Dye-sensitized photoinactivation of the lactic dehydrogenase and acetylcholinesterase from the boll weevil, Anthonomous grandis

PESTICII)~:

BIOCHEMISTHY

AND

PHYSIOLOGY

7, 21-27

(1977)

Dye-Sensitized Photoinactivation and Acetylcholinesterase Anthonomous MICHAEL

Departtnent

of the Lactic from the Boll grandis l

Dehydrogenase Weevil,

F. CALLAHAZI,~ COLEVAN 0. PALMERTREE,~ JOE R. BROOME,~ AND JAMES R. HEITZ

of Biochemistry, Forestry Experiment Received

Mississippi Station,

December

State University, Mississippi State, 8, 1975;

accepted

Mississippi Mississippi

March

Agricultural 39762

and

31, 1976

The lactic dehydrogenase and acetylcholinesterase enzymes of the boll weevil, Anthonomous grads, have been shown to be inactivated by dye-sensitized photooxidation mediated by subst*ituted xanthenes. The efficiency of the photooxidation reaction was correlated with the degree of halogenation of the dye molecule, the efficiency of the dye in singlet oxygen formation, and the strength of binding to lactic dehydrogenase. Changes in the in viva levels of these enzymes due to ingestion of rose bengal by adult weevils are not further modified in the presence of light.

at the subcellular level by investigating the inactivation of acetylcholinesterase (AChE) Insects have recently been shown to be (5, 6). To date, however, the target(s) in clxtrcmcly susceptible to dye-sensitized the cell specifically responsible for the photooxidation. Mortality has been dedeath of the insect has not been idcntificd. scribed for the house fly, Afusca domestica This report deals with t’he effects of (I, 2), the imported fire ant, XoZerlopsG dye-sensitized photooxidation on the lactic &hteri (3), and the boll weevil, Anthonodehydrogenase (LDH) and AChE of the mous yranclis (4). Attempts have been boll weevil. These enzymes were chosen made to explain the mechanism of action because of the Manic paralysis associated with mortality (3). Both in vitro and irz vivo 1 This work was supported in part by funds from Mississippi State University made available through experiments were performed to determine Dr. Lewis It. Brown, Associate Dean of t.he College the involvcmcnt of inactivation of these of Arts and Sciences, and from the Mississippi two enzymes as a causative agent in Agricultural and Forestry Experiment Station. The mortality due to dye-sensitized photoallthors also thank Dr. James Frazier, Dr. Howard oxidation. Chambers, and Dr. Thomas Kellogg for critically INTRODUCTION

reviewing No. 3154. 2 Present Laboratory, Tennessee 3 Present Tiniversity, 4 Present hIemphis 38152.

the

manuscript.

MAFES

Publication

MATERIALS

address: Comparative Animal liesearch University of Tennessee, Oak Itidge, 37830. address: School of Dent,ist,ry, Emory Atjlanta, Georgia 30322. address : Department of Biology, State University, Memphis, Tennessee

AND METHODS

Adult boll weevils used in this st’udy were products of the Robert T. Gast Rearing Facility of Mississippi State University. In vivo effects of rose bcngal were studied on newly emerged adult insects fed 5 X 1OP N dye-impregnated artificial diet in the dark 21

CoyyrighL All rights

0 1077 by Academic l’rcsu, of repro&ct.ion in any form

Inc. reserved.

