Photolysis of metarhodopsin II: rates of production of P470 and rhodopsin

Yidon

Res.

Vol.

8, pp. 1457-1466.

Pcrpmon

PHOTOLYSJS OF PRODUCTION

Press

1968.

PriBtd

in Great

Mtain.

METARHODOPSIN OF P470 AND

II: RATES RHODOPSINl

OF

THEODOREP. WILLIAMS Departmentof BiologicalScienceand Instituteof MolecularBiophysics, FloridaStateUniversity,Tallahassee, Florida32306

(Received 8 August 1968) INTRODUCTION IT HAS long

been known that the prosthetic group of unbleached rhodopsin is based on retinal and that absorption of light by rhodopsin yields the all-trans isomer (Hunnm and WALD, 1952). The system is rendered unstable by this act and “dark” (thermal) reactions follow, which result in the loss of red color. Equally well-established is the phenomenon of photoreversal of bleaching. This reversibility leads to the estab~s~ent, at low tem~ra~es, of phot~q~b~a among thermally stable and thermally unstable chemical species (COLLINSand MORTON, 1950; WALD et al., 1950; YOSHIZAWAand WALQ 1963). It further provides the explanation for (a) the observed upper (50 per cent) limit on bleaching with short, intense flashes (HAGINS, 1955; HUBBARDand K.ROPF, 1958; WILL-, 1964), (b) deviations from the Bunsen-Roscoe law (WILLIAMS,1965), and (c) a quantum efficiency of bleaching that varies with time AILS and BR~L, 1968). Thus, in the reactions of rhodopsin, light can play a dual role: It can start the bleaching process and it can stop it, these apparently antithetical acts being based on the photoisomerization of the prosthetic group. MATTHEWSet al. (1963) combined their results with those of many other workers and proposed the mechanism of bleaching shown in Fig. 1. R is rhodopsin (1 l-&s), I is iso-rhodopsin (9-&s), P is pre-huni, L is huni and MI and MII are tautomeric forms of metarhodopsin. The P465 is an un-named species absorbing ma~mally at 465-470 mn, Straight arrows represent thermal reactions and wavy arrows are photo-reactions. The P, L, and M species are thermally unstable and decay to products. They are unstable presumably because they possess the all-rrans chromophore and they can be re-stabilized only if they absorb the additional light which re-isomer&s retinal to the appropriate (9. or ll-) cis configuration. The P465 species is perhaps the 134s isomer (cf. MATTHEWSet al.) and is unstable. A previously unanswered question, and one to which this paper is addressed, is whether or not thermal or “dark” components exist in the photoreversal steps. In a general way, the existence of these dark components has been proposed by Wald and several of his co-workers (YOSHIZAWA and WALD, 1963; MA~WS et al., 1963). In the present work the rates at which meta II is converted into rhodopsin and the 465 pigment 11&s

This workwas supportedby USPHS Grant NB-07140. 1457

THEODORE P. WILLIAMS

1458

Fro. 1. Proposed mechanism of rhodopsin bleaching adapted from Matthews et al. Wavy arrows are photochemical steps and straight arrows are “dark” reactions. Symbols are explained in the text.

have been measured. Previously, these rates had not been measured since, as will be seen, they are extremely fast. Besides providing this new kind of data, this paper presents arguments regarding the nature of the protein-chromophore interactions in photoregenerated rhodopsin and the 465 compound. METHODS

AND MATERIALS

Isolated cattle rods were tanned with 4% alum solutions solutions of different, selected pH.

and were then extracted

with 2% digitonin

TRIG

FIG. 2. Schematic diagram of apparatus which permits the measurement of the rates of conversion of metarhodopsin II into P470 and rhodopsin. Once the meta II in sample, S, is produced by orange irradiation (OR) it is flashed from below with a U.V. flash from PR. Reaction rates are followed by monitoring the intensity of the monochromatic light beam with the photomultiplier, PMT. The apparatus is shown schematically in Fig. 2. To start the experiment, a small aliquot (0- 15 ml) of pigment solution was injected into a semi-micro cuvette, S, and the cuvette was slipped into a closefitting constant temperature block (not shown). When the desired temperature was attained, the sample was irradiated from above with OR, a strong orange light (Wratten W-21 filter over a 6 V auto headlamp). This irradiation was continued for at least 2 but not longer than 3 min. The irradiation time depended upon the original pigment concentration and the temperature-being proportional to the former and inversely related to the latter. Since it is known that the interval between the initial (bleaching) absorption and the photoreversing absorption can strongly influence the extent of photoreversal (WIUIAMS, 1966), the pm-irradiation time was held constant in any given session. After the meta II was produced by this orange irradiation, two measurements of the response of the photomultiplier, PMT (RCA l-P28), were displayed on the scope and photographed: (a) a baseline (no light) measurement with the shutter, H, closed; and (b) a pre-flash measurement, ZO, with the shutter open. These measurements could be made quickly (cu. 2-3 set after the orange light went off) by means of a specially constructed timing circuit (not shown). Then the shutter was opened, but the scope was not triggered until the timing circuit closed a switch that gave the photoreversing flash from flashgun, PR (Honeywell 65-C), situated below the sample. In this case, the scope was triggered by the output from the photo-detector, P, which responds to the onset of the flash and which rises to triggering level

