Kinetic measurements on rhodopsin solutions during intense flashes

Vision Res.

Vol. 8. pp. 171-786.

Peryamon Press 1968. Printed in Great Britain.

KINETIC MEASUREMENTS ON SOLUTIONS DURlNG INTENSE THEODOREP.

RHODOPSIN FLASHES

WILLIAMSand SANDRAJ. BREIL

Departmentof BiologicalScienceand Instituteof MolecularBiophysics, FloridaState University,Tallahassee,Florida32306 (Received 5 January 1968; in revised form 22 February

1968)

INTRODUCZTION WHENa molecule of the visual pigment, rhodopsin, absorbs a quantum of light, a series of thermal reactions is started that results in bleaching, i.e. loss of red color. While this process is, itself, complex, it is further complicated by the fact that bleaching may be halted if certain of the transient intermediates absorb light (HUBBARDand KROPF,1958; WILLIAMS, 1964). Since bleaching thus involves both photic and thermal reactions, either sort (depending on conditions) can determine the rate of loss of red color. Thus, at low temperatures (less than -20°C) bleaching may be arrested at some intermediate stage when, regardless of the intensity or duration of irradiation, the red color is never completely lost. Indeed, a photoequilibrium mixture of pigments and intermediates will be established (WALD,et al., 1950; COLLINSand MORTON, 1950). Low temperatures, then, slow the thermal steps sufficiently for thermal, not photic rates to be the deciding factor in the loss of red color. On the other hand, if the pigment is maintained at room temperature or above, the thermal reactions will be very fast, and the rate at which the quanta are put in may partly or completely determine the bleaching rate (HECHT,1924; DARTNALL,1957). Indeed, at such temperatures, several of the thermal reactions are so fast that special techniques are required to study production of the transient species. Several investigators have used short, intense flashes to study the rates of some of the thermal steps (WULFF,et al., 1958; GRELLMANN, et al., 1962; Run, et al., 1964). In all of these studies the bleaching flash, no matter how short, blinded the monitor photodetector during the time the pigment molecules were actually absorbing the bleaching light. Consequently, in these experiments no observations could be made on the pigment system while it was absorbing light from the bleaching flash, and many questions remain unanswered in this area. For example: (1) Does a photoequilibrium arise during the course of an intense flash? Such equilibria have been demonstrated at low temperatures but not at room temperature in the flash situation. (2) How are the thermal and photic components related at temperatures near room temperature? Can one or the other be shown to be rate limiting in a manner analogous to the low temperature, steady light situation? Details of the kinetics of certain evoked visual responses cannot be solved without this kind of information. This paper presents the first measurements made on the rhodopsin system during an intense electronic flash. These measurements have been made by the kinetic spectroscopic technique (NORRISHand THRUSH,1956). The particular intermediate investigated 777 B

778

THEOWREP. WILLIAMS AND SANDRAJ. BREIL

was metarhodopsin II (meta II) because (I) both photic and thermal reactions are involved in its production, (2) it can be studied during nearly the entire course of the bleaching flash, and (3) its absorption spectrum (A max 380nm) has little overIap with the spectra of other intermediates or with that of rhodopsin. This last feature greatly facilitates the measurements. In this work, the rates of accumulation of meta I1 both during and after a flash have been studied using frog and cattle rhodopsins. Temperature, intensity, and extracting medium have been varied in order to study the relation of photic- and thermai-components of meta II production. MATERLALS AND METHODS

Frog retinas were ground, washed with buffer (M/15 phosphate, pH 6.5), and extracted with I per cent HTA (hexade+zyltrimethykmmonium bromide) or 2 per cent digitonin in M/15 phosphate buffer, pH 6.5. Gattk rod part&s were floated on 40 per cent sucrose, tam& with 4 per cent alum, and then extracted with HTA or digitonln. The rates of accumulation of meta IL were followed using the kinetic spectroscopic apparatus shown schematically in Fig. 1. A bleaching lksh from flash gun B, (Honeywell 65-C) is triggered manually. The F2 VI-

