Synchrotron X-ray studies of biological preparations at low temperatures with optical monitoring of sample integrity

ANALYTICAL

BIOCHEMISTRY

1%4,248-257

(1982)

Synchrotron X-Ray Studies of Biological Preparations at Low Temperatures with Optical Monitoring of Sample Integrity B. CHANCE,* W. PENNIE,*

M. CARMAN,*

V. LEGALLAB,*

AND L. POWERS?

*Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania I91 04; and TBell Laboratories, Murray Hill, New Jersey 17974 Received November 30, 198 1 Increasing use of X-ray absorption spectroscopy (edge and EXAFS) to determine the local structure of active sites of biological molecules has caused greater interest in and attention to the nature of X-ray damage and the integrity of the sample. In a complementary way, efficient X-ray photon collection from the sample is required in order to maximize data acquisition and minimize sample damage. This report describes systems currently in use at Stanford Synchrotron Radiation Laboratory and Cornell High Energy Synchrotron Source, comprising two types of low-temperature techniques for sample protection and fast X-ray photon detection of large solid-angle collection.

X-ray synchrotron studies now comprise a large segment of biological structure determinations (l), particularly where membrane proteins or insoluble proteins, otherwise uncrystallizable, are studied. The basic problem of establishing and maintaining sample integrity under X-irradiation becomes more pressing as more intense synchrotron radiation has become available with dedicated operation of otherwise parasitic storage rings (SSRL, CHESS) or with the new national light source synchrotron operation, NSLS. This article describes new developments aimed, on the one hand, toward protecting the sample by low temperatures and monitoring its properties with online optical and offline EPR techniques and, on the other hand, toward technological developments for minimizing the incident photon flux upon the sample by maximizing the capability of fluorescence signal detection. This article is in two parts: first, appropriate cryostats are described for maintaining the biological sample at such a temperature that migration of hydrated electrons is minimized; second, development of crite0003-2697/82/120248-10$02.00/0 Copyright Q 1982 by Academic Press. Inc. All rights of reproduction in any form reserved.

248

ria for increasing the efficiency and speed of photon collection from the sample is discussed. Examples of the use of this equipment at SSRL are provided. MATERIALS

AND METHODS

EXAFS system. The complete system for studies of biological materials of low metal content with fluorescence detection and optical sample monitoring systems is illustrated in Fig. 1. The monochromatic X-ray beam is further collimated by a slit, and the incident beam intensity (Z,) is measured by an ionization chamber. Immediately after the I,, chamber is the cryostat (Types I and II are described here), which has double Mylar windows to minimize thermal gradients and moisture accumulation, and beneath them is a low-temperature chamber in which, for beamline 11-3, is located two elliptical 5 X 1 l-mm sample holders-one irradiated, the other an unirradiated reference sample. The X-ray beam is aligned with the sample area so that there is no elastic scattering from the sample holder. While fluorescence has a spherical distribution from the sample, the elastic scatter-

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249

MONITORING

x- ray Beam

FIG. I. Diagram of X-ray cryostat detection and optical monitoring components of the Johnson Foundation edge/EXAFS system.

ing has a donut-like distribution whose minimum lies along the direction of polarization of the X-ray beam (horizontal). The detector array was centered at right angles to the beam in the horizontal direction where a maximum ratio of fluorescence to elastically scattered radiation is obtained (1). A filter which selectively absorbs the elastically scattered radiation was used to enhance this ratio (2). The filter material contains an absorber having an atomic number of one less (2 - 1) than the absorber in the sample (2) so that its K absorption edge falls between the energy of the elastically scattered radiation and the Ka fluorescence of the sample, selectively absorbing elastically scattered radiation. The signal-to-noise ratio of such a method is given by

I S’N = (Zf,f+ $‘+ Zfl)“2 ’