Photolysis of Metarhodopsin II: Rates of Production of P470 and Rhodopsin

14159

in cu. S+ec. The shutter was closed quickly. It was opened again and the &al intensity of the monitor beam, ZZ,was measured 5 set after the llaah. The total density change is logie(Z,lZf), and the density change at any time is logio(ZO/Zt)where Zr is the intensity at that time. Tests showed that the monitoring intensities did not saturate the photom~tip~er nor bleach the pigments. The monitoring beam is obtained from a high-pressure xenon arc, Xe. It passes first through monochromator 1 whiih isolates a broad band in the desired region of the spectrum and then through the sample. Monochromator 2 is set at the particular wavelength bemg monitored (band pass co. 10 run) and the emergent beam is caused to fall on the photomultiplier. In order to irradiate meta II selectively and at the same time prevent large photoreveming flash-artifacts, a filter, FZ (combination of Corning filters 7-54 and 5-B), was placed over the &t&gun. FZ isolated a broad band (half-width of 25 nm) centered at 380 mn and was, therefore, appropriate for selectively irradiating meta II. Since F2 transmits below 430 nm, photo~ve~ng flail appear on the records when ~~hro~tor 2 is set to monitor wavelengths shorter than this. It was found that pigment densities of at least 1.0 (so0 nm, 1 cm path) were needed to give good results; this is partly a function of the intensity of the photorevendng Sash at 380 nm. If the flash had delivered more than its present number of quanta (cu. 2x 101s quanta/flash/sample area, integrated over a 25 nm bandwidth), lower initial pigment concentrations could have been used. As in an earlier study (WILLUMSand E&EIL,1968) the density vertically through the solution was mhdmimd by using depths of only O-3 cm. This was done to prevent serious concentration gradients which might otherwke have been caused by uneven absorption of the photoreveming flash. The photolysis of meta II was studied over the temperature range, 4-26” C. Dry air was directed across the optical faces of the cuvette to prevent fogging at low temperatuns.

FIG. 3. Oscilloscope record of changes in monitor beam intensity. Sweep speed 02 m&m, monitoring wavelength 470 nm. A fast decrease in intensity is complete in co, 2 ms. A slower and smaller decrease occurs after the Bash.

Figure 3 shows a record, obtained with a solution at pH 4.4, and lo-O0 C, measured at 470 nm. The sweep speed was O-2 ms/cm; therefore, a single trace represents cu. 2 ms. Note that the I, trace shows a rapid increase in density at this wavelength during the flash. (Ninety per cent of the flash output is dissipated in cu. 1.3 ms). This rapid change is virtually over in the 2 ms shown. However, the 5 set measurement shows that a slower increase in density occurred after the flash. Measured at this wavelength, the slow change is much smaller than the fast change. The slow change is better demonstrated if the monitoring wavelength is set at 500 nm; a relevant record is shown in Fig. 4. This record was obtained under different conditions: pH 4.6, 7-O’ C, sweep speed 0.5 mslcm. (The high frequency noise is pickup from the timing circuit.} This solution was more concentrated and the density changes were larger than those in Fig. 3. The fast change is still the larger but at this wavelength the slow change is nearly equal to it. In order to determine which pigments are being produced by these fast and slow processes, complete spectra were determined between 430 nm and 570 nm. One such

1460

FIG.4. Oscilloscope record of changes in monitor beam intensity. Sweep speed 0.5 ms/cm, monitoring wavelength 500 nm. Fast and slow changes are still apparent but are more nearly equal at this wavelength.

