiological roles of the PER isozymes. A number of roles have been proposed for plant peroxidases; e.g. [2-51; however, the precise roles of the isozymic forms remain unclear. Peroxidase-catalyzed reactions are either peroxidations or oxidations [6]. Peroxidation (2) uses H 2 0 ,and oxidation (3) uses 0,. 01 73-0835/92/0102-00-82 $3.50-t.25/0
E/~Ylrol)hOrE.FiF1992,
PER + H,O,
13, 82-86
- + - +
+ 2AH,
PER + 0, + 4AH,
PER
PER
Achromatic bands produced by peroxidase in gels stained for malate dehydrogenase
2H,O
2H,O
+ 2AH'
+ 4AH'
(2)
2 Materials and methods
(31
NAD, PMS and horseradish peroxidase (HRP) were obtained from Sigma (St. Louis, MO). NBT was obtained from Aldrich (Milwauke, WI). The standard assay reagent was the gel staining reagent [l]except that the surfactant was omitted and the PMS concentration was reduced. It contained 0.3 mM NAD,0.25 mM NBT,20 PM PMS, and the substrate (50 mM sodium malate produced by reacting DL-malic acid and Na,CO,) in 0.1 M Tris buffer, pH 7.0. The standard reagent was modified by omitting PMS and/or NAD, and H,O, and HRP were added either alone or together. All reaction mixtures contained 4 mL of standard or modified assay reagent plus 1.0 mL of 0 mM, 5 mM or 10 mM H,O, and 0.1 mL of 0, 1, 10,25,50 or 100% dilutions of an HRP stock solution (0.08% weight/volume HRP, approximately 20 PM). The main experiment examined the effects ofPMS,NAD,H,O,,andHRP onNBTreduction.The components were mixed and the reduction of NBT, at room temperature under low light intensity, was measured by recording A,,, each hour for the first 4 h and again after 14 h. The AS6,data measured two parameters. For reactions in which the reduction of NBT occurred ( i e . , in which the background color developed), the rate of reduction of NBT to NBTH, was constant for at least the first 3 h. However, for some reaction mixtures the reaction rate began to slow down by the fourth hour. By 14 h no further increases in NBTH, were detected in any of the assays. The A,,, reading at 14 h, therefore, measured the total production ofNBTH, when the reaction was allowed to go to completion.The increase in AS6,during the first 3 h measured the initial (maximal) rate of reduction on NBT. The second experiment examined the effects of adding H,O, and HRP after the reaction had gone to completion. Standard and modified assay reagents were mixed at time zero and the reduction of NBTwas allowed to go to completion (14 h). H,O, and HRP were then added and A,,, was recorded each hour for the next 4 h and after an additional 14 h. Only the rates ofNBT reduction are reported for this experiment. All assays were run in duplicate and the data were analyzed by analyses of variance.
Both peroxidation and oxidation involve peroxidase Compounds I and I1 as intermediates and both generate free radicals of the hydrogen donor. The free radicals either interact (4) or enter a free radical chain (5) which uses O,, generating H,O, via superoxide radicals (with some exceptions, notably the oxidation of indole acetic acid [6]).
2AH' AH' .t
-+ A
AH,
+ AH, + 0,
-+ A
(4) AH' I
+ H,O,
83
(5)
It is therefore possible that reactions which appear to be peroxidase-catalyzed oxidations (because they occur without the addition of H202)are, in fact, peroxidations which are initially mediated by traces of H,O, and, thereafter, by the H,O, generated by the free radical chain. In fact, this type of mechanism has been proposed for the plant peroxidase isozymes which are involved in lignification [5].These isozymes catalyze the NADH-dependent formation of H,O, . The reaction is a peroxidation and is accelerated by the addition of H,O,. NADH acts as the hydrogen donor but the initial H,O, for the peroxidation is apparently generated by the auto-oxidation of NADH. The specific objectives of the present study were (i) to determine whether the achromatic bands are produced by a peroxidation or an oxidation reaction and (ii) to determine which of the possible reduced compounds produced during the formation of the background coloration are used as the hydrogen donor in the peroxidase reaction. The possibility that the NADH might be the hydrogen donor was of particular interest. If so, the achromatic bands might be useful for examining the different PER isozymes, and the isozymic forms in different genotypes and tissues, in terms of their potential for involvement in lignification without having to isolate und purify the isozymes first. The effectiveness of peroxidase in suppressing the formation of the background coloration in the presence and absence ofboth H,O, and NAD was examined. At the outset of the experimentation, gels were stained using modifications of the staining reagent and the achromatic bands produced by plant tissue extracts were examined. However, it was found that the results seen on the gels could be simulated by using an assay procedure and by examining the effects of including horseradish peroxidase (HRP) in the reaction mixture.The results reported are from the assay procedure.The emphasis of the study was to examine the effects of HRP on NBT reduction and not the mechanism for NBT reduction per se, which has already been extensively investigated [7-lo]. Nevertheless, in interpreting the results, it was necessary to consider the mechanism which resulted in the generalized reduction of NBT and gave rise to the background coloration. The results appeared to be consistent with a proposed mechanism for this reduction which has been described by others [8-10].This mechanism is discussed later in the context of results obtained here.
