Experimental studies of some moderately fast processes initiated by radiation

0146-5724/89 $3.00+0.00 Copyright © 1989PergamonPress plc

Radiat. Phys. Chem. Vol. 34, No. 4, pp. 633~46, 1989 Int. J. Radiat. Appl. Instrum., Part C

Printed in Great Britain. All rights reserved

EXPERIMENTAL STUDIES OF SOME MODERATELY FAST PROCESSES INITIATED BY RADIATION J. BUTLER, B. W. HODGSON, BRIGID M. HOEY, E. J. LAt,a3, J. S. LEA, ELIZABETH J. LnqDLEY, F. A. P. RUSHTON and A. J. SWALLOW Paterson Institute for Cancer Research, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, U.K. Abstract--Numerous improvements have been made to the Paterson Institute linear accelerator since its installation in 1967. New light sources, improved light guidance, smaller cells and a wider range of photo-detecting devices are now in routine use. Data are collected and processed by a computer-based method which has'replaced the original osciUoscope-basedsystem. Processes taking place over more than a few seconds can be studied with an arrangement combining pulse radiolysis with an ordinary spectrophotometer and arrangements for "single-shot" studies of faster processes are now being designed. Detection methods are also available which do not rely on transmission of light, and transient changes in conductivity can be measured. Among the systems which have been extensively studied are the Fricke dosimeter, in which measured overall yields can now be quantitatively correlated with the rate constants of 34 individual reactions taking place. Studies have also been conducted with peptides and proteins in which electrochemically-driven charge transfers have been demonstrated between methionine, tryptophan, tyrosine and cysteine/cystine units. Free radical reactions in Mitomycin C have been elucidated which are consistent with pulse radiolysis observations and the formation of radiolytic-products as determined by HPLC. Adriamycin reduction has also been studied: the Adriamycin semiquinone is unusually stable with respect to dismutation but its lifetime is limited by a decomposition process in which daunosamine is expelled. The expulsion is followed by a further rearrangement. Many of the reactions investigated require tens or hundreds of seconds to reach essential completion.

between 8 and 12 MeV and variable pulse widths from 5 ns to 5 ps in steps of factors of 2 and 2.5. A photon can traverse a molecule in less than 10 -18 s. Typically, peak currents are around 5 A for the 5 and Subsequent processes may be regarded as "fast" if 10 ns pulses (in the short pulse stored energy mode) they take place too quickly for unassisted observation and are routinely set at 500 mA for pulse lengths by man, say in less than a few seconds. Most of the between 100 ns and 5 #s although beam currents up work in our laboratory has been concerned with to 800 mA are easily available. Single pulses are processes taking place over periods between mi- normally used, but repetitive pulses can be given at croseconds and hundreds of milliseconds. We are also a frequency which can be varied up to a maximum of exploring processes taking place over periods be- 50 p.p.s. tween about a second and a few minutes, too slow for A high power klystron and its power supply consticonventional pulse radiolysis, but too fast for easy tute a major part of the equipment. The original study where initiation is produced by conventional klystron was replaced by one from Thomson CSF in radiation sources. Our techniques are developed 1973, requiring modifications in the wavegnide confrom original apparatus described in Keene (1964). nection to the accelerator and the installation of a They have undergone significant improvement since different focus coil power supply. Klystrons are rethey were last presented comprehensively (Keene, placed approximately every 6 yr or 15,000 h of oper1972a, b) and are still being developed. This paper ation. They are rated at 20 MW peak power but run describes the techniques in their current state and at 10 MW for increased life. gives examples of recent results obtained with them. As well as high current short pulses, a particular requirement is very low dark current. We have reduced the dark current to that of a single 7 ps RF LINEAR ACCELERATOR pulse from the main klystron by pulsing it and the Our facility relies on an "L" band (1300 M Hz) triode electron gun continuously at 50 Hz to give linear accelerator installed in these laboratories stability, but delaying both RF and gun drive pulses (Keene, 1972a) in 1967 (Fig. 1). Similar accelerators relative to the main klystron 180 kV pulse. A single have been installed at the Hahn-Meitner Institute, beam pulse is produced by bringing all three pulses Berlin, and the Instituto di Fotochimica e Radiazioni into time synchrony only when a single beam pulse is di Alta Energia, C.N.R., Bologna. The accelerator required. The trigger generator to do this was deproduces a pulsed electron beam with an energy of signed in-house (Hodgson and Keene, 1976). INTRODUCTION

633

634

J. BUTLERet al.

Fig. 1. Paterson Institute linear accelerator. Solutions are moved by gas pressure from a reservoir (inside the box seen in the lower foreground) into the pulse radiolysis cell (in the holder above). The housing at the upper right hand side of the picture contains the lamp.