taken at 4” C and pH 6.5 is shown in Fig. 5. The density changes due to the fast process were measured from the onset to the end of the photoreversing flash (i.e. O-O-2.18 ms) and those due to the slow process from 2.18 ms to 5 sec. The former shows a maximum at 470 nm and the latter at 500 nm. The conclusion is that the “465” pigment of MATTHEWSet al. (here a “470” pigment) is produced very quickly when meta II absorbs light. But rhodopsin is produced only slowly-definitely after the flash is over. Other sets of data were analyzed using different criterion times for the presumed durations of the two processes, e.g. O-5 ms and 5 ms-5 sec. The fast density changes were still maximal at 470 nm and those for the slow process at 500 nm. The ratio of density changes, AD&ADsss, as shown in Fig. 5, is 3.6. This fact will be considered in more detail later.

o.o- 2.18Ills

Fm. 5. Overall density changes due to photolysis of meta II. The fast changes produce P470 and the slow changes produce rhodopsin.

The rate of rhodopsin production, slow compared with the flash duration, lends itself to conventional study. Slow sweep speeds were used on the oscilloscope and the density changes vs. time were determined at several temperatures. The process is firstorder as shown in Fig. 6. The straight lines do not pass through the origin because these slow changes are, of course, preceded by the fast ones. Therefore, the slow changes were not measured until it was quite clear that the fast processes were over. The rate constants obtained from these two lines are O-16 and O-022 ms -1 at 20’ and 5” C respectively. An Arrhenius plot, made from other data, is given in Fig. 7. The open circles

Photolysis of Metarhodopsin II: Rates of Production of P470 and Rhodopsin

I

&

20

I

40

.

I

60

1461

80

FIG. 6. First-order rate plot of production of rhodopsin at two temperatures. Do’ is the density measured after the fast process is over, D,, the density at any time during the slow process and D/the final density.

0.10 t 0.06

; 2 ic-

QO’

I 3.40

I 345

I 3.50

I 3.5 5

I 3.6

1000/T FIG. 7. Arrhenius plot of rates of rhodopsin production. Open circles: pH 6.5; closed circles: pH 4.5. The temperature range is S-21” C. The activation energy is 18 kcal/mole.

are the rates in solutions at pH 6.5 and the filled circles are the rates at pH 4.5. The activation energy is 18 kcal/mole and there is no dependence of the rate on pH over this range. The spectral properties of the intermediate that produces photoregenerated rhodopsin can be measured. The intermediate is relatively long-lived, does therefore accumulate and, despite the fact that it absorbs within the envelope of the flash output, can be observed because the flash is well over during its lifetime. The slow process shows a density loss maximal at 380 nm-precisely where meta II absorbed its light. This is shown in Fig. 8.

THEODOREP. WILLIAMS

1462

I

I

360

I

1

370

380

1

I j

390 400

FIG. 8. Absorptionproperties of the long-lived intermediate which produces rhodopsin. Since the flash is well over, this substance must have ll-cis retinal already. yet it absorbs maximally at 3&onm.

The rate of production of P470 was measured at several temperatures and was found to be independent of this parameter. Figure 9 shows four sets of data taken at 4”, 13’.

1, ms

FIG. 9. Rates of formation of P470 from the photolysis of meta II at 4”. 13”, 17” and 22” C. Individual points are not shown for clarity but all cluster within the vertical bars, thus indicating no temperature depcndexxe of the rate. The solid line is the integrated quanta1 output of the photolysis flashgun. The close correspondence between the bars and this curve shows that P470 is produced from meta II as fast as the quanta arc absorbed.

17’ and 22’ C. The individual points cluster entirely within the spread of the vertical bars and for clarity are not shown separately. The solid line is the integrated quanta1 output of the flasher, and it is obvious that P470 appears as fast (on this time scale) as the quanta are put in. There was no dependence of the AD~,~/AD~~ ratio on pH ; it remained at 3-5. Other measurements of this ratio showed that it was independent of temperature, also. This is shown in Table 1. Note that the rate constant k, for the production of rhodopsin varies by a factor of 5 over this range of temperature, yet the ratio is constant. Since the rate of P470 production is independent of temperature, these results indicate that the ratio of P470 to rhodopsin is determined by relative quantum efficiencies of a