FIG. 1. Diagram of the kinetic spectroscopic apparatus. Bleaching is initiated by flash gun, concentrations are dete&~& by the use of test flash T. Filter, Fl, renders B,andmetaII the bkachlng flash devoid of U.V. light; F2 transmits only U.V. This combination of filters prevents blinding of the photomultlplkr tube. (Other details are given in the text.) light from this flash is transmitted through titer Fr. to the sampk of photopigment, S, which is situated in a cunstant tempera&e alumlnium block (not shown). Filter, Fl (Wratten 2-E). rendezs the bleaching flash devoid of U.V. light but does not appreciably a&t the flash output above 4X&n. Photocell, P, responds to the onset of the bkaching flash and tm the timer which, in turn, has barn preset to trigger the teat strobe, T, at the preserlbed time. (The output of P rises to tr@ring level in about 5 w). T is the General Radio “Strobotac”. The filter FZ (Corning 7-54 plus S-58) transmits the appropriate wavelengths of the test flash (maximum intensity at 38Onm) that are strongly absorbed by meta II. The test light then passa through the sample, another Fz combination, and a focusing lens (not shown). This second F2 filter transom‘ts the teat fksh e&k&y but blocks the bkachlng flash. In this way, the [signal] : fbkaching artifact] ratio is made very large. T&e test Bash then falls on the p~t~ultip~ tube, PMT, (RCA lP-28) producing a response that is displayed on an oscilloscope and photographed. Reaponsee were shown to be proportional to the intensities of the test flashes maching the PMT. Three different measurements of the intensity of the teat flash transmitted by a sample were recorded: (1) a mt of intensity, lo, before the bkach; (2) a measuremen t of intensity, Zt, at some time after the onset of the bkaching flash; and (3) a mt of the intensity, Zj, 5-10 seconds a&er the bleaching lksh. The optical density change log10 (Zs/Zz)is a measure of the meta II accunmkted at time, t, and logto (Zolrf) of the total meta II. To facilitate Ernst of expe&ents the amount of meta II present at time, t, is expressed as a percentage of the total meta II produced. An example of the three intensity measurements is given in Fig. 2. Note that Ze > Zt > Z/which indiites an accumulation of meta II with time. In this particular example the test Bashwas given 0195 msee after the onset of the bleaching flash. The bleaching artifact is sometimes scc~l as a small transient depr*lsion in the base line of the Zt trace, but in this case it is absent.

Kinetic Measurements on Rhodopsin Solutions during lntense Flashes

719

The bleaching flash has a fairly long duration compared with that of a typical kinetics experiment. It rises to a peak in its output in about Q35 msec and has a decay halftime of about 0.5 msec. Ninety per cent of its output is dissipated in 1.3 msec.

FIG. 2. Oscillogram of test flash measurements. Intensity incmases downward. Is is the test ilash intensity before bleaching; Zr is during the bleaching ilash; Z/is 5-10 set after the bleaching flash. Zs > Zt > ZJ, indicating the accumulation of meta II. In this example, the test ffash was given 0 195 msec after the onset of the bleaching flash. (The rise time of the test flash output is 20 tLstc_-too fast to permit it to show up under the conditions used for this picture.) The bleaching flash output was calibrated at several wavelengths using rhodopsin solutions as actinometers. Neutral dursity filters were used to attenuate the intensity so that a flash caused no more than 20 per cent bleachiig. Under these conditions it can be assumed that virtually no double&its on any one molecule occur. Hence, all molecules, which bleach, absorb only one quantum each (WILLIAMS,1964). Finally, assuming the quantum efficiency for bleach@ to be 1-O at all wavelengths (DART+J& et al., 1936; Ww 1965; although see P. LXEBMAN, 1%5), the qua&d output is calculated. This is shown in Fig. 3. Integrating this curve over the region, 6oonmAOOnm, it was calculated that the maximum &her output is 1.7 x 1017quanta/flash/sample area. IO

8 ”

F

h6 X

t

II

360

-1

420

1

460

I

I

SO0 540 A, nm

I

CSO

I

620

FIG. 3. Calibration of the bleaching flash output. Rhodopsin solutions were used as actinometers and were bleached with Rashes at the wavelesrgths shown. The flashes were attenuated in order to prevent doublahits on individual molecules.