Is&m

ow-3575

100

dChE \\-a~ measurfid as prccviously roported (6). Boll weevil homogc!natc~s \v(lre shown t,o contain up t.o 30°j, nonspecific &erase by eserine sulfate> inhibition. These enzymes which are capable of hydrolyzing 50 acetylthiocholine also seem to bo ~usc~cpt,iblc to dye-sensitized photooxidation. LDH was assayed on a Gilford Model 3400 E rapid sampling spectrophotomct.rr. R(l:tc*tion mixtures contained 5..5 X lo-* iI/ pyruvntc,, 1.7 X 10d4 M KADH, and 4.G X lo-” dII TIME (MINI phosphate buffer, pH 7.5, in :t total volume FIG. 1. Dctermin.ation of first-or&w rate conslank of 1.4 ml. Reactions were initiatcbd upon of inactivation jar LDH. Data arc plottcrl as log addit,ion of a O.l-ml aliyuot. of homogenuto prrcent activity as a junction 0s time of illumination for the following concentrations of rose bengal: ()o, and werp fo1lowc.d by ahsorbancac, d(bcdrcnse non,e; (A), 2.39 X IF M; (0), 3.49 X 1P M; at S40 nm. ( l ), 4.7% x lo-7 M; (CT), 5.&’ x 1~3-7 M. /,/‘ght ItL vitro determit~atiot2s. \~‘hok body intensity was 14,500 pW/cm2. homogenates of untreated 1~111 lvctlvils for 4 days postcmergence (3). In. UZ?IYI (30-50 inscl&,/lO ml buffer) lv(:r(’ prcpart>d as doscaribfsd ubovc and frozrn until nccdt>d. effects of rose bcngal and the other xanthcne In studying the time-d(lpmdcnt, inhibition dyes were studied on homogenates of insect,s of AChE by dye-sc>nsitizcd photooxidation, fed only the artificial diet for 4 days Pyrex incubation tuhrs containing 4 rng of postemergence. protein, varying concentrations of dycb> In vivo determinations. Follo\z-ing 4 days and buff(>r to a final volume of 5.0 ml, of feeding on the rose bengal-impregnated w(‘rcb placed in a w&r bath at 30°C and artificial diei, weevils wpre divided into illuminated with 14,500 pW/cm? light from two groups: those fed dye but not exposed a Sylvania Sun Gun. A 5-min prcincubation t’o t’he fluorescent lights, and those put into of the homogenate protein at 30°C was used petri dishes (20 insects each) and exposed to 3800 pW/cm2 fluorescent light’. Control prior to addition of dye and buffer. D~rc addition served to initiak the rc~nction, and insects, fed only the artificial diet, were after various times of incubation, the treated identically. As t,he symptoms of the remaining AChE activity wna mc:lsurod lethal light-catalyzed reaction occurr(Ad, (0). In st’udying the time-dcpcndont inhibiaffected individuals were combined into tion of J,DH, the same proccldurc \I:IS samples of 10 insects each. Control insects, followed cxc*ept that 2.5 mg of protcki both light-exposed and non-light-exposed, wd in :L ;S.O-ml t(~t31 voiumt~. and rose bcngal treakd non-light,-expos(,d boll lvecvils wcrc’ cbombincd into samples at, At t,imed intervals, 0.2 ml of the inc~ubation mixture \V:IS removed and add(bd to st:tnthe same time, as there were no dclctcrious symptoms in those populations. Tissue dard assay mixtures containing 1.6 X lo-” preparations were made with a motorill NADH, 5.2 X 10M4 d1 pJ-ruv:ctcl, :Lnd driven all-glass homogenizer in t,hc dark 4.3 X lo-’ :1/ phosphate bufftlr, pH 7.5, in :I I.+ml total volume. J,DH activity was using a buffer of 0.2 M sodium phosphatedetermined from the decrease in 340 nm 0.25 M sucrose at pH 7.2. Homogenatcxs containing the whole insects and G ml of absorbance on ‘a Beckman Actn V ,sp(~~~trobuffer were centrifuged at 50009 for 15 photometer. For both AChE and J,DH, t,hc rnzymr activit’y at time> zero inruhation min at 4°C to remove crll debris. Prot,cin conc:c~ritratiorIs \serc &~t~~rmin(~d by the s;cyrvcd :ts the! b:& for lOOa/l, :Ic*tiYit y :rt, method of Lowry (7). each dye concentration. The timr:-ind(~l)c~n-

PHOTOINACTIVATION

OF

dent inhibition of LDH in the absence of illumination was studied according to the methods of Lineweaver and Burk (8), and Dixon (9). Assay mixtures contained 1.8 X 1O-4 M NADH, varying concentrations of pyruvate, and dye, in a 3.0-ml total volume of 4.3 X lo-* M phosphate buffer, pH 7.5. Addition of 1.7 mg of protein homogenate initiat’ed the reaction which was followed by decrease in 340 nm absorbance in a Beckman Acta V spectrophotometer. RESULTS

Both the AChE and the LDH from the boll weevil were shown to be susceptible in vitro to dye-sensitized photooxidation. Figure 1 shows the decrease in LDH activity as a function of time of illumination of the enzyme with various concentrations of rose bengal. As the dye concentration is increased, the inhibition reaction proceeds more rapidly. Less than five percent variation was recorded throughout the dye range for the time zero illumination indicat’ing the lack of a time independent inhibition at those dye concentrations. The linear response is indicative of a first-order dependency on the enzyme concentration. A first-order rate constant of inactivation (k,) may be determined for each line by the following equation : ICI = In 21th

x-

I

0

/

2

I

4

6

ROSE BENGAL (x 16~ M) FIG. 2. Determination of second order rate constant o.f LDH inactivation by rose bengal. Values of kl ure plotted aga.inst rose bnngal cotwvhution. !l’he slope of the line is equal to k,.