In its early years of operation the oxide-coated gun cathodes failed because of poor emission after only a few hundred hours of operation or less. This problem was associated with a poor waveguide vacuum. After curing the leaks with the help of a built-in mass spectrometer leak detector, and thorough degassing, lifetimes were improved to several thousand hours of operation (between 1 and 2 yr). Reliability was improved by using ceramic rather than quartz RF windows to isolate the 1.4 x 105 Nm -2 air-filled feed and exit waveguide from the high vacuum ( ~ 5-10 x 10 -6 Nm -2) in the accelerating waveguide. Among other improvements, the RF drive system has been redesigned to enable the drive klystron to be tuned while operating so as to optimise gain at 1300 MHz and the pre-buncher excitation controls have been relocated outside the accelerator room to permit optimisation of beam parameters with the beam running. With general improvements to the control and protection circuits, careful daily running up of the machine with routinely recorded parameters and regular preventative maintenance including two main services per year, the down-time is less than 5% and there is a high order of both short and long-term stability. This high reliability is an important feature of the installation, in that it permits experiments with scarce or expensive materials to be planned and executed with confidence.

OPTICAL PULSE RADIOLYSIS Although several different properties can be used to detect the transient changes produced by a pulse of radiation, changes in optical absorption are the most generally informative. Figure 2 shows the present arrangement of our facility as developed from the system already described (Keene, 1972b). (i) L i g h t sources

Characteristics of the lamps used in most of our experiments are listed in Table 1. The 250 W xenon arc lamp and the tungsten and deuterium lamps are still in current use as described previously (Keene, 1972b). The Noblelight (NL132) high stability xenon arc lamp is now used to monitor species which absorb in the u.v. over periods up the hundreds of milliseconds. It is a point source lamp with 0.4 mm arc gap and is operated at a constant current (3 A) which gives least noise and ripple interference. Its output has less than 0.1% peak to peak noise and drift. It does not require a cooling fan but instead an integrated heat sink is used to dissipate excess heat. The absence of a fan helps to reduce instability in light intensity caused by vibrations. A high pressure xenon arc lamp with a sapphire window is available for longer wavelengths, 2000-3500 nm. It can be used in the pulsing mode

Studies of moderately fast processes initiated by radiation

l I'/////////+

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/////////////A Biological shielding

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Fig. 2. Schematic diagram of the pulse radiolysis facility. The electron beam goes through a secondary emission chamber M, producing a charge which is proportional to pulse dose, and then into the pulse radiolysis cell holding the sample, S. (Hodgson and Keene, 1972) where fast detection ( < 10/~s) is required.

(ii) Light guidance systems Between the light source and the irradiation cell there is a remote controlled C o m p u r Electronic M shutter which has replaced the previous mechanical device to minimise photolysis of the sample. The aperture of the electronic shutter has a diameter of 29 m m and fastest aperture time of 5 ms but can also be operated in m a n u a l mode if desired. The m o n o c h r o m a t o r is situated inside the Faraday cage (Fig. 3). A Kratos G M 252 high intensity quarter metre m o n o c h r o m a t o r with four interchangeable gratings has superseded the previous monochromator. It is stepper-motor driven with 0.1 n m resolution and controlled by a custom-built wavelength selector. The spectral range covers 180-3000 n m and the scattered light constitutes less than 0.35% at 500nm. A new system of twelve Schott circular glass filters in a filter wheel is positioned before the monochromator and is also stepper-motor driven. The filters include ultraviolet absorbing, infrared transmitting and glasses suitable for use in different areas of the visible region. There are also bandpass filters for work in the ultraviolet, i.e. 235-340 n m dropping to < 0.001% transmission at 220 and 360 n m and narrow bandpass filters for 206, 214 and 228 n m with

Type Xenon arc Xenon arc Tungsten halogen Low pressure deuterium arc

Manufacturer Wotan Noblelight Thorn Sylvania

A2 = 23 nm, falling to < 0.01% transmission outside the passband. Both the m o n o c h r o m a t o r and the filter drive can be linked to a D I - A N control interface system which allows the wavelength and appropriate filter to be selected from the computer keyboard. When using wavelengths greater than 2000 nm, focusing lenses are removed and optical windows are replaced by sapphire.

(iii) Irradiation ceils Flow-through capillary cells are in general use (Fig. 4). They are composed of high purity fused silica cylindrical microbore tubing, internal diameter 3 mm, with Spectrosil quartz windows. Optical path lengths from 0.5 to 10 cm are available. The capacity of the smallest cell is 35 m m 3, allowing detailed spectral information (about 20 wavelengths) to be obtained from a sample of less than 4 cm 3. The purging and flow systems are essentially the same as in the original apparatus. Solutions are normally contained in reservoir vessels of capacity 25-100 cm 3. With scarce or expensive materials, samples as small as 1 cm 3 can be contained in a motor-driven syringe programmed to deliver exact volumes of fresh solution through the capillary cell before each pulse.

(iv) Photo-detection Characteristics of the photodetectors in c o m m o n use with their associated circuitry are given in

Table 1. Lamp characteristics Manufacturer's Power Stability over Comments designation (W) Intensity Wavelength ms or more XBO 250 250 High All Fair Can b¢ pulsed NL 132 50 High All Good -A1/209 100 Medium Visible Good -and IR DE 50A 50 Low Lrv Good --

636

J. BUTLERet al.

Fig. 3. Detection equipment. The monochromator and filters can be seen to the left of the operator's head. The filter and wavelength selectors are above the HP 9876A printer (left). The Tektronix 7612D digitiser and HP 9836S computer are on the right.