780

THEODOREP. WILLIAMSAND SANDRAJ. BROIL

In contrast to the bleaching flash, the test flash rises to its peak in 20 psec and can therefore provide an almost instantaneous sampling of the meta II concentration at any time after the onset of the bleaching flash. The meta II concentrations were measured at times ranging from 0.04 msec to several seconds. Rate measurements were made at three temperatures (5, 26, and 37~5°C).

RESULTS

Figure 4 shows the rate of meta 11 accumulation at room temperature in flashed cattle pigment extracts in 1 per cent HTA and 2 per cent digitonin. The bleaching flash profile is shown as the dashed curve to permit comparison of the meta II production with the

0

FIG. 4. Rate of accumulation of meta II (MII) in HTA and digitonin at room temperature. The dashed line is the bleaching flash output (ordinate in arbitrary units). In HTA, 100 per cent meta II is present within the flash duration. This indicates little or no thermal limitation on the rate. In digitonin, it takes 10 msec for 100 per cent meta II to appear. Since the flash is finished by this time, it means that there is a thermal bottleneck in the production of meta II in the digitonin solution.

flash output. It is obvious that, in HTA, all of the meta II is present well within the flash duration. This means that a photoequilibrium concentration of meta II exists in the HTA preparation during the flash. Indeed, integrating the quanta1 output from the flash and comparing this with the rate of meta II accumulation in HTA shows that the meta II appears nearly as fast as the quanta are put in. This means that there is little or no thermal limitation on the meta II production in HTA at this temperature. On the other hand, it takes 10 msec for all of the meta II to appear in digitonin preparations; this indicates a thermal limitation on the rate of meta II production in digitonin. If these interpretations regarding thermal and photolimitations are correct, the effects of intensity and temperature should be predictable. Thus the rate of accumulation of meta II at 26°C should depend on intensity in HTA but not in digitonin. At high temper-

Kinetic Measurements on Rhodopsin Solutions during Intense Flashes atures, however, the meta I1 production

781

rates should be intensity dependent in both media. Finally, at low temperatures, the meta II rates should be thermolimited, and no intensity dependence should appear in either HTA or digitonin. Some results that verify one of these predictions are shown in Fig. 5. These data were obtained with frog pigment in digitonin solutions at an elevated temperature (37*5”C). Clearly the rate at which meta II accumulates depends on the intensity of the flash at this temperature indicating that the thermal processes are not rate limiting. Figure 5 also shows that a phot~qui~brium ~n~ntration of meta II arises during the highest intensity flash. This condition probably does not obtain during the two lower intensity flashes. (More consideration will be given to this in connection with Table 1). The overall results of varying temperature and intensity in cattle pigment solutions are summarized in Fig. 6. The reciprocal of the time required for 50 per cent meta 11 to appear, i.e. l/t,, is taken as a measure of its production rate. This rate parameter can be considered to be primarily a measure of the meta I- meta II equilibration rate because the other thermal reactions of meta II are too slow to be considered here (MATIHEWS, et al., 1963) and the use of the Fr filter prevents the usual photo reactions of meta II. Figure 6 shows that, at 37_5”C, there is an intensity dependence in both HTA and digitonin solutions; this means that the thermal processes are not controlling the rate of meta II production. At YC, no intensity dependence is evident in either medium; therefore, the thermal processes completely control the meta II rate. At the intermediate temperature, 26”C, an intensity dependence exists in HTA solutions but not in digitonin; therefore, thermal processes are not limiting in HTA but they are in digitonin. Thus, the predictions regarding photic and thermal libations are subs~tiat~ by these results. It is of interest to notice that the use of HT’A simulates the use of elevated temperatures. That is, the rates in HTA are always greater than in digitonin at any given temperature and intensity. Furthermore, the transition from thermal control to photic control occurs at a lower temperature in HTA solutions. TABLE 1.