BOLL

WEEVIL

23

EMYMES

TABLE Second-Order

1

Rate Constant for Xanthene

Dye

(kt) of Znactivation Dyes

kz (litjers/mole Boll

weevil

min) Fire

ant.”

-LI>TI

AChE

AChE -

Rose Bengal Phloxin B Erythrosin B Eosin Yellowish Fluorescein Rhodamine B o Uat#a taken

42,500 10,810 25,650 5,880 0 0 from

56,800 15,100 8,300 7,050 39 0

208,000 33,000 28,000 4,050 320 0

(6).

where t; is the time required for SOY0 inhibition. The contribution of the dye concentration to the rate equation for enzyme inhibition can be determined as shown in Fig. 2. The value for kl is plotted as a function of the dye concentration used to generate that value. The slope of the resulting line is k2, the second-order rate constant of inactivation, and is used in the following equation : v = k, (dye)

(enzyme).

In this equation, v is the velocity of the inhibit’ion reaction. Values for k, were determined for six xanthene dyes against both I,DH and AChE and are reported in Table 1. Also recorded in Table 1 for comparison purposcls are li, values for the AChE from the imported fire ant (6). With the exception of the erythrosin B-sensitized inactivation of boll weevil LDH, there is a decrease in k2 as one proceeds down the table. For a given dye, the k2 values were generally higher for the fire ant AChE than for the boll weevil AChE. Boll weevil LDH exhibited the lowest k, values of the enzymes tested. No k2 was calculated for the fluorescein-sensitized inactivation of boll weevil LDH due to a Iack of inhibition at concent,rations as high as 1.22 X 10e3 M over a 30-min incubation period. Lil;e\vise,

I/PYRUVIC

ACID

CM-’ x lCj3)

FIG. 3. Linwwvrr-Burk plot of rose bcngal inhibition of LDH. I’!ymvic acid concentrations were varied from 6.67 X 1P to .?.OO X iO-4 M. The control line (0) contained no rose bengal and the inhibitrd line ()o contained 4.47 X 10-S M TOW bengal. Reactions wm’ initiated by addition sf 1.7 mg of prokin homogenate.

rhodaminc B cauwd no inact,ivation uf boll weevil LDH, at 1.02 X 1O-4 A[, or AChE, at 1.61 )( 1OF 171,over a 30-min incubation pc+od. Viguro 3 she\\-s a I,incirvcavcr-Burk plot of t)hc rose bcngal inhibition of the I,DH from the boll wwil. A true dissociation const’ant in the form of an inhibition constant, h’r, of 3.33 x 10-S nr, \vas calculated for t,his interaction. In a comparison study, a Dixon plot was gerwratcd which yielded a h’l of 3.X0 X 10-j nl for the rose brngal inhibition of boll w\-cwil I,DH (E’ig. 4). Table 2 shows the lir valuc~s calculat~cd for t’hc dyw uwd in t,his study jvith boll \\wwil I,DH. The affinity of t’ho dycls for 1,DH roughly parallels tho ~fficic,ncy of photo-oxidation as listed in Table 1. After quantitating the rwcrsihle and irrcversiblc in ~ttw rcwtions between t)he V:lri(JUS dycas and cnzymcs, the irk viva dffY!tS Of t,hft ItlcJSt ~~ff~YhW dye!-SCTISitiZcT, row bcngal, on the enzymes were studied. Table 3 shows the levels of the LDH and AChE enzymes in boll weevils after treatment with rose bengal. Sinre rose bwgal consumptic)n affwts the protein concentration of adult weevils, enzyme

wtivitiw (1111!) arc csprrssed as :I functicon both of homogenato volume (ml) and protein cwnwntrntion (mg). Overall J,DH and AChE activity is dcprc>sscd in dyrt trc>atcd insects as can bc srrn by hhc (mu/ml) rows. Thcrc was no difference between light-exposed and non-light-exposed LDH activities but thcrcb was a significant difference (P < 0.01 I~vcl) in the AChE values. If the 1,DH and AChE lrwls arc prcsentcd as spccifio activities (m’I_‘/mg), the large decrease in protein concentration causrs a rovcrsal in the data. It would then apprar that thwc is more enzyme activity in treated insects and that light has no rffect on the in P~POonzymc lewls. J)ISCUSSION The chnractwistics (Jf the xanthenc dye-wnsitizrd photooxidation of J,DH and AChE: cnzymrs frcJm thr boll \\-evil arc consistrnt with current thcoriw of the gonclral mwhanism of dycwcnsitizcld photooxidation. The involvcmc~nt of thcl tripkt cwitcld state of the rlyc-wnsitizcbr is included in the primary t~hwriw rolatcd to this prowss (10). Yigurc 5 shows the mechanism by which light rnergy is absorbed by the dye molwulc and the possible routw of subseqwnt energy mowmcnt. The dye, rose bengal for insknw,