Table 2. The Hamamatsu silicon photodiode, which programmable digitiser (Fig. 2). The digitiser covers has a quartz window, is useful when using u.v. light sample rates from 1Hz to 200MHz (i.e. 1 s - 5 n s to study sub-microsecond processes. It is used in intervals). Its memory can hold 2048 8-bit words per conjunction with the pulsed xenon arc lamp. The channel giving a resolution of 1 part in 256 in the response time is quoted at about 1 ns at 100 V reverse vertical direction. Typically only the first 500 words bias into 50 fl and the effective area of the diode is of data are used. The memory can be partitioned to 13.2 mm 2. The diode has better signal/noise charac- give samples at differing rates though this feature has teristics than the photomultiplier-based systems espe- not yet been used. Other analog inputs (recording cially at longer wavelengths where tube sensitivity back-off, dose, etc.) required in digital format for access via the controller are converted using a D I - A N falls rapidly. The detector circuit incorporates an automatic microsystems data acquisition controller (for simplicback-off system (Keene and Bell, 1973) which allows ity not shown in Fig. 2). Various plug-in options changes in transmission to be observed by feeding include the DMS 230 high speed 12-bit (11-bit + sign) back a signal equal and opposite to that produced by analog to digital converter (ADC) and DMS 233 the photodetector anode current prior to the electron analog multiplexer (MUX), the combination of pulse, thus maintaining the anode within a few mV of which gives a typical A D C conversion time of 35 #s though speed here is not essential. This arrangement zero. allows digitisation and transfer of up to 16 singleended or 8 differential analog inputs in the range DATA COLLECTION AND PROCESSING ___5 mV to ___10.28 V at 12 gain settings, all under (i) Data collection system programme control. Also present are a DMS 211 The most important change in the pulse radiolysis 16-bit digital input module and a DMS 221 16-bit equipment has been the replacement of the original output module that are used to set wavelength, to oscilloscope system (Keene, 1972b) by a data collec- select appropriate filter, and communicate status tion system based on a dual channel Tektronix 7612D information under software control.

Studies of moderately fast processes initiated by radiation

637

Fig. 4. Capillary cells. Optical path lengths of the cells shown are 2.5, 1.0 and 10 era.

Processing and program control is provided using a Hewlett Packard (HP) Series 200 model 9836S computer. Standard features include two 5¼ in. disk drives, a keyboard mounted rotary " m o u s e " , an eight-slot backplane for expansion cards and a 12 in m o n o c h r o m e C R T display. The V D U is capable of displaying 25 lines of text at 80 characters per line and graphics with a resolution o f 512 × 390 pixels, either independently or simultaneously. R A M includes

128 K bytes on the processor board and two 256 K bytes via plug-in boards, a total of 640 K bytes. Also included is a built-in H P - I B parallel port used to communicate with all the peripheral devices described here. An HP-98626A card is fitted to provide an RS232 serial link to a P D P 11/34. Using this link and the Kermit software protocol, the 9836S is able to emulate a P D P 11/34 terminal. In this mode data can be transferred to take advantage of the increased

Table 2. Photodetector characteristics Manufacturer's

Type Photomultiplier Photomultiplier Silicon photodiode Silicon photodiode Germanium photodiode

Manufacturer EMI EMI UDT Hamamatsu Philco-Ford

designation 9558 QA 9783 R PIN- 10UV S1722-02 1.,4521

Wavelength(nm) 180-850 160-700 200-1070 190-1060 500-1800

Rise time (ns) 100 2-3 200 <5 10

Workingload (f/) 10 k 50 10 k 50 50

638

J. BUTLERet al.

processing power provided by a mini computer for more complex analysis. Printed output is provided by a HP 9876A thermal graphics printer which generates very fast printing speeds. The full graphics screen is reproduced in 9 s.