QUANTUMEFFICIENCY OF META II PRODUCTION DURING DI~~TONIN EX~RAC’IS OFFROQRODSAT 37*5’c

LogI

00

-0.8

-1.6

TilllC (msec)

Mokcuks

0112

17.2

:z 0391 0 593 0.917

z2’ 400 41.5 44.0

Et!

36.9 18.5

0590 0935 1400

38.7 47.5 51-l

0202 0394 0593 0.933 1.420

4.7 8.1 14.7 16.0 21.3

Quanta

0.011 @020 QO28 0039 O+Iw

A FLASH.

Quwltum eflkkmy

043 8: O-41 048

782

THEODORE P. WILLIAMS

AND

J. BWIL

SANDRA

I00

80

* s S

60

40

20

0

1,ms. Fro. 5. Rate of meta II production at 37.S”C. The thermal reactions are enhanced su~iently at this temperature for there to be no apparent thermal limitation on the rate. Consequently, the quanta1 fluxes are limiting and an intensity dependence exists.

I

f 5

I IO

I

15

I

t

I

/

20 temp..T

2s

30

35

Fro. 6. Interaction of thermal and photo-components of meta II production from cattk rhodopsin in HTA and digitonin. Circles are log I = GO: triangles, -@8; squams, -1.6. The half-times of meta II production are intensity dependent at high but not at low temperatures. This is becausa high temperatures remove thermal limitations on the rate.

Kinetic Measurements on Rhodopsin Solutions during Intense Flasbcs

783

Several attempts were made to identify the point at which the meta II accumulation undergoes the transition from thermal to photic dependence but there was no sharp “breakpoint” in the meta II accumulation rate. Results similar to those in Fig. 6 were found with frog pigment with two minor exceptions: (1) In frog pigment solutions meta II rates are not strictly controlled by thermal processes at 26C” in digitonin; thus, a slight intensity dependence exists at this temperature; and (2) the rate of meta II accumulation is considerably greater at 5°C in frog pigment preparations than with cattle pigment (rt for frog is 10 msec, for cattle it is 500 msec). These measurements on the meta II accumulation provide an interesting view of the dynamic events occurring during the course of a flash. For example, early in a flash, quanta are utilized efficiently to transform rhodopsin into meta II (and other intermediates). However, if a photoequilibrium is approached or attained later in the flash, additional quanta will not be used efficiently to produce a net increase in the meta II concentration, but will simply interconvert cis and tram isomers of retinal. Thus, the ratio [meta II molecules] : [quanta absorbed] should decrease with time during an intense flash. Table 1 verifies this for the case of a maximum flash intensity (quanta per sample= l-7 x 1017). The meta II/quantum ratio falls from 0.069 to O-031 between O-112 and 0.917 msec. Lower intensity flashes yield ratios which are (1) larger and therefore indicate more efficient conversion to meta II ; and (2) change less during the flash. In fact, results for the lowest intensity flash show that nearly one out of every two quanta absorbed produce a meta II molecule, and this is constant over the entire ilash period. DISCUSSION