I -4

I -3

I -2

I -I

0

I ROSE

2 BENGAL

3

4 (~16~

5 Ml

FIG. 4. Dixon plot of rose bengal inhibition of LDH. Rose bengal concentration was varied up to /,.91 X lPs M. Pyruvic acid concrntrations ~tscd ‘wrre: (X \, 6.67 X 10-6 M ; (O), 1.00 X 1Cr’ M. I;r~ccclions u~re initiated bu additiorb of OS.5 ma of wrotrain homooorotc.

PHOTOINACTIVATION

OF

absorbs a photon (a) in t’he ground singlet state (So) and, depending on the energy of the photon, is excited to the first excited singlet state (81) or some higher excited singlet state (82). From the higher excited states, the excess energy is rapidly lost as heat (h) to the first excited singlet stat’e. At this point the energy has three possible fates : (h), lost as heat; (f) photon emission as fluorescence; (h), intcrsystcm crossing to the first excited triplet state (2’1). From the first excited triplet state, there are three main fates: (h), lost as heat,; (p), photon emission as phosphorescence ; (a), scnsitization of a second molecule such as oxygen. In this case, the oxygen molecule would be excited to the first excited singlet stat’e (81). The triplet-kiplet intcract’ion between excited-st’ate dye and ground-state oxygen is favorable. Previous experimental evidence exists to support the hypothesis that the population of the triplet excited state of the dye is important to dyesensitized photooxidation of enzymes. Wade and Spilws (11) studied the phot’oinactivation of trypsin scnsitizcd by halogen&cd fluorescein derivatives, and Callaham el al. (6) used several of the same dyes in a study of the AChE from the imported fire ant. The data included in this report concerning the dye-sensitized photooxidation of the LDH and AChE from the boll weevil correlate well with the previous findings.

BOLL

Treatment

of Rose Bengal

on Enzynu

Activities

Dye

II-1 (10-4

n Data expressed as mean value =I= * Value significantly different from c Value significantly different from d Value significantly different from

i i f i i 37

Al)

LineweaverBurk

I)ixon

Composite

0.33 1.28 2.48 3.21 0 0

0.38 0.96 2.2.; 2.96 0 0

0.36 1.12 2.37 3.09 0 0

Rose Bengal Phloxin B Erythrosin B Rosin Yellowish FluoresceirP Rhodamine Ba a Concentration of 6.76 measurable inhibition.

X 10e4 AI

dye

gaye

3 in Boll

W’ewils

at 4 Days

I’ostcwwrgcnce

Rose Bengal

0.17” 6.01 3.55 0.53 0.26

1 SD. control cont,rol control

no

In each case, an incwasc in the second-order rate const,ant of inactivation was correlated with an increase in the population of the triplet excited state of the dye (6). The affinity of the various substituted xanthcnc dyes for the J,DH enzyme \VBS investigated by I,incwcaver-Burl< and Dixon analysis (Table 2). All dyes capable of inhibiting I,DH were shown to be competitive w&h respect to pyruvic acid. This follows the observation of Glazer (13) in which it was proposed that dye-protein interactions occur with a much higher frequency at the active site of the enzyme. The strength of binding betwen dye and

Control

2.33 140.32 62.14 9.32 4.43

2

Drtermination oj Inhibitor Constants for Boll Weevil LDH

Light Protein (mg/ml) LDH (mu/ml) huh) AChB (mu/ml) (mu/w) Replications

2.5

ENZTItIES

TABLE

TABLE EJcct

WEEVIL

exposed

0.93 f 0.0.~” 81.81 zk 4.02” 96.92 zt 7.51” 6.43 f 0.2.2 7.56 f 0.w 27

at 0.001 level. at 0.02 level. at 0.1 level.