3 Results The results from the main experiment are shown in Fig. 1. Figures l a and l b give the amounts of NBTH, produced at the completion of the reaction. Figures l c to If give the rates ofNBT reduction.The graphs for25 and 50% HRP are not shown in Figs. lc-f but fell, as expected, between those for 10 and 100% HRP. The analyses of the data for this experiment demonstrated the following significant effects.
3.1 The effects of NAD and PMS on NBT reduction
In the absence of both H,O, and HRP, PMS was required for NBT reduction and the addition of NAD, at the concentration used here, had no effect on the amount of NBTH, produced (0 mM data, Fig. la) or on the rate of NBT reduction (0 mM, 0% HRP data, Figs. 1c-f).
84
Electrophoresis 1992, 13. 82-86
M. A . Fieldes
3.2 The effects of adding H,O, but not HRP
These effects can be seen in Fig. l a and by comparing the 0% HRP plots in Figs. lc-f. The addition of H,O, induced NBT reduction even when no PMS and NAD were present. Adding either NAD or PMS increased both the amount of NBTH, produced and the rate ofNBTreduction.The effect of NAD was less than that of PMS. The effects of NAD and PMS on the amount of NBTH, produced were additive; however, their effects on the rate of NBT reduction were not additive and were dependent on the concentration of H,O,. At 5 mM H,O,, adding NAD in the presence or absence of PMS did not alter the rates of reduction. At 10 mM H,O,, however, adding NAD increased the rate of reduction in the absence of PMS but decreased the rate of reduction in the presence of PMS.
tration. The effectiveness of HRP in suppressing the amount of NBTH, produced can be seen by comparing Figs. l a and lb. It suppressed the amount produced in the absence of NAD and PMS and it was more effective if PMS was present but less effective if NAD was present. Similar effects of HRP on the rate ofNBT reduction can be seen by comparing the 0 and 1% plots in each of the Figs. lc-lf. HRP was much less effective in suppressing the rate of NBT reduction when NAD was present, which can be seen, in particular, by comparing corresponding plots in Figs. l c and Id. When no PMS was present, the rate of H,O, induced NBT reduction at each HRP concentration increased if NAD was present and these increases were greater than the slight increase seen when no HRP was present. A final point was that the effects of HRP on both the rate of NBT reduction and the amount of NBTH, produced increased, linearly, with increasing H,O, concentration.
3.3 The effects of adding HRP Table 1 summarizes the results obtained from the second experiment. Addition of HRP alone after the reaction had reached completion had no effect. In contrast, addition of H,O, to the reaction mixtures, after the initial reduction of NBT was complete, caused further NBT reduction'and HRP suppressed this additional reduction. Again, the effectiveness of HRP in suppressing the additional reduction was reduced if NAD was present.
In the absence of H,O,, HRP concentrations of 10% or above fully suppressed the PMS-induced reduction of NBT (0 mM data). In the presence of H,O,, increasing the HRP concentration decreased (suppressed) the rate of NBT reduction and the amount of NBTH, produced. However, as can be seen in Fig. lb, the H,O,-induced NBT reduction was not fully suppressed even at the highest HRP concen-
a
Amount of NBTH? produced with n o HRP
-
',"
r .~~~~~, f
A m o u n t of NBTHz p r o d u c e d w i t h 100%HRP 2.0
f
0 N
0)
"
c
$0.5 4
0.0
0
5 Concentt-ation of HzOz (mM)
Rate of NBT reduction
~
10
0
!l.oL 1 .5
0
W 1 0 ~ c ._ 4
10
Rate of NET reduction - w i t h NAD
no NAD o r PMS
1.5-
5 Concentration of HzOz (mM)
[
1
._
4. :o
100
0.0
5 Concentration of HzOz
0
Rate of NBT reduction
(mu)
~
lb
0 10
6 0.5
I 0 0.0
0
5
10 100
Concentration of H202 (mu)
Rate of NBT reduction - NAD a n d PMS
w i t h PMS
0 1
1.5,
.:t
II
I 1 .o
._ 10
6 0.5 Y
100
10 100
0.0
0
5 Concentration of HzOz (mM)
l10 o
Figure 1.Comparative plots ofthe responses to increasing H 2 0 2concentration seen when the assay reagent was modified and when HRP was added to the reaction. (la) and (lb) give the amounts of NBTH, produced at the completion of the reaction for assays containing no NAD or PMS (0),only NAD (N), only PMS (P), and both NAD and PMS (PN) when no HRPwaspresent (a) and when 100VoHRPwas present (b). (lc) to (If) give the rates of NBT reduction when 0, 1, 10 or 100% HRP was added to assays containing no NAD or PMS (c) onlyNAD (d),onlyPMS (e),and bothNAD and PMS (f).The average standard errors were 0.0802 for the means in (la) and ( lb) and 0.0386 for the means (lc) to (If).
Achromatic bands produced by pcroxidase in gels stained for malate dehydrogenase
H e c i r o p h o r e s i . ~1992, 13, 82-86
Table 1. Effects ofadding 10 mM H z 0 2and/or 100% HRPlo reduced NBT after the inilial reaction had reached completion
NoHRP NO H202
Rates of NBT reduction NoHRP HRP NoHz02 H202
HRP H202
Initial assay reagent
0.01 -0.01
PMS and NAD PMS (no NAD)
0.81 0.83
0.05 0.02
0.33 0.17
a) Mean (n = 2) values for the change in ASho(rate of NBT reduction). Based on the differences between duplicates, the average standard error for the means was 0.0378.