(ii) Software techniques HP Basic 3.0 provides the software environment generally. Though a wholly interpreted language, the speed of execution and simplicity of programming preclude the need to resort to compiled languages. Basic also provides useful system features such as softkey interrupts (below), interrupts from the rotary mouse, live keyboard useful for calculations and debugging even during program execution, and an extensive graphics command library. HP Pascal 3.1 is also available, in particular for running Kermit to transfer data files. With this language combination (both written in MODCAL) processor intensive routines (which are relatively slow in Basic) may be compiled in Pascal and referenced in Basic thus taking advantage of optimised compiler speed. Though not employed so far, this could be useful in the future. Ten keys are set aside to interrupt program flow when software-enabled. Upon activation interrupt service routines then take appropriate action. The bottom two lines of the display are also set aside in inverse video and divided into ten 14-character fields, one corresponding to each key. They can be labelled by software to prompt the user with corresponding action. Each program and subprogram therein maintains its own set of interrupts and labels. They are used interactively, for example, to adjust parameters from displayed menus. The keyboard rotary mouse can be programmed to interrupt program flow. A parameter used in its set-up initiates a timer which counts the degree of rotation for the interval specified. The count may be accessed by the servicing routine to mimic real time feedback. It is used, for example, for graphics cursor movement. A pre-drawn skeleton graticule maintained in R A M in digital format can be transferred to display R A M and subsequently displayed very quickly ( ~ 100 ms) thus dispensing with slower draw statements each time. Global variables used in the pulse radiolysis software are allocated a common area of R A M which is never reinitialised once defined. Consequently other chained programs declaring the same structure and type of variables will have access to these. This further implies that unless a particular parameter is changed its previous value prevails. This technique minimises keyboard input. Pulse radiolysis data are split into three categories. First there are relatively static data i.e. pulse length, photomuitiplier box type, monochromator type, lamp, lamp filters, etc. Secondly there are data the computer accesses directly, i.e. digitised trace, reading

from the secondary emission chamber proportional to dose (SEC), and the pre-pulse light intensity expressed in mV(I0). Finally there are other relatively dynamic data, i.e. wavelength, bandwidth, description of the sample being irradiated, etc. The software formats and initialises disks ready to receive data. Two files are maintained on each disk. One (file RECORD) contains a pointer to the last record of the data stored. The remainder of the disk is taken up by data (file DATA), a file comprising 260 records each 1020 bytes in length. Each record contains parameters for each trace followed by the trace itself. 500 digital points would normally occupy 1000 bytes of disk space but prior conversion to ASCII bytes (since each is an integer in the range 0-255) reduces this by half. Data are stored in record sequence but once written may be retrieved randomly by its record identifier printed out with each record stored.

(iii) Typical sequence of operation The resident boot ROM first performs a series of internal checks then searches for a language system to load. The operator now inserts a 5 ¼in. disk containing core Basic. Since this occupies virtually one whole disk, special binary extension files (supplied separately by HP) enable the operating system to be tailored to the needs of the software. To automate this process a special user written autostart (file AUTOST) program is searched for, loaded and run. Such things as card drives (in particular for HP-IB), error messages, matrix algebra etc., are loaded. This then chains to another program (file SYS CHECK) which sets up global variables with default values, sets the system clock (via the operator) and checks for minimum configuration (digitiser, printer, DI-AN). The main pulse radiolysis program is then loaded and run. At this point the Basic system has taken 412 K bytes of RAM and the pulse radiolysis program (plus global variables) 91 K bytes leaving 137K bytes available for local usage. While operating, the pulse radiolysis software needs a suitable data disk. A menu-driven screen displaying the current accelerator parameters follows. A response from the operator removes the display when the data are correct though access is available at most times via a softkey interrupt. The digitiser is initially set up to sample at 5 ns per point. It is armed to receive a trigger and displays a skeleton graticule along with time and amplification factors. The accelerator is fired via a push button which then cascades other events set up from the master trigger generator. The digitiser is finally triggered and flags the computer accordingly when a full complement of data is ready to be transmitted via its status byte which is continually monitored. The digitised trace is transmitted, followed by the prepulse light intensity, I0, and the dose reading, SEC. The data are then plotted out and labelled with I0 and SEC. The operator can now either press the button

Studies of moderately fast processes initiated by radiation

639 10/2/88 131

Date Record

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720 nm 10 n m

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: tungsten

Lamp filter Photomultiplier

: none : PM BOX

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:

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:

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Fig. 5. Typical print-out. The trace shows a sharp decrease in optical transmission at 720 nm due to the formation of the semiquinone of Adriamycin followed by a partial recovery of transmission, almost complete by 200/J s, due to the attainment of an equilibrium with the hydroquinone and the parent, neither of which absorbs at 720 nm. Further changes take place over longer periods (see Fig. 9).

for another shot or signal that he wishes to retain the present data. In the latter case the softkey interrupt branches to another menu-driven display to update the more dynamic parameters. In particular a field designated " R E M A R K " is used to note shot-to-shot peculiarities. On receipt of a subsequent shot this field is erased. Having signalled the information as correct the trace is stored on disk a n d a printed copy is produced. Figure 5 shows a typical result. Data can be signal averaged when required, by successive accumulation of traces. Each accumulation is displayed on the standard graticule using appropriate re-scaling. When the signal-to-noise ratio is acceptable the final result stored is found by dividing the accumulated data by the number of accumulations. In this manner loss of significant digits resulting from division with integers is reduced to a minimum. Data for spectral analysis are logged and stored in a similar way. The first trace accepted is used to set up a mask for subsequent time slices (up to four presently allowed). This is possible because trigger jitter is less than one sample period even at 200 MHz ( < + 2 ns). The converted data only are stored internally in arrays ready for graphical presentation. In many cases the chemistry of the system is such that the time course of the change in absorbance would be expected to fit first or second order kinetics.