(1)

where Z,, and Zssfare the fluorescence and elastic scattering, respectively, from the sample transmitted by the filter and Zrris the

fluorescence from the filter caused by absorption of both elastically scattered and fluorescence radiation from the sample. Since the latter also has a spherical distribution, the filter size was the minimum required to intercept sample fluorescence and elastic scattering that would normally strike the detector array and was positioned as far from the array (or as close to the sample) as possible to minimize the solid angle of collection of this filter fluorescence (3). The thickness of the filter or filters was detecmined from the above equation to give a maximal signal-to-noise increment over that obtained for the particular sample without filter(s). (See Tables 1 and 2 below.) Clearly the larger the solid angle collected by the detector array, the more fluorescence counts measured. However, the elastic scattering contribution increases with a solid angle; the maximum signal-to-noise increment is obtained with collection of a cone of revolution of ~40’ from the horizontal. Finally, only radiation from the sample which has passed the filter should reach the

250

CHANCE

FIG. 2. Illustration of the effect of freezing on water proton movements as determined by nuclear magnetic resonance, courtesy of J. S. Leigh, Jr.

detection array; thus shielding by lead, for example, for iron absorption studies, is required, and not aluminum as used by Cramer and Scott (4) since the latter contains iron impurities which contribute to the background. Three scintillation counters of the detection array out of a total group of seven are shown in Fig. 1. The signals from the photomultipliers are delayed by multiples of 25ns increments to avoid pulse pileup from response to an X-ray pulse (1) into a diode adder, which further avoids loading of one photomultiplier by another. A wide-band amplifier of gain 10 drives the signal through a long cable out of the hutch to a leadingedge discriminator and the hex scaler controlled by the PDPl l/O3 computer. This arrangement avoids the costly duplication and time-consuming adjustment of amplifiers and discriminators generally used (4) and provides low noise amplification of the signal variable through the dynode string of the photomultipliers. The sample is monitored optically by a split-beam spectrophotometer affording two channels for illumination (5), one for the Xirradiated, measured sample, and one for the unirradiated reference sample, both of which are contained in the cryostat and illuminated by the same wavelength of light. The reflectance from these two signals is then timeshared onto the detector, synchronously demodulated, and presented as an absorbance difference as a function of the wavelength.

ET AL.

Cryostats: Type I. The effect of X-irradiation on biological samples has been reported earlier (6); with fluxes of 10’“-lO” photons/s, hydrated electrons (e,) are produced in the sample at -2.5 PM/S, leading to reduction of a 1 mM sample of cytochrome oxidase at the rate of 0.2%/s at room temperature. This rate of production of hydrated electrons remains the same as the protein concentration in the sample is lowered. Thus, a single 7-min X-ray absorption measurement cannot be made before a 1 mM Fe sample is completely reduced. This production rate is difficult to change but one simple method for altering the effective rate of acceptance of hydrated electrons by the sample is to immobilize them by freezing (7). Figure 2 illustrates freezing of the water protons in a solution of 35% ethylene glycol and water. It is seen that motion of the water molecules becomes NMR undetectable at -80°C leading to a diminution of the diffusion of molecules in water of lo’- to 108-fold (8). Thus, mobility of (e,) would be expected to decrease in a similar manner (3). Under conditions of limited diffusion of the hydrated electrons, the efficiency of their utilization by the iron and copper centers of the metalloproteins is now acceptably small, and the stability of the samples is vastly increased to the extent that optical methods are unable to detect changes of the sample properties in excess of 10% over several consecutive shifts, i.e., 24 h of X-ray exposure. Thus, cryostats have been developed to operate in the region of -100°C and to afford at the same time the following features: (a) ready sample changing (b) rapid thermal equilibrium (c) wide-angle acceptance, transmission, and emission of X-irradiation (d) convenient mounting of filters for minimizing elastic scattering (e) X-ray-transparent windows usually made of Mylar films (f) exposed surfaces which operate for many hours without accumulating frost