4 Discussion The results indicate that the generalized background coloration of the gels results from the oxidative reduction of NBT by PMSH, but that the NAD present in the normal gel staining reagent would have little, if any, effect on the intensity of the background coloration. The photochemical reduction of NBT is known to be enhanced by increasing the concentration of PMS [7] and, as expected, the background coloration of gels increased when either the light intensity or the PMS concentration was increased and also when the substrate (malate) concentration was increased. From the assay results, the reduction of NBT appeared to involve an equilibrium reaction because in most cases it reached completion well before the total available NBT was fully reduced and, after it was complete, the addition of H,O, induced further NBT reduction. There were two points which had to be considered before the effects of peroxidase on the reduction of NBT could be interpreted. First, H,O, increased both the rate and the extent of NBT reduction and, secondly, NAD did have an effect on the reaction if H,O, was also present. In the reaction mechanism, proposed by others [8-lo], for the oxidative reduction of NBT by PMSH,, reaction (6), generates free NB-tetrazolinyl radicals which may then produce reduced NBT via reaction (7). PMSH,
+ 2NBT
2NBTH'
-
-
PMS
+ 2NBTH'
NBT + NBTH,
(6) (7)
Under aerobic conditions and at low NBT concentrations, however, there is also an equilibrium reaction (8) between tetrazolinyl radicals and 0, which generates superoxide radicals and interferes with the production of NBTH,.
+ 0, * NBT + H' + 0;
85
[9, lo]. The equilibrium reaction is the pivotal reaction. It is dependent on the presence of oxygen and displacing it to the right prevents NBT reduction [lo]. Displacing it to the left would increase NBT reduction. These results indicate that the addition of H,O, increased the PMSH,-mediated reduction of NBT. However, it also induced NBT reduction in the absence of both NAD and PMS and this indicated that it was directly affecting the concentration of tetrazolinyl radicals either by preventing the equilibrium reaction, or by shifting it to the left, or by increasing their production so that reaction (7) occurred in addition to reaction (8). In considering these possibilities, a number of other reaction conditions were examined. Under anaerobic conditions, the rate of reduction of NBT increased; this was expected since reaction (8) is prevented [lo], and it was found that H,O, further increased this rate of reduction. That is, the effect of H,O, was independent of the equilibrium reaction. However, when the substrate was omitted from the reaction some reduction of NBT occurred (which was suppressed by HRP) but there was no effect of H,O,. That is, a reaction between H,O, and the substrate or a component of the substrate reduced NBT. It would also, therefore, reduce PMS generating PMSH, for reaction (6) and appears to reduce NAD since NAD only induced NBT reduction when H,O, was added. The increased reduction induced by NAD in the presence of H,O, agrees with the observation [7] that NADH can substitute to some extent for PMSH, in reaction (6). However, ifPMS was also present, NADH would mediate its reduction, providing additional PMSH, for reaction (6). Interpreting the effects of HRP in the context of the basic reaction mechanism, it was clear that peroxidase produced the achromatic bands in gels by using either NBTH, or PMSH, as the hydrogen donor in a reaction which was probably a peroxidation. The fact that peroxidase also used NADH as a hydrogen donor was not, initially, as obvious. In the absence of NAD and PMS, HRP presumably used NBTH, directly as a hydrogen donor. However, it also suppressed the PMSH,-mediated reduction of NBT and PMSH, appeared to be the preferred hydrogen donor. There were a number of indications that both reactions were peroxidations. The effectiveness of H R P in suppressing NBT reduction was enhanced by increasing the concentration of H,O,. HRP had no effect if it was added after the initial reaction was complete unless H,O, was also added. Furthermore, H R P was equally effective in suppressing NBT reduction under both aerobic and anaerobic conditions.
