Kinetics of quinone inactivation of Tulare apple mosaic virus

VIROLOGY

33, 609-612

Kinetics

(1967)

of Quinone

Inactivation

of Tulare

Apple

Mosaic

Virus’

Research

and Extension

G. I. MINK2 Department of Plant

Pathology,

Washington Center,

State University Irrigated Agriculture Presser, Washington 99660 Accepted

August

8, 1967

A direct assay technique was used to measure the kinetics of o-quinone inactivation of Tulare apple mosaic virus (TAMV). Inactivation occurred at rates measurable in seconds, minutes, or hours depending upon the particular quinone tested and the ratio of benzoquinone equivalents per milligram of virus used. The inactivation rate appeared independent of the absolute virus concentration. Inactivation rates were not linear at any quinone concentration measurable. However, inactivation data plotted according to the equation l/1 = (b/a)(l/t) + l/a where I is the percent inactivation, a and b constants, and t exposure time, gave linear curves from which initial inactivation rates (percent virus inactivated in the first second) and minimum exposure times (times required to cause 160$& inactivation) could be determined. INTRODUCTION

Tulare apple mosaic virus (TAMV) is rapidly and irreversibly inactivated by exposure to many oxidizing agents including a wide variety of quinone derivatives (Mink, 1965; Mink el al., 1966). During earlier studies, it appeared that inactivation occurred more rapidly in the presence of some oxidizing agents than in others. However, because TAMV was exposed to oxidants for lo-15 minutes and then removed from solution by centrifugation prior to assay, during which time the virus continued to be exposed to the oxidizing agents, meaningful studies on the rate of virus inactivation in the presence of various oxidants could not be made. In addition, ultracentrifugation of treatments introduced a potential error into kinetic studies due to changes in the sedimentation coefficient of partially oxidized virus (Mink, 1965). Conl This investigation was supported in part by funds provided for biological and medical research by State of Washington Initiative Measure No. 171andbyNationalScience Foundation GrantGB5639. Scientific paper No. 2964 Washington State University College of Agriculture, Pullman, Project No. 1887. 2 Associate Plant Pathologist.

609

sequently, a technique was developed to determine the kinetics of oxidative inactivation by direct assay. MATERIALS

AND

METHODS

Preparation and assay of TAMV. Purified virus was prepared from frozen, systemically infected Nicotiana tabacum L. ‘Havana 423’ as previously described (Mink et al., 1963) except that tissue was macerated by hand in a mortar. For kinetic studies, solutions adjusted to contain 150-300 pg of virus per milliliter in 0.02 1M pH 5.0 sodium acetate buffer were divided into four 0.2ml aliquots. Equal volumes of various quinone derivatives previously adjusted to the desired concentration were added to each of the four aliquots. As rapidly as possible (usually within 5 seconds) enough sodium dithionite dissolved in acetate buffer to ensure complete quinone reduction was added to one aliquot of the virus-quinone mixture. This solution was considered the zero time treatment and used as the basis for calculating the percentage of inactivation obtained with the remaining treatments. After preselected intervals, the remaining virus-quinone aliquots were reduced in turn. Inasmuch as all four treatments were

MINK

610

. 1050

/ 25

-

/

./ c a

k!

20

3.4.5 TMQ / 60

120

SECONDS FIG.

1. Inactivation

curves of TAMV

180

10

5

15

MINUTES

RESULTS

E$ect of quince and virus concentration on inactivation When more than 250 Fg of benzoquinone equivalents of TCQ were added per milligram TAMV and sodium dithionite was added immediately thereafter, no infectivity could be detected in any treatment. At TCQ concentrations below 250 pg and at all concentrations of partially substituted qui-

I

2

3

HOURS

exposed to three o-quinones

identical except for the length of time TAMV was exposed to oxidizing agent, the treatments were assayeddirectly on primary leaves of Phaseolus vulgaris L. ‘Bountiful’ under conditions previously described. Preparation and use of o-q&none derivatives. Tetrachloro-o-benzoquinone (TCQ) ; 4 methyl-o-benzoquinone (4MQ) ; 3, 4, 5trimethyl-o-benzoquinone (3, 4, 5-TM&), and 3, 4, 6-trimethyl-o-benzoquinone (3,4, 6-TM&) were prepared as described previously (Mink et al., 1966). Weighed amounts were dissolved in 0.3 and 0.5 ml of absolute alcohol and diluted immediately before use to desired concentrations with 0.02 M pH 5.0 acetate buffer. For comparative purposes the actual weights of the o-quinone derivatives were converted to equivalent weight of o-benzoquinone (Mink et al., 1966). Consequently all quinone concentrations cited in this report refer to the micrograms of benzoquinone equivalents present in solution per milligram virus.

I

1

at various quinone-to-virus TABLE

ratios.