Photolysis of Mttarhodopsin II: Rates of Production of P470 and Rhodopsin

1463

TABLE1. Temp. ’ C 22 14 7

AD4701

Aboo

3.5 3.5 3.4

kXl/W

o-173 @076 O-035

photochemical step and not by relative rates of thermal reactions. If rates of thermal reactions determined the ratio it would decrease with increasing temperature. In order to check the total amounts and distribution of all the compounds involved in these reactions, the following experiment was carried out: The density changes at 500 nm and 380 nm, caused by the orange pre-irradiation, were measured and in one run were found to be 0.718 and O-651 respectively. These were taken as measures of the loss of rhodopsin and the gain of meta II respectively. Since the absorption coefficients of these two substances are known, the densities can be converted to concentrations, viz 1.71 x 10 -5 and 155 x 10-s moles/l. respectively. The difference between these two values was assumed to be the concentration of meta I, i.e. 1.6 x 10 -6 moles/l. The meta Imeta II equilibrium constant obtained from these concentrations is 9.7 which is in good agreement with the data (extrapolated) in Fig. 3 of Matthews et al. for a solution of pH 45 and (in this case) 4’ C. Then, upon giving the U.V. flash, the sum of the density changes at 500 nm and 470 nm was 0.178 while the total loss at 380 nm was only 0.146. At first sight, it would appear that more rhodopsin and P470 were produced than meta II lost. (Matthews et al. suggest nearly equal absorption coefficients for all three compounds.) However, it must be remembered that meta I will partly compensate for the loss of meta II by re-establishing the equilibrium between the two of them. Using the value of 9.7 for the equilibrium constant, it is calculated that 0.017 o.d. units would be added to the meta II reading by such a mechanism, there being more than enough time in 5 set to do so (WILLIAMS and BREIL, 1968). Thus the meta II lost does equal the sum of rhodopsin plus P470 to within 6 per cent. A final experiment was carried out to ascertain that double hits on meta II were not occurring and thereby complicating the observations. This was done simply by producing a known amount of meta II, flashing it and determining that 25 per cent was lost in one flash. A statistical calculation showed that, at most, 5 per cent of the meta II absorbed two quanta during the Ilash. In fact it is likely that not even this number of double hits occurred since the calculation assumed no loss of absorber (meta II) during the flash duration. This is a poor assumption given the quick conversion to P470. DISCUSSION

These experiments verify that meta II can be converted by light into both a 470 pigment and rhodopsin. The density changes at and near 500 run were too small to permit an assessment of the possible presence of iso-rhodopsin. Therefore, it will not be considered in the rest of this discussion. The mechanism which these results suggest is given in Fig. 10. Meta II absorbs a photon and enters an electronically excited state, indicated by the asterisk. The molecule emerges from this excited state as either P470 (13-cis?) or M II’ (1 I-cis). The quantum

1464

THEODORE

P.

WILLIAMS

Fro. 10. Proposed mechanism for the production of P470 and rhodo~~ from meta II. Meta II absorbs a photon and enters the excited state indicated by the asterisk. P47O and M II’ are produced with relative quantum efficiencies of 3-S and 1.0. M II’ slowly (in msec) becomes rhodopsin.

efficiencies are in the ratio 35:1-O, respectively. M II’, whose lifetime is measured in msec, decays to rhodopsin in the dark. Why is it that the color associated with P470 appears immediately but that of rhodopsin only slowly ? Spectral shifts of these 90-120 nm can be associated with either, or both, of the following: (a) Re-protonation of the Schiff’s base linkage (cf. Matthews et al.); and/or (b) the establishment of a close fit between opsin and the newly-formed isomers 13-c&(?) and 1l-c&. In the case of P470 it seems unlikely that major conformational changes in opsin would occur fast enough to yield the new pigment. Therefore, the conformational changes, if they occur, must be fairly subtle and the opsin of P470 probably closely resembles that of meta II. Leaving aside for now the problem of whether or not protonation occurs and contributes to the spectral shift, let us propose that a major reason for the shift is that the opsin of meta II is already in a conformation which can accept the 13-&s (?) isomer without much rea~angement and provide a fairly close fit. Then the conclusion, regarding the production of rhodopsin, is that 1I-& does not find the opsin in a suitable conformation and the “dark” reaction is the time necessary for opsin to conform to the new geometry. The reason a protonation mechanism is not invoked is that it will not work equally well for the production of both P470 and rhodopsin. It would work for the production of P47O-the only requirement being that it be fast enough to make quanta1 absorption the rate-limiting step. However, if protonation were that fast, and if it occurs in the production of M II’, why does M II’ absorb at 380 nm? Furthermore, why is there no dependence on pH of the decay of M II’? Surely it must be that steric interactions, not protonation, determine the rate-limiting step in the spectral shift from M II’ to R. Of course, it could be that the two paths are different-the spectral shift in one case, P470, being determined by protonation while in the other, rhodopsin, by confo~ational changes. The present results cannot provide an answer to this problem. One cannot calculate from the present results the extent of the overall conformational changes that occur upon production of rhodopsin. It is possible, however, to calculate an entropy of activation for the process. For example, at 22” C, k,=O*I6 ms -1 (c$ Fig. 6); this gives an entropy of activation, nSt, of + Il.4 e.u. This is rather small for protein conformational changes and, surprisingly, it is positive. A straightforward interpretation of this result would be that conformational reorganization is not too extensive and is in the direction of more randomness. If meta II is a disorganized substance and rhodopsin an organized one, (Matthews et al.), it is not obvious