The peroxidase-mediated oxidation of PMSH, would reduce its participation in reactions (6) and (10). Preventing The superoxide radicals may undergo spontaneous dismu- reaction (6) should decrease NBT reduction but preventing tation (9) or may be scavenged by PMSH, (10). In each case reaction (10) should increase NBT reduction by changing the position of the equilibrium reaction. As expected, H,O, is produced. PMSH, clearly participated in reaction (6) in preference to 0; + 0; 2H' H,O, + 0, (9) reaction (10) since the net observed effect was a decrease in NBT reduction. The reverse situation did not, however, exPMSH, 20; + 2H' PMS + 2H,O, (10) plain the fact that HRP was less effective in suppressing NBT reduction when NAD was present. Primarily because, The main point of this proposal is that, under aerobic condi- even if HRP completely prevented NADH from participattions, the superoxide radicals are generated by the equilib- ing in the reduction of NBT, the amount of NBTH, prorium reaction rather than by the auto-oxidation of PMSH, duced should not exceed the amount produced when no NBTH'
+
+
-
-
(8)
86
Elecrrophuresis 1992, 13, 82-86
M A Fielde\
NAD was present. Peroxidase apparently mediated the oxidation of NADH but this oxidation increased the amount of NBTH, produced, and it was clear that the effect of this oxidation on NBT reduction differed from the effect of the peroxidase-mediated oxidation of PMSH,. In fact, the results suggest that the peroxidase-mediated oxidation of NADH generates superoxide radicals which shift the equilibrium reaction to the left, and/or H,O, which causes reduction ofNBT.This is consistent with the mechanism proposed for the peroxidase-catalyzed, NADH-dependent formation ofH,O, in which the NAD radicals produced entera free radical chain [ 5 ] . In contrast, the results indicate that the PMSH radicals produced during the peroxidasemediated oxidation of PMSH, do not enter a free radical chain and this supports the contention that the superoxide radicals produced during the PMSH,-mediated reduction ofNBTare not generated by the oxidation ofPMSH, [9,10]. In conclusion, it should also be possible to use the observation that peroxidase is less effective in suppressing NBT reduction when NAD is present in order to examine the peroxidative oxidation of NADH by the peroxidase isozymes of other plant species directly on electrophoresis gels. In each case, the general protocol would involve comparing the corresponding achromatic bands formed on gels stained in the presence and in the absence ofNAD. However,for each species or isozyme the cofactor requirements should be examined since, unlike HRP, some of the peroxidases [5] which are thought to be involved in lignification require cofactors to mediate the oxidation of NADH.
This study was supported by a grantfrom the NaturalSciences and Engineering Research Council of Canada. The technical assistance provided by Mrs. Sylvia Cantin and the comments and suggestions of a reviewer are acknowledged with thanks. Received April 15, 1991
5 References Fieldes, M.A. and Gray, T.J., J. Espt. 501.1988, 39, 499-509. Sembdner, G., Gross, D., Liebisch, H.-W. and Schneider, G., Encyclopedia of Plant Ph-vsiology, Volume Y ? Hormonal Regulation ofDevelopment I : Aspects o f f l a n t Hormona, Springer Verlaz, New York 1980, pp 281-444 Zeroni, M. and Hall, M.A. ibid. 1980, pp 511-586. Metraux, J.-P., Plnnr Hormones nnd their Roles in Plant Growth and Development, Kluwer Academic Publishers Group, Boston 1987, p p . 297-3 17. Mader,M. and Amberg-Fisher,V., Plant Physiol. 1982,70.1128-1 131. Gaspar, T., Penel, C., Thorpe,T. and Greppin, H., Pernxidases 1970/ Y N U , Universitk de Geneve, Center dc Botanique, Geneve 1982, pp 9-59. Van Noorden, C.J.F. and Tas, J., J. Histochem. Cytochem. 1982, 30, 12-20. Auclair, C., Torres, M. and Hakim, J., FEBS Lett. 1978, 89, 26-28. Picker, S.D. and Fridovich, I., Arch. Biochem. Bioghys. 1984, 228, 155-158. Van Noorden, C.J.F. and Butcher, R.G., Anal. Biochem. 1989, 176, 170-1 74.
E/rctrophorr\i.\ 1992. 13. 82--86
M . A. Ficldrs
Mary Ann Fieldes Department of Biology, Wilfrid Laurier University, Waterloo, Ontario
An explanation of the achromatic bands produced by peroxidase isozymes in polyacrylamide electrophoresis gels stained for malate dehydrogenase When plant tissue extracts are electrophoresed on polyacrylamide gels 2nd the gels are stained for malate dehydrogenase by the standard NAD-dependent dehydrogenase reaction, terminating in the formation of reduced Nitroblue Tetrazolium (NBT), achromatic bands, in addition to the expected chromatic bands, are observed. The achromatic bands are seen when the staining conditions favor a generalized background staining of the gel and have been shown, in a previous study, to be caused by peroxidase isozymes [l].The present study examined the mechanism by which peroxidase produced the achromatic bands using horseradish peroxidase (HRP). The generalized background staining resulted from the phenazine methosulfate (PMS)-mediated reduction of NBT. This reduction was enhanced by H 2 0 2and suppressed by HRP. Peroxidase apparently catalyzes the peroxidative oxidation of reduced PMS, which suppresses the generalized reduction of NBT in gel regions containing peroxidase isozymes producing the achromatic bands. In contrast, however, H R P also appears to catalyze the peroxidative oxidation of reduced NAD, but this reaction increases the reduction of NBT. The results are discussed in the context of the mechanisms proposed by others for the PMS-mediated reduction of NBT and for the peroxidase-catalyzed NADHdependent formation of H,O,. This peroxidase-catalyzed reaction has been proposed for the plant peroxidases involved in lignification.