SYNCHROTON

X-RAY

STUDIES

(g) possibility for optical observation via reflectance spectrophotometry, usually of the back side of the sample holders. The loss of fluidity by freezing requires that chemical changes of the sample be carried out in a series of steps, each step is trapped at a particular point in the reaction. This procedure is, however, consistent with the long time currently necessary for the acquisition of synchrotron X-ray data (- 10 h) at concentrations of -1 mM. The cryostat is cooled by cold dry nitrogen gas in the same way as the “triple-trapping” cryostats used in low-temperature spectrophotometry (9). This cryostat gives a nearly 180” viewing of the sample as indicated by the hemispherical Mylar temperature shield that enables the central space to be maintained cool and pressurized by the flow of cooling nitrogen. The intermediate space is pressurized by a flow of warmer nitrogen at a pressure of several centimeters of water, and the external face is warmed by one or more polyethylene tubes held in place by Mylar tape near the point of incidence and emergence of the Xray beam. The flow of gases through the chambers can be adjusted to avoid significant frost accumulation over an 8-h period in high humidity. A drawing of the connections from the liquid nitrogen Dewar flask through the transfer line and the cold spaces of the cryostat containing the reference and measured sample holders is indicated by Fig. 3. The front-face view illustrates how the gas passages encircle the samples and exit via the monitor thermocouple. The top view on the right illustrates how the samples are illuminated by the X-ray beam through the Mylar windows, how the warmer gas space modulates the thermal gradients from the cold space to the atmosphere, and how room temperature nitrogen impinges upon the external face to give frost-free surfaces for the X-rays. The construction of the cryostat minimized thermal losses by using Styrofoam whenever possible.

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251

A plug in the back of the cryostat admits the bifurcated, 2-m-long light guide for reflectance spectroscopy. Light in the region of 400-900 nm is time-shared between the two samples. The fibers for reflectance measurements are interwoven among those for illumination, and thus illuminate the photomultiplier. The back of the sample itself is viewed optically through its back Mylar wall. Both fluorescence and transmission modes of X-ray absorption measurement can be used with the cryostat design. For transmission, the cryostat face is normal to the Xray beam, and the above-mentioned aperture for the monitoring spectrophotometer light guide is fitted with a similar plug containing a Mylar window. An ionization chamber then measures transmission through the sample ( 10). For use in fluorescence modes, the cryostat is turned to an angle of 45”, and the X-ray fluorescence is collected at 90” to the incident beam (Fig. 1). When this cryostat is used with a filter for absorbing the elastically scattered photons, the filter is mounted on a lead-covered projection from the detecting scintillation counters. When a LiF lens is used (1 l), the filter becomes unnecessary. Cryostats: Type ZZ. The second design is a more sophisticated one (Fig. 4) in which the windows are only the size for excitation and optimum fluorescence detection. This configuration affords a solid angle viewed from the detector that only slightly exceeds the angle which is covered by the available detector array. The gas passages are similar to those of Type I. The cryostat is especially adapted for use with readily removable filters of appropriate size and has a filter location that is as close as possible to the sample. Thus, in this case, no projection from the scintillation detectors is necessary to mount the filter; nevertheless, as the diagram shows, shielding of the detectors from any radiation not passing through the filter is necessary and is provided by a lead-foil “snout” that makes contact with the cryostat

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CHANCE

ET AL.

FIG. 3. Diagram of cooling gas flows in Type I cryostat.

window. This cryostat may be run indefinitely at -130°C without any evidence of frost accumulation on the windows provided dry Nz gas is used. Detectors. Figure 5 illustrates the general connections and geometry in the detector system, amplifying and enlarging upon Figure 1. The X-ray signal emergent from the cryostat is filtered (2), and an array of seven detectors is placed six to eight inches from the sample, allowing fluorescence collection of a cone of revolution of -40” around the horizontal. The detectors are EM1 Type 4813B 1Cstage photomultipliers (EMI Gencorn, Plainview, N. Y.) provided with Pilot B scintillating plastic (Nuclear Enterprises, Edinburgh) of 1%ns lifetime and having sufficient thickness that >90% of the X-ray photons at 7-9 KeV are absorbed (9). Light sensitivity is avoided by layers of aluminized

Mylar which are sealed tightly to the housing containing the scintillator, photomultiplier, and magnetic shield. The dynode circuit is resistive through the first seven dynodes and dynodes 7 to 14 are provided with a voltage-regulated circuit in order to amplify with fidelity the signals occurring at high count rates to avoid their rejection by the leading-edge discriminator. The loo-ohm output of the photomultiplier is connected to the amplifier via a Shottky diode (Fig. 6) so that when the negative output due to a single-pulse output from the photomultiplier is observed at the anode of the photomultiplier, the current is drawn through the 50-ohm summing resistor. The characteristic impedance seen by any one photomultiplier is only the 50-ohm resistor and is not paralleled by the capacitance of the cables to the other photomultipliers. Thus, the diode adder allows matching of

SYNCHROTON

X-RAY

STUDIES

WITH OPTICAL

Reference

Sample

Measured

Sample

pi&b;

, .