1

EFFECT OF ABSOLUTE VIRUS CONCENTRATION THE RATE OF TAMV INACTIVATION BY

ON

TCQa ConcentraT&$%1) 50 loo

Exposure time (seconds) 30

90

30b 28

78 74

a Each solution contained gram of virus. b Percent inactivation.

150 93 95

25 pg TCQ per milli-

nones, inactivation occurred at a measurable rate (Fig. 1). The rate of inactivation was dependent on the particular quinone tested and the ratio of benzoquinone equivalents per milligram of TAMV used. Even though the quinone-to-virus ratio remained constant among experiments, assays were sometimesmade at different virus concentrations in different experiments. Results of an experiment performed with two virus concentrations at a single quinone-to-virus ratio indicated the inactivation rate was independent of the absolute virus concentration (Table 1). Determination of Initial

Inactivation

Rates

Loss of infectivity did not occur at a constant rate regardless of the quinone derivative or concentration used. Graphically the data in Fig. 1 resemble the early

KINETICS

OF QUINONE

INACTIVATION

611

,.

. TCQ

I

4MQ

I 1

o 14.6

I ;

3

.l

.2

RECIPROCAL

.3

I .05

TIME

X 1O-2

TMQ .15

.lO

2. Reciprocal plots of TAMV inactivation curves. Experimental data shown in Fig. 1 are plotted according to Eq. 2. In addition TAMV inactivation in the presence of both 3,4,5- and 3,4,6-TM& are compared at 500 pg and 25 pg. FIG.

stages of tyrosinase oxidation (see Nelson and Dawson, 1944). Reactions of this type can be expressed mathematically as Eq. (1) where I is the amount of inactivation, a and b are constants, and t is the time in seconds. Plots of the reciprocal equation (2) yield a straight line. Linear curves were obtained with the experimental data for TCQ, 4MQ, and two trimethyl quinones (Fig. 2).

TABLE INITIAL

o-Quinone derivative

TCQ 4M0

3,436TM& 3,475

(2) When experimental data are plotted according to Eq. 2 the reciprocal of the slope of the linear curve represents the initial reaction rate, that is, the reaction rate during the first second. The initial rates of virus inactivation in the presence of four o-quinone derivatives were calculated from the slopes in Fig. 2 (Table 2). The initial reaction rate of TCQ was 100 times greater than that of either trimethyl quinone.

RATES VARIOUS

TM&

(1Percentage first second.

2

OF INACTIVATION OF TAMV 0-QUINONE DERIVATIVES

BY

o-Quinone conInitial centration tested inactivation rate4 Cdw TAMV) (% se4 25

1.0

1025

0.28 0.28 0.01 0.05

500 25 500 25

of virus inactivated

0.01 during

the

Prediction of Inactivation Times Since inactivation data were expressed as percentages, the intercept of the linear curves in Fig. 2 and the 100% inactivation line along the II: axis represents the reciprocal of the time required to obtain total inactivation at a given quinone concentration. Consequently, it was possible to predict from a few points determined experimentally, the minimum time required to in-

612

MINK

activate all virus present by any quinone derivative. Similar values were also extrapolated from the experimental data by plotting percent inactivation versus the logarithm of the exposure time. DISCUSSION

In previous studies on oxidative inactivation of TAMV it was necessary to select exposure times to quinones empirically. These earlier studies demonstrated the necessity for using minimal quinone concentration for minimum exposure periods to avoid spurious results in studies on the possible role of RNA in virus inactivation (Mink, 1965). Kinetic studies indicate that minimal exposure times to give complete inactivation at any quinone concentration can be predicted graphically from a limited number of tests. Although several viruses appear to be sensitive to quinone inactivation (Fulton, 1966), it is not known whether viruses in

this group vary among themselves in their relative sensitivities. The direct assay technique described here may be useful in making direct quantitative comparisons among viruses on their relative sensitivity to compounds such as TCQ. Such knowledge is essential to meaningful studies on the mechanism of quinone inactivation of plant viruses. REFERENCES R. W. (1966). Mechanical transmission of viruses of woody plants. Ann. Rev. Phytopathol. 4, 79-102. MINK, G. I. (1965) Inactivation of Tulare apple mosaic virus by o-quinones. Virology 26,700-707. MINK, G. I., BANCROFT, J. B., and NADAKAVUKAREN, M. J. (1963). Properties of Tulare apple mosaic virus. Phytopathology 53.973-978. MINK, G. I., HUISMAN, O., and SAKSENA, K. N. (1966). Oxidative inactivation of Tulare apple mosaic virus. ViroZogy 29,437~443. NELSON, J. M., and DAWSON, C. R. (1944). Tyrosinase. Advan. Enzymol. 4,99-153. FULTON,