1 Introduction In this laboratory, we use polyacrylamide gel electrophoresis to separate the malate dehydrogenase ( M D H ) isozymes in plant tissue extracts and stain the gels using the standard NAD-dependent, MDH-catalyzed conversion of malate to oxaloacetate. The staining procedure involves the malate/ NAD/phenazine methosulfate (PMS) electron transfer reaction (l), which terminates with the reduction of yellow Nitroblue Tetrazolium (NBT) into its purple-blue in soluble formazan (NBTH,), and the M D H isozymes are detected as purple-blue bands.
ir
MDH+ I oxaloacetate
NAD
PMSH,
2
ir
NBT
3
!L NADH 11PMS
+
JLBTH,
(1) However, PMS-NBT mixtures tend to be unstable and considerable background coloration can occur under certain staining conditions, e.g. bright light, alkaline pH, or elevated temperature. When we stain our electrophoresed plant extracts under conditions which favor background staining, achromatic bands are observed in addition to the chromatic M D H bands. By comparing the banding patterns, relative band intensities, and relative mobilities of the peroxidase isozymes in specific tissue extracts with Correspondence: Dr. Mary Ann Fieldes, Department of Biology, Wilfrid Laurier University, Waterloo, Ontario, Canada, N2L 3C5 Abbreviations: HRP, horseradish peroxidase: MDH, malate dehydrogenase; NBT, Nitroblue Tetrazolium; NBTH2, reduced Nitroblue ‘Ietrazolium; PER. peroxidase; PMS, phenarine methosulfate; PMSHZ,reduced phenazine methosulfate
0VCH Verlag~gesells~liaftm h H , D-6Y40 Weinheim,
1992
those of the achromatic bands,we have shown [l]that, with one exception, these achromatic bands are caused by peroxidase (PER; EC 1.11.1.7).The exception is a band detected only in seedlings, which is possibly caused by catalase or by a superoxide dismutase with high specificity. In practical terms, there are two consequences ofthe achromatic bands. First, the banding patterns for the two enzyme systems in some types of tissue extract overlap and, at some gel concentrations, the achromatic bands disrupt the M D H banding pattern. This occurs even when the staining conditions d o not generate background staining and could result in misinterpretation of the M D H banding pattern. Secondly, we sometimes find it useful to obtain data for PER isozymes from the achromatic bands. Our studies involve screening large numbers of tissue extracts from individual plants at different developmental stages and, particularly in the case of leaf and seedling studies, the extract volumes are relatively small. We normally use the guaiacol/H,O, staining procedure [I] to examine PER isozymes. However, if the volumes of the extracts are limited we can stain for M D H , generating a moderately stained background, and obtain relative mobility data for the PER and M D H isozymes, simultaneously, from the achromatic and the chromatic bands. The present study was conducted for two reasons. The first was that there has been considerable discussion of the mechanisms involved in the PMSH,-mediated reduction of NBT under aerobic conditions. The mechanism by which peroxidase suppresses this reduction is, therefore, of general interest.The second was because we are also interested in the physiological roles of the PER isozymes. A number of roles have been proposed for plant peroxidases; e.g. [2-51; however, the precise roles of the isozymic forms remain unclear. Peroxidase-catalyzed reactions are either peroxidations or oxidations [6]. Peroxidation (2) uses H 2 0 ,and oxidation (3) uses 0,. 01 73-0835/92/0102-00-82 $3.50-t.25/0
E/~Ylrol)hOrE.FiF1992,
PER + H,O,
13, 82-86
- + - +
+ 2AH,
PER + 0, + 4AH,
PER
PER
Achromatic bands produced by peroxidase in gels stained for malate dehydrogenase
2H,O
2H,O
+ 2AH'
+ 4AH'
(2)
2 Materials and methods
(31
NAD, PMS and horseradish peroxidase (HRP) were obtained from Sigma (St. Louis, MO). NBT was obtained from Aldrich (Milwauke, WI). The standard assay reagent was the gel staining reagent [l]except that the surfactant was omitted and the PMS concentration was reduced. It contained 0.3 mM NAD,0.25 mM NBT,20 PM PMS, and the substrate (50 mM sodium malate produced by reacting DL-malic acid and Na,CO,) in 0.1 M Tris buffer, pH 7.0. The standard reagent was modified by omitting PMS and/or NAD, and H,O, and HRP were added either alone or together. All reaction mixtures contained 4 mL of standard or modified assay reagent plus 1.0 mL of 0 mM, 5 mM or 10 mM H,O, and 0.1 mL of 0, 1, 10,25,50 or 100% dilutions of an HRP stock solution (0.08% weight/volume HRP, approximately 20 PM). The main experiment examined the effects ofPMS,NAD,H,O,,andHRP onNBTreduction.The components were mixed and the reduction of NBT, at room temperature under low light intensity, was measured by recording A,,, each hour for the first 4 h and again after 14 h. The AS6,data measured two parameters. For reactions in which the reduction of NBT occurred ( i e . , in which the background color developed), the rate of reduction of NBT to NBTH, was constant for at least the first 3 h. However, for some reaction mixtures the reaction rate began to slow down by the fourth hour. By 14 h no further increases in NBTH, were detected in any of the assays. The A,,, reading at 14 h, therefore, measured the total production ofNBTH, when the reaction was allowed to go to completion.The increase in AS6,during the first 3 h measured the initial (maximal) rate of reduction on NBT. The second experiment examined the effects of adding H,O, and HRP after the reaction had gone to completion. Standard and modified assay reagents were mixed at time zero and the reduction of NBTwas allowed to go to completion (14 h). H,O, and HRP were then added and A,,, was recorded each hour for the next 4 h and after an additional 14 h. Only the rates ofNBT reduction are reported for this experiment. All assays were run in duplicate and the data were analyzed by analyses of variance.