FIG.

,

-~

y

‘Mn

Fluorescenl

Filler

4. Diagram of Type II cryostat.

the output of all of the photomultipliers as long as they do not conduct simultaneously, since the shunt capacitance of the Shottky diode is negligible at these frequencies. In view of the time structure as proposed for NSLS at Brookhaven or as now exists at SPEAR at Stanford, the 150- to 400-ps bunch of electrons could possibly elicit excitation from the sample of more than one

X-ray fluorescence photon. While the probability of more than one photon striking the same detector is small, pulses could arrive at the diode adder simultaneously from different tubes, creating problems of pileup and bad impedance matching. A delay line of 50 ohm characteristic impedance is inserted into the anode of each one of the photomultipliers so that a multiple of 25 ns delay 5oJ-l

Schottky

Diodes

L

f

t

Amp&f w 1

m

,

I 1

I 1

MCA

Hex Scaler External

Gain adj

-24 KV FIG.

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MONITORING

5. Connection diagram of scintillation detectors.

Units

E&%3

254

CHANCE

intervenes between the output of each one. The total delay is 170 ns, corresponding to a possible repetition frequency of 6 MHz, as compared to 1.28 MHz for the SSRL ring frequency. A leading-edge discriminator (50 MHz counting capability) serves to eliminate lowenergy output from the photomultipliers, and a limit on the high-energy outputs is not required, since higher-energy signals are eliminated by the low-pass X-ray filter. The hex scaler then receives the output of the leading-edge discriminator, which is monitored by a rate meter. Band width of circuit. The rise and fall of the output of the photomultiplier as viewed through the wide-band amplifier occur in 5 ns. This is consistent with the 50 MHz bandwidth obtained in the discriminator and the hex scaler. Thus, the discriminator/counter deals adequately with the pileup problems in the seven detectors and is more than adequate for the average count rates per tube of several megahertz as obtained with 10 or 20 mM myoglobin on beamline II-3 at SSRL. Ringing. Matching of the coaxial cables with their characteristic impedance (12) is essential to avoid reflections and ringing from poorly matched cables. The task becomes empirical as multiple units of various input admittances are connected in parallel, with the result that the simplest assembly of items is easiest to manage, as shown in Fig. 6. For example, auxiliary test equipment, monitors, etc. may be operated from

6

FIG.

6. Diode-summing circuit for photomultipliers.

ET AL. TABLE

1

REPRESENTATIVE OUTPUTS OF DISCRIMINATOR FUNCTION OF LEVEL SE-ITING

Discriminator

50 eV below edge (Is) 100 eV above edge (IA) signal/noise*

AS A

level (mV)

0

20

50

100

241” 313 140

200 274 145

172 237 150

145 200 150

Note. Measurement of Cu component of 1 mM cytochrome oxidase (-130°C). SSRL beamline I-5, 3.0 GeV, 74 mA, Cu EXAFS. a In (X10-r) cps. b For a l-s counting interval.

a buffer amplifier with a narrower bandwidth and is the assembly employed effectively when the count rates are 10.1 MHz. Elastic scattering background. In each experiment, the elastic scattering background is observed below the absorption edge, for example, copper or iron, at l-2 mM concentration. The increase of the count rate is measured as the energy is increased above the edge (see Tables 1 and 2) in which count rates for typical samples are given in kilocounts per second (see Tables 1 and 2) a measure of the elastic scattering and fluorescence signal. Usually, this corresponds to 0.1-0.3 MHz with the background equal to or greater than the edge jump (the condition of beamline II-3 had deteriorated in the most recent run when only 0.1 MHz of edge jump was obtained in a signal of 0.4 MHz for a 5 mM iron sample). Setting of the discriminator level. The leading-edge discriminator was set by comparison of the count rate above and below the edge. The discriminator level was raised until the count rate loss above the edge was more than that below, indicating that signal and not background was being rejected. In this manner it was possible to obtain the profile of discriminated counts above and below the edge for a specific sample and optimize the operating conditions, as shown in Tables 1 and 2.