Non-light 1.06 78.62 79.48 7.82 8.11

exposed z!z O.OBh 31 5.0.ih f 6.7.ic f 0.39” zlz 0.20” 20

S, i” 15 \ \

ROSE BENGAL

FIG. 5. General mechanism tion of singlet oxygen induced

To OXYGEN for rose bengal sensitizaby visible light.

enzyme is directly related to the number and size of the halogen substituents in the dye molecule. Wade and Spikes (11) made a similar interpretation of data based on differences in spectra obtained upon titration of trypsin with several halogenated fluoresceins. However, they did not find a correlation between the strength of binding and the ability to sensitize photooxidation in their system. Brllin and Yankus (13) suggested that dye binding may even decrease the dye-sensitizing ability in some systems. As can be seen by a comparison of Tables 1 and 3 there was a direct correlation bctwern the strength of binding of the six xant’hcnc dyes to J,DH and their ability to sensitize photoinactivation of the same enzyme. Although the increase in halogenation of this dye series has been shown to increase the population of the first excited triplet state and; thcrcforc, the ability of the dye to scnsitizc photoinactivation of an enzyme, it may not be true that enhanced binding by the dye to an enzyme n-ill have a pwdict,ablc effect on t,h(>rcsultant photooxidation waction. Behavioral alterations have bwn observed upon cxposurc of light to dye fed house flies (5), imported fire ants (3), and boll weevils (4). The indicat,ion x$-as ihat tlw effect of light and dye on t,hctinswt ~1s ncurotosic in nature. The: AChE lcvcl

of inswts killed in this mannffir, li(owcwr, have failed to show a significant, change (.j, 6). The insrcta 11sct1in those studks wrr fed for a shorter time period and the AChE lcvcls reported in terms of specific activity (mU/mg). As rworded in Tablr 3, the protein concentration of dye treated boll weevils is significantly Iowcr than controls. Light does not have a clisccrnible cffcct. If the enzyme activities are presented in t,crms of the homogenate volume, then a large decrease in enzyme activity is observed for both LDH and AChE. It is only when the lower protein concentration is divided into the loxvcr mzymc activity that the resulting ratio indicatw that dye treated enzyme levels are higher than controls. Throughout, the effects of light are negligible. Row bclngal may (XUSC a decrease in overall enzyme acstivity, but due to its effect, on total protcGn thcbrc would appraar to bc only an incwaw in activity. The in vi~ao target of dxc-scnsitized photooxidation remains as yet mldescribed. REFERENCES

1. T.

2.

3.

4.

5.

6.

P. Yoho, L. Butler, and J. E. Weaver, Photodynamic effect of light on dye-fed house flies : Preliminary observations of mortality, J. Econ. Entomol. 64, 972 (1971). T. P. Yoho, J. E. Weaver, and L. Butler, Photodynamic action in insects. I. Levels of mortality in dye-fed light-exposed house flies, Environ. Entomol. 2, 1092 (1973). J. I:. Broome, 32’1. F. Callaham, L. A. Lewis, C. X. Ladner, and J. R. Heitz, The effects of rose bengal on the imported fire ant, Solmopsis rich&-i (Forel), Camp. Hioch,cm. Phgsiol. 51c, 117 (197.i). 11. F. Callaham, J. I:. Broome, 0. H. Lindig, and J. R. Heit,z, Dye-sensitized photooxidation reactions in the boll weevil, Anthonomous grandis, Environ. Entomol. 4, 837 (1975). T. P. Yoho, The photodynamic effect of light on dye-fed hollse flies, Musca dowwsfica L., Ph.D. dissertation, West Virginia University, 1972. M. F. Callaham, 1,. A. Lewis, $1. E. Holloman, J. 1:. Broome, and J. R. Heitz, Inhibition of the acetylcholinesterase from the imported fire ant, Solenopsis richteri (Forel), IJ~ dyescrlsitized photoosidat ioIl, (‘o/,rlj. IIiwhr m. Physiol. SIC!, 123 (1975).

PHOTOINACTIVATION

OF

7. 0. IT. I,owry, N. J. Rosebrough, A. L. Farr, and I:. J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265 (1951). 8. H. Lineweaver and D. Burk, The determination of enzyme dissociation constants, J. Am. Chcm.

9. M.

Sot.

56, 6%

(1934).

Dixon, The determination of euzyrne inhibitor constants, Biochem. J. 55, 170 (1953). 10. J. D. Spikes, Photodynamic action, in “Photophysiology” (A. C. Giese, Ed.), Vol. III, p. 33, Arademic Press, New York, 1968.

BOLL

WEEVIL

ENZYMES

27

11. M. J. Wade and J. D. Spikes, The efficiency of halogenated fluoresceins as sensitizers for the photodynamic inactivation of trypsin, Pkotothem. Photobiol. 14, 221 (1971). 12. A. N. Glazer, On the prevalence of “nonspecific” binding at, the specific binding sit,es of globular proteins, I’roc. ili’al. Acud. Sci. L’SA 65, 1057 (1970). 13. J. S. Bellin and C. A. Yankus, Influence of dye binding on the sensitized photooxidation of amino acids, Arch. Biochem. Biophys. 123, 7X (1968).