Fitting routines are available, based on earlier work (Gilbert C. W., unpublished data). Equations that can be fitted are: AOD(t) = A + B exp(-kt);

and 1

A O D ( t ) = A + -)--'~(Bk +t

;

where O D ( t ) = lOgl0 ( ~ )

;

and y ( t ) = mV deflection at time t after the pulse. The parameters can be fitted iteratively to ~ 5 0 equally spaced samples, after an initial approximation, using the method of least X2 until subsequent improvements are within 5% (usually taking 3-4 iterations). The estimated parameters are used to generate a theoretical curve which is plotted over the initial data for direct visual comparison. Printout of the parameters and their errors is available. An additional linear transform for either model is available for more accurate comparison between theoretical and measured data. Emission data are treated in a similar manner. External routines are provided for more complex kinetics and for subsequent manipulation of data files.

J. BUTLERet al.

640 Final mirror of l i n a c o p t i c s

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Fig. 6. HP 8451A diode array spectrophotometer connected to linac optics by fibre optic link. "SINGLE-SHOT" PULSE RADIOLYSIS Our pulse radiolysis equipment enables data to be collected at a single wavelength over a chosen period of time after each pulse. Information at other wavelengths is obtained by selecting each further wavelength on the monochromator, replacing the irradiated sample with a fresh one with the aid of the flow system, and delivering another pulse. For studies at times greater than about ten seconds, variations of absorbance with time and absorption spectra at chosen times can be obtained by delivering a single pulse to samples contained either in capillary cells or in quartz spectrophotometer cells. To do this, the pulse radiolysis optical system is interfaced with a Hewlett-Packard HP8451A spectrophotometer using a fibre optic light guide. The experimental arrangement is shown in Fig. 6. The pulse radiolysis analysing light is transmitted into the Faraday cage via the normal optical system. Before reaching the monochromator, the light is focused using a Comar silica lens ( f = 100mm, ~b = 25 mm) into a 1 mm flexible light guide (Schott uv transmitting fibre bundle). The light emerging from the other end of the light guide is aligned with the diffraction grating of the HP 8451A. The pulse radiolysis analysing light effectively replaces the internal deuterium lamp of the HP 8451A which is masked out while the spectrophotometer is used in this mode. In the normal "absorption" and "transmission" modes of the HP 8451A, the background (dark current) is automatically subtracted from each spectrum. The dark current is measured by closing a shutter between the internal lamp and the sample. This measurement is made immediately after the spectrum has been collected. When the interface is used, closing this shutter has no effect so the background subtraction is incorrect. It is therefore necessary to use the HP 8451A in the "intensity" mode and store the raw data in local memory or on disk, for manipulation after the experiment is completed. An additional

problem arises if the analysing lamp used with the interface is not a deuterium lamp. The deuterium spectrum contains a number of spikes (particularly at around 660 nm) and in the regions where these occur the diodes have been masked to avoid saturation. This produces unwelcome dips in the recorded spectra which have to be accepted as artefacts. The changes in absorbance of the sample at time t after the pulse (AOD(t)) is calculated using:

AOD(t) = log10 (Io/1(0); where l(t) is the intensity of light transmitted through the sample at time t. Both I0 and I(t) are corrected for the dark current before the absorbance is calculated. When the interface is used, the dark current is measured by closing the shutter between the pulse radiolysis analysing lamp and the sample. This gives a background that includes any stray light that has leaked in via the fibre, although in practice this is negligible compared to the thermally generated background. The HP 8451A can be programmed to make spectroscopic measurements over a given wavelength range at a series of predetermined times. The minimum time interval, and the maximum number of spectra that can be collected depend on the wavelength range and the mode of data storage used. Sampling intervals can range from a few tenths of a second to many tens of seconds, with the lower limit set by the HP 8451A and the upper by the stability of the experimental conditions. Once the data have been collected, and the dark current measured, it is possible to display either the variation in absorbance with time at a given wavelength or the difference absorbance spectrum of the sample at selected times. The data can be saved on disk, or transferred to a larger computer for more detailed analysis. We are currently designing a new workstation which, following a single pulse of electrons, will allow data to be collected simultaneously at several wave-

Studies of moderately fast processes initiated by radiation lengths at sampling intervals of between a few tens of ns and a second. The present system will remain operative, and may be improved upon, for handling certain experiments. These will include experiments where it is essential to have either very fast data collection (sampling at intervals of less than about 20 ns) or very good wavelength resolution (better than 5 nm). The new workstation will be situated in the existing Faraday cage. The change in the optical path can easily be accommodated with the present optical components. A "polychromator" (i.e. a flat-field monochromator) will be used to disperse the light. The range of wavelengths and the resolution obtained in a single run will depend on the diffraction grating used. Light at the wavelengths selected for the measurement will be guided from the spectrum to a series of detectors. The signals produced by the photodetectors will be treated as above. At the end of the experiment the data from each detector will be collected and processed by the HP computer. Apparatus of this type will be invaluable for pulse radiolysis experiments on samples which are difficult to prepare in large quantities. It should also prove useful for preliminary experiments with new systems as it will allow the regions of interest in the spectrum to be identified quickly. SPECIAL DETECTION TECHNIQUES