Photolysis of Metarhodopsin II: Rates of Production of P470 and Rhodopsin

1465

why the transition state between meta II and rhodopsin should be even more disorganized than meta 11. Perhaps this result simply reflects that a measurement of the entropy of activation need not always reflect the thermodynamic entropy change. To what extent is photoregenerated rhodopsin like freshly extracted rhodopsin ? An earlier attempt to measure this, using ORD, was only partly successful (WILLIAMS,1966). A study of the thermal stabilities of rhodopsin, photoregenerated rhodopsin, P470 and meta II is reported in an accompanying note (BAKER and WILLIAMS,1968) and contributes some additional information on this matter. It appears that the 11-cis chromophore holds photoregenerated rhodopsin together as tightly as it does freshly extracted rhodopsin. (The importance of ll-cis retinal in stabilizing opsin has already been pointed out by HUBBARD, 1958.) Furthermore, the lfcis (?) chromophore of P470 seems to hold the system together more than does the all-truns chromophore of meta II. Matthews er al. have stated that their P465 is produced from meta II by thermal as well as photic means. Apparently, a slow thermal isomerization of the all-truns retinal can occur which leads to P465. OSTROYet al. (1966), on the other hand, believe that P465 is an all-truns species. If this is so, what is the action of light on the meta II as shown in the work of Matthews et al., and in the present study? Is it possible that P470 is a mixture of cis and tram isomers ? Obviously more work is needed before this question can be answered. REFERENCES BAKER,

B. N. and WILLJAMS, T. P. Thermal decomposition of rhodopsin, photoregenerated rhodopsin and P470. Vision Res., this issue. COLLINS,F. D. and MORTON, R. A. (1950). Studies in rhodopsin. 3. Rhodopsin and transient orange.

Biochem. J. 47, 18-24. HAGINS,W. A. (1955). The quantum efficiency of bleaching of rhodopsin in situ. J. Physiol. 129, 22. HUBBARD, R. (1958). The thermal stability of rhodopsin and opain. L gen. Physiol. 42,259-280. HUBBARD, R. and KROPF, A. (1958). The action of light on rhodopsin. Proc. Not1 Acad. Sci. 44, 130-139. HUBBARD, R. and WALD, G. (1952). Cis-truns isomers of vitamin A and retinene in the rhodopsin system. J. gen. Physiol. 36, 269-315. MATTHEWS, R. G., HUBBARD,R., BROWN,P. K. and WALD, G. (1963). Tautomeric forms of metarhodopsin. J. gen. Physiol. 47, 215-240. OSTROY,S. E., EtU%UT,F. and An RAHMISON,E. W. (1966). The sequence of intermediates in the thermal

decay of cattle metarhodopsin in vitro. Biochim. Biophys. Actu 112, 265-277. WALD, G., DARRELL,J. and ST. GEORGE,R. C. C. (1950). The light reaction in the bleaching of rhodopsin. Science, N. Y. 111, 179-181. WILLUMS,T. P. (1964). Photorevemal of rhodopsin bleaching. J. gen. Physiol. 47, 679-689. Wruu~s, T. P. (1965). Rhodopsin bleaching: relative effectiveness of high and low intensity flashes. Vision Res. 5, 633-638.

Wruu~s, Wm,

T. P. (1966). Induced asymmetry in the prosthetic group of rhodopsin. Vision Res. 6,293-300. T. P. and B~EIL, S. J. (1968). Kinetic measurements on rhodopsin solutions during intense

flashes. Vision Res. 8,777-786. YOSHZAWA,T. and WALD, G. (1963). Prelumirhodopsin and the bleaching of visual pigments. Narurc, Lond. 197, 1279-1286.