The present results provide information on the relation between the photic and thermal components of bleaching during a fiash and it has been shown that photo-driven equilibria can exist during the flash. Since the kinetic spectroscopic technique has not been used before in the study of visual pigments, some discussion of it should, perhaps, be given. Thus the test flash filter, Fz, transmits a rather broad band (25nm) and the possible effects of this should be considered. Fortunately, the density measurements do not seem to be grossly distorted for, when matched against the narrow band (ca. 4nm) of a spectrophotometer, the total density changes at 380nm were found in one experiment to be 0.173 in the kinetics apparatus and 0.185 in the spectrophotometer. Perhaps a more serious reservation about the measuring procedure concerns whether density changes at 380nm are related solely to changes in the meta II concentration. First, the meta II absorption spectrum is undoubtedly overlapped, even at 38Onm, by that of meta I and, to a lesser degree, rhodopsin, itself. Unfortunately, this is inherent in the system, and the results must be viewed with this qualification. Second, free retinal absorbs maximally at about 387nm and, if produced within 10 set after the flash, could distort the measurements. This is unlikely, however, because the hydrolysis of meta II to retinal is a slow process (MATTHEWS,ef al., 1963). Furthermore, if the bleaching flash is followed by a U.V.flash from another source there is considerable (ca. 16 per cent) photo-regeneration of stable pigment. This shows in a qualitative, though not quantitative way, that it is predominantly meta II, not retinal, which is being measured (MA-, et al., 1963). Finally, support for the proposal that the species being measured here is really meta II derives from some of the measurements of MA-S, et al. They measured the half-times for equilibration (of meta I and meta II) at low temperatures (cf. their Fig. 6). When an

784

THF.ODORE P.WILLIAMSAND SANDRA

J. BREIL

Arrhenius plot is made from their data and extrapolated to 25”C, a value for t+ of 1*Omsec is obtained. This is exactly the value we find (Fig. 6). The possibility exists that the bleaching Sash sets up a concentration gradient which, when viewed at right-angles, could cause distorted density readings, This difhculty can be circumvented if either (1) the flasher is situated so as to surround the cuvette or (2) the optical density along the bleaching (i.e. vertical) path is made low, ideally 0*2 or less. An approximation to the latter method has been employed here because the geometry of the bleaching fhtsher does not lend itself to the former. Thus, the solutions that were used had densities (vertically through the solution) of no more than 0.3, This figure represents a compromise between minimizing any concentration gradients and obtaining measureable density changes with the test flash. In several experiments, the fllter, Fi, was removed and meta 11 was allowed to absorb light from the bleaching flash. This had no observable effect on the rate of meta II accumulation. However, the flash output is low below 400nm even without an Fi filter. Thus, adding or su~a~ng the intensity of these wavele~~s would be expected to have little effect. Also, removal of the Fr filter naturally caused large bleaching artifacts, therefore any subtle differences which might exist would be lost due to an unfavorable [signai] : [artifact] ratio. It is possible, also, that the meta I - meta II equilibrium is fast enough in some cases for removal of one or the other meta- species by photo-reactions to be (partly) compensated by a re-shifting of the thermal equilibrium. In Table 1 the ~culation of the number of quanta absorbed during the flash was made by multiplying the fIasher output at all wavelengths (interpolated in many cases, cf. Fig. 3) by the percentage absorption of the sample at those wavelengths. It was assumed that the absorption spectrum of all absorbing species could be approximated by the rhodopsin spectrum. A more accurate procedure would require a knowledge of the concentrations of all species present at all times during the f&h, and such info~tion is not available. HTA is a cationic detergent which, in concentrations greater than ea. 7 - 8 per cent, immediately denatures rhodopsin in our usual preparation (although compare WILLIAMS, 1968). These detergents cause denaturation because they “wet” the hydrophobic structures of proteins. This, in turn, weakens the protein fabric and causes widespread conformational changes. Since such conformational changes occur upon the production of meta II (MA-~THBWS,et al., 1963), HTA could enhance the meta II production rates because it facilitates these changes. One of the objectives of this work was to elucidate the relation between the photic and thermal steps so that the kinetics of certain evoked visual responses might be better understood. For example, PAK and EBREY (1965) have studied some of the kinetics of the early receptor potential, ERP. Their Arrhenius plot of the rate of generation of the “Ri response’* shows that at 5°C the production of Ri becomes almost entirely photically not thermally limited. This is to say that at 5°C the rate of rise of Rr depends almost entirely on the rate of rise of their flash output and not upon the rate of some thermal reaction. Thus, their data points at 5°C should not be included in their “activation energy” analysis. Another point is that they took the reciprocal of the time to the peak of 1pr as the measure of the rate of response production. This will obviously not give a true rate constant for the process. Perhaps a better procedure for correlating the ERP production with particular chemical reactions would be to evoke the response (in an excised retina, say) while at the same time monitoring the color changes by kinetic spectroscopy.