Both peroxidation and oxidation involve peroxidase Compounds I and I1 as intermediates and both generate free radicals of the hydrogen donor. The free radicals either interact (4) or enter a free radical chain (5) which uses O,, generating H,O, via superoxide radicals (with some exceptions, notably the oxidation of indole acetic acid [6]).
2AH' AH' .t
-+ A
AH,
+ AH, + 0,
-+ A
(4) AH' I
+ H,O,
83
(5)
It is therefore possible that reactions which appear to be peroxidase-catalyzed oxidations (because they occur without the addition of H202)are, in fact, peroxidations which are initially mediated by traces of H,O, and, thereafter, by the H,O, generated by the free radical chain. In fact, this type of mechanism has been proposed for the plant peroxidase isozymes which are involved in lignification [5].These isozymes catalyze the NADH-dependent formation of H,O, . The reaction is a peroxidation and is accelerated by the addition of H,O,. NADH acts as the hydrogen donor but the initial H,O, for the peroxidation is apparently generated by the auto-oxidation of NADH. The specific objectives of the present study were (i) to determine whether the achromatic bands are produced by a peroxidation or an oxidation reaction and (ii) to determine which of the possible reduced compounds produced during the formation of the background coloration are used as the hydrogen donor in the peroxidase reaction. The possibility that the NADH might be the hydrogen donor was of particular interest. If so, the achromatic bands might be useful for examining the different PER isozymes, and the isozymic forms in different genotypes and tissues, in terms of their potential for involvement in lignification without having to isolate und purify the isozymes first. The effectiveness of peroxidase in suppressing the formation of the background coloration in the presence and absence ofboth H,O, and NAD was examined. At the outset of the experimentation, gels were stained using modifications of the staining reagent and the achromatic bands produced by plant tissue extracts were examined. However, it was found that the results seen on the gels could be simulated by using an assay procedure and by examining the effects of including horseradish peroxidase (HRP) in the reaction mixture.The results reported are from the assay procedure.The emphasis of the study was to examine the effects of HRP on NBT reduction and not the mechanism for NBT reduction per se, which has already been extensively investigated [7-lo]. Nevertheless, in interpreting the results, it was necessary to consider the mechanism which resulted in the generalized reduction of NBT and gave rise to the background coloration. The results appeared to be consistent with a proposed mechanism for this reduction which has been described by others [8-10].This mechanism is discussed later in the context of results obtained here.
3 Results The results from the main experiment are shown in Fig. 1. Figures l a and l b give the amounts of NBTH, produced at the completion of the reaction. Figures l c to If give the rates ofNBT reduction.The graphs for25 and 50% HRP are not shown in Figs. lc-f but fell, as expected, between those for 10 and 100% HRP. The analyses of the data for this experiment demonstrated the following significant effects.
3.1 The effects of NAD and PMS on NBT reduction
In the absence of both H,O, and HRP, PMS was required for NBT reduction and the addition of NAD, at the concentration used here, had no effect on the amount of NBTH, produced (0 mM data, Fig. la) or on the rate of NBT reduction (0 mM, 0% HRP data, Figs. 1c-f).
84
Electrophoresis 1992, 13. 82-86
M. A . Fieldes
3.2 The effects of adding H,O, but not HRP
These effects can be seen in Fig. l a and by comparing the 0% HRP plots in Figs. lc-f. The addition of H,O, induced NBT reduction even when no PMS and NAD were present. Adding either NAD or PMS increased both the amount of NBTH, produced and the rate ofNBTreduction.The effect of NAD was less than that of PMS. The effects of NAD and PMS on the amount of NBTH, produced were additive; however, their effects on the rate of NBT reduction were not additive and were dependent on the concentration of H,O,. At 5 mM H,O,, adding NAD in the presence or absence of PMS did not alter the rates of reduction. At 10 mM H,O,, however, adding NAD increased the rate of reduction in the absence of PMS but decreased the rate of reduction in the presence of PMS.
tration. The effectiveness of HRP in suppressing the amount of NBTH, produced can be seen by comparing Figs. l a and lb. It suppressed the amount produced in the absence of NAD and PMS and it was more effective if PMS was present but less effective if NAD was present. Similar effects of HRP on the rate ofNBT reduction can be seen by comparing the 0 and 1% plots in each of the Figs. lc-lf. HRP was much less effective in suppressing the rate of NBT reduction when NAD was present, which can be seen, in particular, by comparing corresponding plots in Figs. l c and Id. When no PMS was present, the rate of H,O, induced NBT reduction at each HRP concentration increased if NAD was present and these increases were greater than the slight increase seen when no HRP was present. A final point was that the effects of HRP on both the rate of NBT reduction and the amount of NBTH, produced increased, linearly, with increasing H,O, concentration.
3.3 The effects of adding HRP Table 1 summarizes the results obtained from the second experiment. Addition of HRP alone after the reaction had reached completion had no effect. In contrast, addition of H,O, to the reaction mixtures, after the initial reduction of NBT was complete, caused further NBT reduction'and HRP suppressed this additional reduction. Again, the effectiveness of HRP in suppressing the additional reduction was reduced if NAD was present.