Radiation-chemical processes within opaque and highly scattering systems, such as often occur in biology, can be studied by observing changes in diffusely-reflected analysing light. Our apparatus can be adapted to carry out diffuse reflectance pulse radiolysis experiments (Wilkinson et al., 1984). Analysing light from the pulsed xenon arc lamp is reflected off a sample through the optically transparent back face of an irradiation sample holder, the normal to whose surface is set at an angle 40 deg to the direction of the accelerator beam. Specular reflected light is blanked off as it contains little transient information. Since the 8-12 MeV electrons are not strongly attenuated on passing through the typically ~ 1 mm thick samples, the back-face analysis does not require major modification of the pulse radiolysis apparatus as designed for normal transmission detection. First experiments were performed on opaque samples of organic microcrystals which had already been shown to give transient intermediates on diffuse reflectance laser flash photolysis (Wilkinson and Willsher, 1984). One of the means by which excited states can decay is via the emission of light, usually phosphorescence in the case of triplet excited states and fluorescence in the case of singlet excited states. With the powerful analysing light sources normally used, such emissions do not usually distort measurements of transient absorptions. However, when the analysing light is switched off these emissions may be amplified and

641

observed over and above the background Cerenkov emission, as in the case of naphthalene fluorescence in benzene or cyclohexane (Land and Swallow, 1968). We have seen "delayed fluorescence" in the irradiation of aqueous solutions of dyes such as acriflavin, rhodamine B and fluorescein even when only one quantum is emitted for every hundred hydrated electrons reacting with the semioxidised form of the dye (Prutz et al., 1966). Intermediates produced by radiation may themselves fluoresce if excited by stimulating light. Simple modification of our apparatus has permitted the observation of the emission spectrum of the excited benzophenone ketyl radical as produced by pulse radiolysis of benzophenone in cyclohexane solution (Hodgson et al., 1975). Equipment is available to study transient phenomena using changes in electrical conductivity rather than changes involving light (Robinson and Rodgers, 1975). Use is made of a Pyrex conductivity cell of rectangular cross-section, the electrode assembly consisting of three thin platinum disks mounted parallel to each other and 0.2 cm apart. The outer pair are interconnected and carry a variable d.c. applied voltage and the centre electrode is earthed through a variable load resistor. The complete apparatus was constructed bearing in mind the problem of elimination of excessive electrical interference from the accelerator modulator, and also the interference produced by passage of the electron beam through the conductivity cell and leads. The leads, as well as the cell itself, are therefore double shielded, the outer shield in effect being an extension of the Faraday cage. FRICKE DOSIMETER

As well as pure water, in which the hydrated electron was seen for the first time (Gilbert et al., 1960), aerated acid solutions of ferrous sulphate were among the first systems to be explored by pulse radiolysis in order to demonstrate the capabilities of the technique (Keene, 1964). It has long been accepted that the mechanism consists of oxidation by OH, HO2 and H2 02. Pulse radiolysis has enabled the individual reactions to be observed, and the rate constants to be measured. Unsuspected intermediate complexes have been discovered, which specify the nature of the reactions in greater detail without invalidating the overall mechanism. When ferrous sulphate solutions are given pulses of such magnitude that radical-radical reactions can take place in competition with radical-solute reactions, the yields of ferric ions became diminished and a satisfactory agreement can be obtained between the measured yields and those calculated from the rate constants (Sehested et al., 1969). Chloride ions are often added to ferrous sulphate solutions to reduce any effect of organic impurities. In such solutions the yields under pulse conditions become diminished further than in the absence of chloride. The origin of

642

J. BUTLERet al.

this reduction in yield is the conversion of hydroxyl radicals to chlorine atoms (via an intermediate C I O H - ) which equilibrate according to: Ci + C1- ~- CI 2 . On attempting to correlate the measured yields with the measured rate constants for all the then-known relevant reactions it became clear that previously unsuspected reactions take place in the system: CI~- + HO2~2C1- + H + + 02, k = 1.1 x 109dm3mol-I s-l; CI2 + HO2~CI~ + H + + 02, k = 1.5 × 109dm3mol -l s -1. These reactions were verified by experiments with solutions which did not contain ferrous ions. Most recently yields of ferric ions have been measured at the Paterson Institute and the Riso National Laboratory, Denmark (Bjergbakke et al., 1987). In solutions containing 10 -3 mol dm -3 ferrous ions and 1 0 - 4 - 1 0 - 2 m o l d m -3 chloride ions, the measured time-course for the production of ferric ions over periods up to 100 s, by which time all reactions are virtually complete, can be satisfactorily accounted for in terms of the known reactions providing (a) some of the published rate constants are adjusted within their error limits, and (b) the reaction of chlorine atoms with ferrous ions: CI + Fe2+ ~C1 - + Fe3+; takes place with a high rate constant. The value required for a good fit is 1.3 × 10~°dm3mol-~s -~ This value is twice as high as previously concluded, but the previous measurement was an indirect one and the errors could be larger than thought. It should also be noted that the reaction may proceed through the formation of FeCI2~" rather than separate ions. PEPTIDES AND PROTEINS