SYNCHROTON TABLE

X-RAY

STUDIES

2

REPRESENTATIVE OUTPUTS OF DISCRIMINATOR AS A FUNCTION OF LEVEL SETTING Discriminator

40 eV below edge 100 eV above edge signal/noiseb

level (mV)

10

30

50

488” 721 950

69 451 1900

14 101 960

Note. Measurement of Fe component of 1 mM cytochrome oxidase. SSRL beamline 11-3, 3 GeV, 90 mA Fe EXAFS (May 1980). LIIn (X 1Om3)cps. b For a l-s counting interval.

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increasing the S/N approx. eightfold; on the assumption that only “white” noise (energy independent off) is present. Obviously, more scans were required of beamline I-5 than II3. Comparison with the work of others on cytochrome oxidase is difficult because of the lack of information on ZB and IA values in most publications ( 15). As sample concentrations are decreased, the magnitude of background counts will become more serious and represent a fundamental limitation to the accuracy of the experiment. DISCUSSION

Sample monitoring. Sample monitoring by optical and EPR techniques has been described in two publications that warn of the dangers of (a) redox alteration; (b) free radical formation; and (c) the destruction of the sample itself (13,14). Continuous monitoring seems to be highly advantageous in some cases and essential in others to safeguard that the sample properties are insignificantly (I 10%) changed by the synchrotron X-ray beam. PERFORMANCE

Tables 1 and 2 show the performance of beamlines I-5 and II-3 at SSRL in measuring the copper and iron components of 1 mM cytochrome oxidase, respectively. Equation [l] above, when converted to the quantities of Tables 1 and 2 gives:

S/N

=

IA - IB ‘\ / 1 v 1+ (zA,z:,

111 - 1

where ZB and IA are the count rates below (B) and above (A) the absorption edge, respectively (see Tables 1 and 2). The S/N values for a l-s counting interval are given. It is apparent that the focused beamline gives better performance in these tests. Usually 30 scans of 2 s/point are obtained

The highly significant problem of obtaining structural and charge density data on the metal atoms of enzymes and proteins that are keys to catalytic, regulatory, and transport function in man and animals has prompted a greatly increased emphasis and dependence upon X-ray synchrotron techniques. A number of key problems have emerged: The first is that general insensitivity of the X-ray detection methods favors the study of high concentrations of proteins of small molecular weight such as were used in the initial experiments on hemoglobin ( 10) and rubredoxin ( 16). For example, hemoglobin, although having a molecular weight of 64,000, has 1 iron/ 16,000 molecular weight subunit (17), and thus is readily obtained at high concentrations, as is the case with rubredoxin. Cytochrome oxidase is a widespread biomolecule in which the concentration of the iron and copper centers is relatively dilute. It is insoluble in water, soluble in detergents, and generally difficult to purify. Even under optimal conditions, 1 iron/60,000 molecular weight is as much as could be expected. For example, the cytochrome oxidase at optimal purity contains 10 nmol iron/mg protein (3,18), while myoglobin and hemoglobin contain over 60 nmol iron/mg protein. Thus, there is an intrinsic factor of 6 in difficulty. Furthermore, and most importantly, it is not possible to obtain