In the absence of H,O,, HRP concentrations of 10% or above fully suppressed the PMS-induced reduction of NBT (0 mM data). In the presence of H,O,, increasing the HRP concentration decreased (suppressed) the rate of NBT reduction and the amount of NBTH, produced. However, as can be seen in Fig. lb, the H,O,-induced NBT reduction was not fully suppressed even at the highest HRP concen-
a
Amount of NBTH? produced with n o HRP
-
',"
r .~~~~~, f
A m o u n t of NBTHz p r o d u c e d w i t h 100%HRP 2.0
f
0 N
0)
"
c
$0.5 4
0.0
0
5 Concentt-ation of HzOz (mM)
Rate of NBT reduction
~
10
0
!l.oL 1 .5
0
W 1 0 ~ c ._ 4
10
Rate of NET reduction - w i t h NAD
no NAD o r PMS
1.5-
5 Concentration of HzOz (mM)
[
1
._
4. :o
100
0.0
5 Concentration of HzOz
0
Rate of NBT reduction
(mu)
~
lb
0 10
6 0.5
I 0 0.0
0
5
10 100
Concentration of H202 (mu)
Rate of NBT reduction - NAD a n d PMS
w i t h PMS
0 1
1.5,
.:t
II
I 1 .o
._ 10
6 0.5 Y
100
10 100
0.0
0
5 Concentration of HzOz (mM)
l10 o
Figure 1.Comparative plots ofthe responses to increasing H 2 0 2concentration seen when the assay reagent was modified and when HRP was added to the reaction. (la) and (lb) give the amounts of NBTH, produced at the completion of the reaction for assays containing no NAD or PMS (0),only NAD (N), only PMS (P), and both NAD and PMS (PN) when no HRPwaspresent (a) and when 100VoHRPwas present (b). (lc) to (If) give the rates of NBT reduction when 0, 1, 10 or 100% HRP was added to assays containing no NAD or PMS (c) onlyNAD (d),onlyPMS (e),and bothNAD and PMS (f).The average standard errors were 0.0802 for the means in (la) and ( lb) and 0.0386 for the means (lc) to (If).
Achromatic bands produced by pcroxidase in gels stained for malate dehydrogenase
H e c i r o p h o r e s i . ~1992, 13, 82-86
Table 1. Effects ofadding 10 mM H z 0 2and/or 100% HRPlo reduced NBT after the inilial reaction had reached completion
NoHRP NO H202
Rates of NBT reduction NoHRP HRP NoHz02 H202
HRP H202
Initial assay reagent
0.01 -0.01
PMS and NAD PMS (no NAD)
0.81 0.83
0.05 0.02
0.33 0.17
a) Mean (n = 2) values for the change in ASho(rate of NBT reduction). Based on the differences between duplicates, the average standard error for the means was 0.0378.
4 Discussion The results indicate that the generalized background coloration of the gels results from the oxidative reduction of NBT by PMSH, but that the NAD present in the normal gel staining reagent would have little, if any, effect on the intensity of the background coloration. The photochemical reduction of NBT is known to be enhanced by increasing the concentration of PMS [7] and, as expected, the background coloration of gels increased when either the light intensity or the PMS concentration was increased and also when the substrate (malate) concentration was increased. From the assay results, the reduction of NBT appeared to involve an equilibrium reaction because in most cases it reached completion well before the total available NBT was fully reduced and, after it was complete, the addition of H,O, induced further NBT reduction. There were two points which had to be considered before the effects of peroxidase on the reduction of NBT could be interpreted. First, H,O, increased both the rate and the extent of NBT reduction and, secondly, NAD did have an effect on the reaction if H,O, was also present. In the reaction mechanism, proposed by others [8-lo], for the oxidative reduction of NBT by PMSH,, reaction (6), generates free NB-tetrazolinyl radicals which may then produce reduced NBT via reaction (7). PMSH,
+ 2NBT
2NBTH'
-
-
PMS
+ 2NBTH'
NBT + NBTH,
(6) (7)
Under aerobic conditions and at low NBT concentrations, however, there is also an equilibrium reaction (8) between tetrazolinyl radicals and 0, which generates superoxide radicals and interferes with the production of NBTH,.
+ 0, * NBT + H' + 0;
85
[9, lo]. The equilibrium reaction is the pivotal reaction. It is dependent on the presence of oxygen and displacing it to the right prevents NBT reduction [lo]. Displacing it to the left would increase NBT reduction. These results indicate that the addition of H,O, increased the PMSH,-mediated reduction of NBT. However, it also induced NBT reduction in the absence of both NAD and PMS and this indicated that it was directly affecting the concentration of tetrazolinyl radicals either by preventing the equilibrium reaction, or by shifting it to the left, or by increasing their production so that reaction (7) occurred in addition to reaction (8). In considering these possibilities, a number of other reaction conditions were examined. Under anaerobic conditions, the rate of reduction of NBT increased; this was expected since reaction (8) is prevented [lo], and it was found that H,O, further increased this rate of reduction. That is, the effect of H,O, was independent of the equilibrium reaction. However, when the substrate was omitted from the reaction some reduction of NBT occurred (which was suppressed by HRP) but there was no effect of H,O,. That is, a reaction between H,O, and the substrate or a component of the substrate reduced NBT. It would also, therefore, reduce PMS generating PMSH, for reaction (6) and appears to reduce NAD since NAD only induced NBT reduction when H,O, was added. The increased reduction induced by NAD in the presence of H,O, agrees with the observation [7] that NADH can substitute to some extent for PMSH, in reaction (6). However, ifPMS was also present, NADH would mediate its reduction, providing additional PMSH, for reaction (6). Interpreting the effects of HRP in the context of the basic reaction mechanism, it was clear that peroxidase produced the achromatic bands in gels by using either NBTH, or PMSH, as the hydrogen donor in a reaction which was probably a peroxidation. The fact that peroxidase also used NADH as a hydrogen donor was not, initially, as obvious. In the absence of NAD and PMS, HRP presumably used NBTH, directly as a hydrogen donor. However, it also suppressed the PMSH,-mediated reduction of NBT and PMSH, appeared to be the preferred hydrogen donor. There were a number of indications that both reactions were peroxidations. The effectiveness of H R P in suppressing NBT reduction was enhanced by increasing the concentration of H,O,. HRP had no effect if it was added after the initial reaction was complete unless H,O, was also added. Furthermore, H R P was equally effective in suppressing NBT reduction under both aerobic and anaerobic conditions.