Free radical intramolecular reactions within peptides and proteins have been extensively studied using pulse radiolysis. This work is of particular value as a help in understanding the mechanisms of radiation damage in proteins. It has been demonstrated that a radical produced on one amino acid residue in a protein can be repaired by another amino acid so that the radical site becomes transferred through the protein structure. Tryptophan radicals, produced by the reaction of azide radicals with peptides, can react with tyrosine units to produce tyrosine radicals: Trp" - TyrOH ~ T r p H - TyrO'. This reaction is driven by the difference in the one-electron reduction potentials of the Trp', H+/TrpH and TyrO', H+/TyrOH couples. Interestingly, the reaction goes into reverse in acid and alkaline solution (Butler et al., 1986).

The process occurs in a variety of proteins. The half life of the reaction depends on the position of the tryptophan and tyrosine units within the protein in that when the amino acids are adjacent to each other the half-life of the transfer can be as short as 10/~s whereas when the amino acids are further apart the half-lives can be as long as 0.1 s (Butler et al., 1982). From work on model peptides with hindered rotation, it has been shown that the intramolecular radical transfer has to involve direct contact between the two amino acids (Prutz et al., 1986). The tyrosine radicals decay mainly in a second order manner to form fluorescent bityrosines. This process has been observed using absorption, fluorescence (Prutz et al., 1983) and (in another laboratory) light scattering (Deeg et al., 1985) techniques. Antioxidants, if present, can prevent the transfer from tryptophan to tyrosine and can also prevent the formation of fluorescent bityrosines by reacting directly with tyrosine radicals (Hoey and Butler, 1984, 1985). Hydroxyl and Br~- radicals are known to react with methionine to form three-electron bonded sulphur radicals (Asmus et al., 1985). These radicals can also take part in intra- and intermolecular radical transfer processes. Methionine radicals produced in small peptides react with tryptophan to give tryptophan cations in the first instance (Prutz et al., 1986). The potential of the couple (TrpH÷'/TrpH) is about 1.4 V (Butler et al., 1982) and hence the methionine radicals must be strong oxidants. Work is in progress to measure their reduction potential. Radical transfer between methionine radicals and tyrosine has been observed in peptides and in ribonuclease A (Prutz et al., 1985). Recent work has revealed the place of cystine and cysteine radicals in the radical transfer process. Hydrated electrons reduce cystine to strongly absorbing anions: Cys(SS)Cys + eaq-~Cys(SS" )Cys. These radicals decay to form cysteine radicals which have been shown to be in equilibrium with tyrosine: Cys(SS" -)Cys + H ÷ -,Cys(S') + Cys(SH); Cys(S') + TyrOH ~- Cys(SH) + TyrO'. Thus, the hierarchy of the radical transfers which have been determined so far with pulse radiolysis is: Met" ~ Trp'--* TyrO" --*Cys(S') --* Cys(SS" -)Cys. However, it should be stressed that this sequence involves only five out of the 21 common amino acids and does not involve the peptide bonds. It is hoped that within the near future several new amino acids will be incorporated into the scheme. QUINONE ANTITUMOUR DRUGS

Several quinones are used clinically in the treatment of cancer including Mitomycin C, Adriamycin

Studies of moderately fast processes initiated by radiation

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the hydroquinone had been formed. Further proof against the reactions ocuring at the semiquinone stage was obtained by analysing the yields for products formed from a dose of radiation delivered from the accelerator (which would favor dismutation of the semiquinone) and a 137Cs source (which would prolong the lifetime of a semiquinone). The yield of products was the same in the two cases, consistent with their being formed from the hydroquinone. The complete mechanism was determined using pulse radiolysis over time scales of between 0.1 #s and 1 min. The scheme which fits the results is shown in Fig. 8. The intramolecular oxidation and rearrangement of the hydroquinone of Mitomycin C forming a modified quinone is especially interesting. This reaction was observed using pulse radiolysis over time scales of 33 s/division. Adriamycin is one of the most successful antitumour drugs to be used in the clinic. It is generally believed that the redox cycling reactions of the drug may be responsible for some of the toxic side-effects of the drug if not for its antitumour activity (for a review, see Butler and Hoey, 1987). When Adriamycin is reduced by one-electron reducing enzymes the daunosamine moiety is liberated. As in the case of the intramolecular reactions of Mitomycin C, it was earlier believed that this process occurred at the semiquinone stage. Pulse radiolysis studies on both Adriamycin and Daunomycin (Land et al., 1983, 1985) have revealed that their semiquinones have several p K o values with corresponding different one-electron reduction potentials. There was no evidence from these studies that the daunosamine is liberated at the semiquinone stage. However with these drugs the semiquinone is in equilibrium with the hydroquinone and the parent even at natural pH values so that loss from semiquinone and hydroquinone would be indistinguishable.