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CHANCE

cytochrome oxidase in a chemically manageable form at such concentrations; detergent, water, etc. are inevitably present so that the real factor of a relative concentration rather than being -6 is probably > 10. On that basis, X-ray exposure times of lo2 longer are necessary to obtain the same signal-to-noise ratio. Even here, complications arise, in that cytochrome oxidase studies actually require as good or better a signalto-noise ratio because of the need to isolate and separate the iron and copper contributions from one another. The result is that instead of, for example, 8 h running time required for a single derivative of hemoglobin, cytochrome oxidase would require over 100 times as long, an impossible figure under present stringencies of beam time allocation. Thus, the efficiency of data collection has been increased so that hemoglobin, even in intact red blood cells, may be satisfactorily recorded by the technique described in this article in one 7-min scan with an accuracy comparable to that obtained in the original experiments (10). Satisfactory data for a single derivative of cytochrome oxidase are now obtained even with 20-25 scans of 7 min each under optimal conditions of beam stability, purity, and efficiency of data collection (3). Concomitant with the increase of X-ray exposure, sample damage of two types arises: ( 1) redox changes due to reduction by hydrated electrons and (2) accumulation of a variety of trapped radicals of concentrations exceeding that of the enzyme itself. These two types of damage are minimized by low-temperature operation. This decreases the mobility of the radicals which otherwise would have migrated to the iron and copper centers of cytochrome oxidase, the most ready acceptor of electrons. Instead, only those radicals generated in the immediate vicinity of the iron and copper

ET AL.

atoms can be used, and this appears to be an acceptably low level, i.e., less than 10% in roughly 24 h of irradiation. The trapped radicals themselves, unlike hydrated electrons, will dismute readily, as is observed by warming of the preparation (6). Thus, the trapped radicals can be estimated only if the sample was maintained continuously at low temperatures. In general, however, any means by which the efficiency of photon collection can be increased and the samples preserved are keys to future studies using these methods. ACKNOWLEDGMENTS The Johnson Research Foundation provided support through NIH Grants GM-27476, GM-27308, HL18708, and HL-28385 and NSF Grant PM-80-26684. SSRL Project 423B was supported by the NSF through the Division of Materials Research and the NIH through the Biotechnology Resource Program in the Division of Research Resources in cooperation with the Department of Energy. The Johnson Foundation Instrument Shops constructed the detection equipment and are prepared to construct similar devices for interested users.

REFERENCES 1. Winick, H., and Doniach, S. (eds.). (1980) Synchrotron Radiation Research, Plenum, New York. 2. Stern, E., and Heald, S. M. (1979) Rev. Sci. Instrum. 50, 1579- 1582. 3. Powers, L., Chance, B., Ching, Y., and Angiolillo, P. (1981) Biophys. J. 34,465-498. 4. Cramer, S. P., and Scott, R. A. (1981) Rev. Sci. Instrum. 52, 395-399. 5. Chance, B., and Lee, C. P. (1969) FEBSLett. 4(3), 181-189. 6. Chance, B., Angiolillo, P., Yang, E., and Powers, L. (1980) FEES Lett. 112(2), 178-182. 7. Chance, B., Moore, J., and Ching, Y. (1982) Anaf. Eiochem., in press. 8. Fletcher, N. H. (1970) Chemical Physics of Ice, p. 160, Academic Press, New York. 9. Chance, B., Graham, N., and Legallais, V. (1975) Anal. Biochem. 67, 552-579. 10. Eisenberger, P., Shulman, R., Kincaid, B., Brown, G., and Ogawa, S. (1978) Nature (London) 274, 30-34.

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11. Marcus, M., Powers, L., Storm, A., Kincaid, B., and Chance, B. (1980) Rev. Sci. Instrum. 51(8), 1023-1029. 12. Chance, B., Hughes, B., MacNichol, E., Sayre, D., and Williams, F. (1949) Waveforms, MIT Radiation Laboratory Series 19, Boston Technical Publishers, Lexington, Mass. 13. Yonetani, T. (1967) in Methods in Enzymology (Estabrook, R. W., and Pullman, M. E., eds.), Vol. 10, pp. 332-339, Academic Press, New York. 14. Powers, L., Blumberg, W., Chance, B., Barlow, C.,

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Leigh, J. S., Smith, J., Yonetani, T., Vik, S., and Peisach, J. (1980) Biochim. Eiophys. Acta 546, 520-538.

15. Scott, R. A., Cramer, S. P., Shaw, R. W., Beinert, H., and Gray, H. B. (1981) Proc. Nut. Acad. Sci. USA

78(2),

664-667.

16. Shulman, R. G., Eisenberger, P., Teo, B. K., Kincaid, D., and Brown, G. (1978) J. Mol. Biol. 124, 305.

17. Theorell, H. (1934) Biochem. Z. 55-63. 18. Yonetani, T. (1960) J. Eiol. Chem. 253, 845-852.