The peroxidase-mediated oxidation of PMSH, would reduce its participation in reactions (6) and (10). Preventing The superoxide radicals may undergo spontaneous dismu- reaction (6) should decrease NBT reduction but preventing tation (9) or may be scavenged by PMSH, (10). In each case reaction (10) should increase NBT reduction by changing the position of the equilibrium reaction. As expected, H,O, is produced. PMSH, clearly participated in reaction (6) in preference to 0; + 0; 2H' H,O, + 0, (9) reaction (10) since the net observed effect was a decrease in NBT reduction. The reverse situation did not, however, exPMSH, 20; + 2H' PMS + 2H,O, (10) plain the fact that HRP was less effective in suppressing NBT reduction when NAD was present. Primarily because, The main point of this proposal is that, under aerobic condi- even if HRP completely prevented NADH from participattions, the superoxide radicals are generated by the equilib- ing in the reduction of NBT, the amount of NBTH, prorium reaction rather than by the auto-oxidation of PMSH, duced should not exceed the amount produced when no NBTH'
+
+
-
-
(8)
86
Elecrrophuresis 1992, 13, 82-86
M A Fielde\
NAD was present. Peroxidase apparently mediated the oxidation of NADH but this oxidation increased the amount of NBTH, produced, and it was clear that the effect of this oxidation on NBT reduction differed from the effect of the peroxidase-mediated oxidation of PMSH,. In fact, the results suggest that the peroxidase-mediated oxidation of NADH generates superoxide radicals which shift the equilibrium reaction to the left, and/or H,O, which causes reduction ofNBT.This is consistent with the mechanism proposed for the peroxidase-catalyzed, NADH-dependent formation ofH,O, in which the NAD radicals produced entera free radical chain [ 5 ] . In contrast, the results indicate that the PMSH radicals produced during the peroxidasemediated oxidation of PMSH, do not enter a free radical chain and this supports the contention that the superoxide radicals produced during the PMSH,-mediated reduction ofNBTare not generated by the oxidation ofPMSH, [9,10]. In conclusion, it should also be possible to use the observation that peroxidase is less effective in suppressing NBT reduction when NAD is present in order to examine the peroxidative oxidation of NADH by the peroxidase isozymes of other plant species directly on electrophoresis gels. In each case, the general protocol would involve comparing the corresponding achromatic bands formed on gels stained in the presence and in the absence ofNAD. However,for each species or isozyme the cofactor requirements should be examined since, unlike HRP, some of the peroxidases [5] which are thought to be involved in lignification require cofactors to mediate the oxidation of NADH.
This study was supported by a grantfrom the NaturalSciences and Engineering Research Council of Canada. The technical assistance provided by Mrs. Sylvia Cantin and the comments and suggestions of a reviewer are acknowledged with thanks. Received April 15, 1991
5 References Fieldes, M.A. and Gray, T.J., J. Espt. 501.1988, 39, 499-509. Sembdner, G., Gross, D., Liebisch, H.-W. and Schneider, G., Encyclopedia of Plant Ph-vsiology, Volume Y ? Hormonal Regulation ofDevelopment I : Aspects o f f l a n t Hormona, Springer Verlaz, New York 1980, pp 281-444 Zeroni, M. and Hall, M.A. ibid. 1980, pp 511-586. Metraux, J.-P., Plnnr Hormones nnd their Roles in Plant Growth and Development, Kluwer Academic Publishers Group, Boston 1987, p p . 297-3 17. Mader,M. and Amberg-Fisher,V., Plant Physiol. 1982,70.1128-1 131. Gaspar, T., Penel, C., Thorpe,T. and Greppin, H., Pernxidases 1970/ Y N U , Universitk de Geneve, Center dc Botanique, Geneve 1982, pp 9-59. Van Noorden, C.J.F. and Tas, J., J. Histochem. Cytochem. 1982, 30, 12-20. Auclair, C., Torres, M. and Hakim, J., FEBS Lett. 1978, 89, 26-28. Picker, S.D. and Fridovich, I., Arch. Biochem. Bioghys. 1984, 228, 155-158. Van Noorden, C.J.F. and Butcher, R.G., Anal. Biochem. 1989, 176, 170-1 74.