Kinetic Measurements on Rhodopsin Solutions during Intense Flashes

785

Acknowle&ement-This work was supported by USPHS Grant NB-07140 and in part by a contract with the Division of Biology and Medicine, U.S. Atomic Energy Commission.

REFERENCES Co-,

F. D. and MORTON,R. A. (1950). Studies in rhodopsin 3. Rhodopsin and transient orange.

Biochem. J. 47, 18-24. DARTNALL, H. DARTNALL, H.

J, A. (1957). The Visual Pigments, John Wiley, New York. J. A., Gocmavts, C. F. and LYI-HGOE,R. J. (1936). The quantitative analysis of the photochemical bleaching of visual purple solutions in monochromatic light. Proc. R. Sot. A X%,158-170. GRELL~~ANN,K. H., LIWNGSKJN, R. and PRAn. D. (1962). A flash-photo&tic investigation of rhodopsin at low temperatures. Nature, J&ad. l93,1258-1260. HECHT,S. (1924). The relation between the intensity of light and the rate of bleaching of visual purple. J. gen. Physiot. 6,731-740. HUBBARD,R and KROPF, A. (1958). The action of light on rhodopsln. Proc. nutn. Acad. Sci., U.S.A. 44, 130-139. L~EBMAN, P. (l%S). Cold Spring Harbor Symp. quant. Bioi. 30, p. 315. Discussion following the paper by Hubbard, Bow&s and Yoshizawa. MA-S, R. G., HUBBARD,R, BROWN,P. K. and WAS_D,G. (1%3). Tautomeric forms of metarhodopsin. J. gett. Physiool. 47,215-240. Noarusx, R. C. W. and THRUSH,B. A. (1956). Flash photolysis and kinetic spectroscopy. Quart. Rev. 10, 149-168. PAK, W. L. and BBREY,T. G. (1965). Visual receptor potential observed at sub-zero temperatures. Nature, Lottd. 205,484-486. PRATT, D.

G., Lrvr~osro~, R. and GRELLMANN,K. H. (1964). Flash photolysis of rod particle suspensions.

Photochem, Photobid. 3,121-127.

WALI), G., D~~ELL, J. and ST. GE~XGE,R. C. C. (1950). The light reaction in the bleaching of rhodopsin. Science, N.Y. 111, 179-181. WILLIAMS, T. P. (1964). Photoreversal of rhodopsin bleaching. J. gm. Physiol. 47, 679-689. WILLMMS,T, P. (1965). Rhodopsin bleaching: Relative effectiveness of high and low intensity flashes. Vision Res. 5, 633-638.

W-MS,

T. P. (1968). On the preparation of very concentrated solutions of visual pigments. Vision

Res. 8, 315316.

WULFF,V. J., ADAMS,R. G., Lw H. and -N, E. W. (1958). Effect of flash i~u~ti~ on rhodopsin in solution. Ann. N. Y. Ad. Sci. 74,281-290.

--Kinetics

ptaKIscoprisuscdtoinv~therateatwhich~odopsinfI (meta II) accumulates during (and, in some caaea, after) @aah photolyais of rhodopain solutions. This process is studied as a function of &ah intermity, temperature, and extract& medium. It is shown that the rate at which meta II aoxmulates is photo-limited at high temperatures but thermo&nited at low ones. Photoequllila are ahown to exist, under certaincircumstances,d~gthecouraeofaflash. TbequantumetE&ncyofmetaII pr~~~isshowntobc~dependgttwb~intcnse~arc\Frcdbutnotwhenweak Saahesareused. It~su~~~at~s my may be useful in investigating tbe chemical basis of the earIy receptor potential.