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Studies of moderately fast processes initiated by radiation 0

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Fig. 10. Reduction of Adriamycin. M o r e recent work has shown the loss of the daunosamine over long pulse radiolysis time scales (Lea et al., 1989). Essentially, after the hydroquinone/ semiquinone equilibrium has been established there is a loss of sugar over a period of several seconds yielding a tautomer of the aglycone (Fig. 9, inset). Over tens o f seconds the resulting tautomer undergoes a rearrangement such that another tautomer (7-deoxyadriamycinone) is formed, which has an absorption similar to that of parent Adriamycin (Fig. 9). Similar processes have been observed using more conventional techniques (Fisher et al., 1985), following rather similar kinetics to those observed here. The mechanism is shown in Fig. 10. Acknowledgements--This work was supported by grants from the Cancer Research Campaign. The authors wish to express their sincere appreciation to the originator of the technique, Dr J. P. Keene, for the numerous further contributions he has made over the years. The late Professor J. H. Baxendale and Drs I. Hamblett and C. Lambert have also made indispensable contributions. REFERENCES

Asmus K.-D., Gobl M., Hiller K.-O., Mahling S. and Monig J. (1985) J. Chem. Soc. Perkin Trans. II, 641. Bjergbakke E,, Navaratnam S., Parsons B. J. and Swallow A. J. (1987) Radiat. Phys. Chem. 30, 59. Butler J., Land E. J., Prutz W. A. and Swallow A. J., (1982) Biochim. Biophys. Acta. 705, 150. Butler J., Land E. J., Prutz W. A. and Swallow A. J. (1986) J. Chem. Soc. Chem. Commun. 349. Butler J., Hoey B. M. and Lea J. S. (1987a) Biochim. biophys. Acta 925, 144. Butler J. and Hoey B. M. (1987b) Br. J. Cancer 55 (Suppl. VIII), 53.

Deeg K.-J., Katsikas L. and Schnabel W. (1985) Helv. Chim. Acta. 68, 2367. Fisher J., Abdella B. R. J. and MacLane K. E. (1985) Biochemistry 24, 3562. Gilbert C. W., Keene J. P., Browne P. F. and Davy T. J. (1960) British Empire Cancer Campaign Report Vol. 38, Part II, p. 498. Hodgson B. W. and Keene J. P. (1972) Rev. Sci. Instrum. 43, 493. Hodgson B. W. and Keene J. P. (1976) Int. J. Radiat. Phys. Chem. 8, 349. Hodgson B. W., Keene J. P., Land E. J. and Swallow A. J. (1975) J. Chem. Phys. 63, 3671. Hoey B. M. and Butler J. (1984) Biochim. biophys. Acta 791, 212. Hoey B. M. and Butler J. (1985) Life Chem. Rep. 3, 80. Hoey B. M., Butler J. and Swallow A. J. (1988) Biochemistry 27, 2608. Keene J. P. (1964a) J. Sci. lnstrum. 41, 493. Keene J. P. (1964b) Radiat. Res. 22, 14. Keene J. P. (1972a) Quaderni dell'Area di Ricerca dell'Emilia Romagna 1, 49. Keene J. P. (1972b) Quaderni dell'Area di Ricerca dell'Emilia Romagna 1, 63. Kcene J. P. and Bell C. (1973) Int. J. Radiat. Phys. Chem. 5, 463. Land E. J. and Swallow A. J. (1968) Trans. Faraday Soc. 68, 1247. Land E. J., Mukberjee T., Swallow A. J. and Bruce J. M. (1983) Arch. Biochem. Biophys. 225, 116. Land E. J., Mukherjee T., Swallow A. J. and Bruce J. M. (1985) Br. J. Cancer 51, 515. Lea J. S., Rushton F. A. P., Land E. J. and Swallow A. J. (1989) In preparation. Lea J. S., Garner H., Butler J., Hoey B. M. and Ward T. H. (1988) Biochem. Pharmac. 37, 2023. Prutz W., Sommermeyer K. and Land E. J. (1966) Nature Lond. 212, 1043. Prutz W. A., Butler J. and Land E. J. (1983) Int. J. Radiat. Biol. 44, 183.

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Prutz W. A., Butler J. and Land E. J. (1985) Int. J. Radiat. Biol. 47, 149. Prutz W. A., Butler J., Land E. J. and Swallow A. J. (1986) Free Rad. Res. Commun. 2, 69. Robinson J. and Rodgers M. A. J. (1975) J. Chem. Soc., Faraday Trans. I. 71, 378.

Sehested K., Bjergbakke E., Rasmussen O. L. and Fricke H. (1969) J. Chem. Phys. 51, 3159. Wilkinson F. and Willsher C. J. (1984) Chem. Phys. Lett. 104, 272. Wilkinson F., Willsher C. J., Warwick P., Land E. J. and Rushton F. A. P. (1984) Nature, Lond. 311, 40.