Abstract-Metarhodopsin II, produced by irradiation of cattle rhodopsin, is flashed with U.V.light and is found to produce rhodopsin and P470 in the ratio 1 : 3.5. P470 is produced as fast as the light is absorbed while rhodopsin is produced slowly--several msec after the flash. The rate of production of P470 is independent of temperature, but the rate of rhodopsin production has an activation energy of 18 kcal/mole. The ratio, P470 : Rhodop&~, is independent of temperature, suggesting that the relative amounts of these substances are determined by the initial absorption of light by meta II. A new compound, meta II’, is observed. The decay of meta II’ is the thermal (“dark”) reaction in the photoregeneration of rhodopsin. A discussion of the nature of opsin in P470 and photoregenerated rhodopsin is given.

THEO~XXE P. WILLSAMS

1466

R&tutu&-L’irradiation par l’ultraviolet de metarhodopsine II, produite elle-meme en eclairant de la rhodopsine de bovide, produit de la rhodopsine et du P470 dam la proportion 1 : 3,5. L’absorption de lumiere produit tout de suite P470 tandis que la rhodopsine est produite lentement, piusieurs miilisecondes apres lXclair. La vitesse de production de P470 ne depend pas de la tem~~ture, tandis que celle de la rhodopsine a une Cnergie ~activation de 18 kcal/mole. Le rapport de P470 a la rhodopsine est independant de la temperature, ce qui sugg&e que les quantitea relatives de ces substances sont dbtermin&s par l’absorption initiale de lumi&e par la metarhodopsine II. On observe un nouveau compose, la metarhodopsine II’, dont la decroissance est la reaction thermique (“obscure”) dans la photortgent5ration de la rhodopsine. On discute la nature de I’opsine dans P470 et dans la rhodopsine photor&&tbr&. Z~fa~-Metarhodo~in II, das bei Bestrahlung von Rinder-Rhodopsin entsteht wird mit u.v.-Licht geblitzt und ergibt Rhodopsin und P470 im Verhiiltnis von 1 : 3,s. P470 entsteht so schnell wie das Licht absorbiert wird, wiihrend Rhodopsin langsamer produziert wird-mehrere Milliikunden nach dem Blitz. Die Entstehungsgtxchwindigkeit von P470 ist temperaturunabh&ngig, aber die von Rhodopsin hat eine Aktivierungsenergie von 18 cai/mol. Das Verh<nis P470: Rhodopsin ist temperaturunabhangig, was vermuten Ia&, dal3 die relativen Anteile dieser Substanzen durch die u~p~~iche Absorption des Lichtea durch meta bestimmt werden. Eine neue Verbindung, das metaIl’, wird beobachtet. Der Zerfall von metaR ist die thermische (“dunkel”) Reaktion bei der Photoregeneration von Rhodopsin. Eine Diskussion iiber die Natur des Opsins im P470 und im photoregenerierten Rhodopsin wird gegeben. Pexoiwe -

Merapoaorxcmf II, nonyraervsbd npe ocrmuem poxoncma Kpymoro poraTor0 cKoTa,o6~~~c~ ~~~~o~ynbrpa-~~on~oBoroc~Ta;~pu 3ToM Eiblno K~~enO,qTOB~3y~TaTe~Ony~~CbpOnO~~~ P470B OTHO~~~l :3,5. P470 O6pii30BbIBaXSIOWHb 6~xpo BO B~M~nOrnO~eHH~~Ta,BTOB~~~KaKpO~ORC~H yepe3 HeCKOnbKO MEiJtnHceKyHn nocne BcrIblIrlKN. o6pa3oemancrr Mememo CKOpOCTb 06pa~3oaami~ P470He 3aBUCNT OT TeMlIepaTypbl,aCKOpOCTb o6pa3oB;uraa ponOnCHHa EiMeeT 3HeprHH) aKTEIB8UBA PaBHyIo 18 KKWI/MOnb. OTHOiUeHHe P470: PO~OIKliHHe 3aBuCHTOTTeMnepalypM;3TO3a~aan~eTnyMaTb,~TOOTH~~~bffbIe ~emmixibf 3mx muecm 0npexemwoTcff Hawnbsibm kxorno8.uemieM meTa hmapo~~ORCUWOMIr.~~~Hap~H T~K xce A~~~KoM~oI~~HT,M~~~F~o~cIIHI~~ Pacnan Mflapononcnrra

II’ -

TepMssrecKaJl (((~~0~~~~)

po~oncmia.&icKyT~pyeTca~onpoconp~po~eoncmia~

po~oqoncasa.

peazusrx npa +oroperenepa~ P470si~o~opererrnpyeMoro