R&aum&Gn &t&e par spectroacopie cin&lque la teneur avec laquelle la m&arbodopsine II (m&a II) s’accumuk pendant (et, dans cermins cas, apr&s)la pbotolyse par un &Jalr lumineux de solutions de rhodopsine. On &tulle ce processus en fonction de I’intenslte de S&lair, de la temp&tture, et du mllleu d’extractlon. On montre que la teneur ~~~ti~ de r&a II eat sujette B une photo-litnltation aux temperatures &levees et B me thermo-Station aux basses. On montre que des photoequilllres existent, sous certaines conditions, durant l’tclair. On montre que l’cfbcacite auantiaue de la uroduction de m&a II d&end du temns oour des tclalrs intenses et non pour de faibl& On &@re que la spectros&ple &et&e ‘Gut etre utile pour rechercber la base chimique du potentiel prtcoce de r&epteur.

Tmooa~

786

P. WILL&W AND SANDRA J. %EiL

Znsammenfaammg-Mit Methoden der kinetischen Spektroskopie wird die Geschwindigkeit untersucht, mit der sich Metarhodopsin II (meta II) w&rend (und in einigen FNen nach) der Blitz-Photolyse van Rhodopsinii%mgen ansammelt. Dieser Vorgang wird in AbhBngigkeit van der Blitzhelligkeit, Temperatur und dem Auszugsmittel, untersucht. Es wird gezeigt, da6 die ~~~ mit der sich meta II ansammeit bei hohen Temperaturen lichtbegt~W und bei &dr4gen w&mew ist. Die Existenz van Lichtgleichgewichten kann unter m Umat&den w&hrend einea Blitzabiaufs nachgewieaen werden. Die Quantenau&e&e der meta II-Pro&Won ist zeitabh&&, WCM starke Blitze verwendet werden, bei schwa&en jedoch n&t. Es wird vorgea&@en, da0 die kinetische Spektroskopie nut&r&end bei der Unterauchung der chemischen Grundlapn des frUhen Rezeptorpotentials einges&t w&en kann. KEEB cnexTpoCxoxI%W 6azna xm10~~53o~a~a AJIR foro, ~06~ HCCJIe~OBaTb CKOj?ocTb C XOTOpOtk MCTapOAOIlCHH 11 (MeTa II) aXXyMyJIlipyeTCSl B TweExie (a, B HelcoTopHx cnysarrx noCne) @OTOJilWiCtl, xIpoH3Box&iM0r0 BCnbHLIKoi2 cwa B pacrmpe polxoaceura. ~TOT nporrccc H3yneH xax &Hnuis HHTeIsxBHocTH B, TeIWUC~Typhl R 3mCHTti. ~OKiuaaO, ¶TO CKOpCCTb C KOTOpOii MeTa 11 Tehcmeparypax, aKxyMyJIlipyeTcn OIxpeJxeJIxeTC~CBCTOEWMH ycJIoBE%MH qxf wscouix HO TapMwacrr Omaha IZpE HH3KHX. i-iOiW%iHO, ‘JTO CyU&CTByeT CBeTOBoe ycxo~mx, 3 TewiHe wxbnum. fIopaBHoWuzc, IKpsi WKoTopWx orIpe7 Ra3aK0, VT0 KlWITOBidi BbIXOA IIpOAyKUliH Mora II388KCKT. OT BPeMeHH, eCJIH yIIOTpe6mfl HHTOHcHBlIa$? BCIIbIlIIILa, HO He 3aBHCHT OT HeTO, eCJ!H HCIIOJIb3yeTCSI c~1a6an BCIIMIIIK& ~[~~OJI~WTC~, ‘iT0 KHHCTH’fWKU CRel’iTpOCKOIMII MOXeT 6bITb IIOJIe3IiOti IIlJE HCf2C~OlJllHH.ti XHMH’ICClCO# OCHOBbI PaHHiW0 ~WtITOpHOrO IlOTeH-

Pe3mle -

PeMa.