Photolysis of metarhodopsin I: Rate and extent of conversion to rhodopsin

Vldon

4~. Vol. 11, pp. 449458.

Pe~amon Prass 1971. Printed in Great Britah.

PHOTOLYSIS OF METARHODOPSIN I: IbiTE AND EXTENT OF CONVERSION TO RHODOPSIN’ BARBARA N. BAKERand THEO~~~RE P. WILLIAMS Department of BiologicalScienceand Institute of Mokcular Biophysics,

norida State Wniversity,Tallabassq Florida 32306,U.S.A. (Receiued

28 September

1970)

INTRODWCTrON RHODCW%& the visual pigment of rods, has as its chromophore, 1l-c& retinal. The absorption of light by rhodopsin results in the isomerization of the 11-c& to the all-trmrs configuration (HUBBARDand WALD, 1952) and this act introduces a series of thermal reactions that leads to the eventual dissociation of the chromophore from the protein, opsin. Since the reaction sequence leads to the loss of red color, it is termed bleaching The bleaching scheme as proposed by MATTFXEWS, HUBSARD,BROWNand WALD (1963) and modified by WIL~~S (1968, 1970) is given in Fig. 1. R is rhodopsin, P is pre-lumi, L

(5oo)

R

-

ILID’ 1380)

P 470 FIG. 1. Schana

of rhodopsin bkaching as proposed by MATTHEWS et al. (1963) and modified by W(1970).

is lumi, MI is meta I, and MI’ is the recently demonstrated form of meta I, indistinguishable from MI spectrophotometrically, but having little or no photoreversibility. MZZis meta II, MZZ’is the intermediate in the production of rhodopsin from MZZand P4,0 is probably the same pigment observed by MATTHEWSet aZ. (1963), and recently named paru-rhodopsin by WALD (1968). Wavy arrows represent photoreactions and straight arrows are thermal reactions. P, L, MZ, MI’ and MZZare produced thermally following photon absorption by 1This work was supported by USPHS Grant 9 ROl EYOO479-03 and in part by contract AT (40.1>2690 of the Atomic EnergyCommission. 449

450

BARBARAN. BAKERANDTHE~WREP. WILLIAMS

rhodopsin and are unstable, presumably because they contain an all-tram configuration of the chromophore. The intermediates produced in the bleaching sequence are capable of photon absorption. When such absorption occurs, a cis isomer may be produced and a stable pigment may be generated (HUBBARDand KROPF, 1958; WILLIAMS,1964). This phenomenon is known as photoreversal of bleaching When photoisomerization of the all-truns intermediate yields the 1I-cis configuration, the resulting photoregenerated pigment is rhodopsin. If the 9-d isomer is produced, the photoregenerated compound is isarhodopsin (HUBBARDand KROPF, 1958). (No further consideration will be given to iso at this time.) If any other isomer is produced, the resulting pigment is unstable and decays with time to the component opsin and retinal. In view of the established existence of thermal components in the bleaching of rhodopsin, it is of interest to inquire whether dark reactions occur in the photoreversal steps, Such thermal steps were implied by YUSHZAWAand WALD (1963). However, the question has only recently been investigated. WILLIAMS(1968) reported that the production of rhodopsin from photolysis of metarhodopsin II does indeed involve a slow thermal component. It was found that the isomeri~tion of the meta II chromophore to an 1l-c& or 9-cis configuration resulted in the production of a cis-meta II (meta II-prime), still absorbing maximally at 380 nm, which was converted by thermal means to rhodopsin. The major product of the photolysis of M11 was shown to be an unstable pigment of h,,, 470 nm. The ratio of PbTOto rhodopsin was a constant, 3*5/l, from 5” to 22” and over the pH range 48. Aside from these results on meta II, the photoreversal of other intermediates in the bleaching sequence has not been studied. Consequently, the present investigation was undertaken to determine both the rate and the extent of the conversion of metarhodopsin I to rhodopsin. The extent of the conversion was of particular interest in view of the newly proposed intermediate, meta I’, which according to WILLIAMS(1970) would have the spectrum of meta I but little or no capacity to regenerate rhodopsin upon photic absorption. METHODS AND MATERIALS Rod outer segmentswere isolatedfrom cattle retinas (Ho-1 Co., Austin, Minn.) by sucrose tloatation. Rhodopsin was extracted with 2% diejtonin in 0.066M phosphatebuffer of pH 6-S.The pH of the rhodop sin solutions was varied by addition of either dilute solutions of phosphoric acid or sodium hydroxide. The light sources employed in the experiments were of two types: a steady orange one, for producing meta I from rhodopsin, and a blue strobe fIash, for pho~~~~ing the meta I once it was made. The former was a 6 V automobile headlamp equipped with a Coming 3-66 sharp cut-off filter. The latter was a Honeywell 65-C strobonar delivered through a combination of Corning S-60 and 3-72 filters. The transmission of this combination was maximal at 460 run and was less than 1 per cent at wavelengths shorter than 440 nm and wavelengths longer than 515 ML The photoreversing flash was, therefore, rather selective for irradiating meta-rhodopsin I. The duration of the flash was 29 msec (defined as time for 99 per cent dissipation of the light energy) and its output was found to be 5.8 X lOI5 quanta, integrated over the band described above. In order to start the experiment of the rule of meta I photoreversal, an aliiuot of 0.2 ml of the rhodopsin solution was placed in a 1 cm pathlength cuvette. The sample was transferred to a thermostatted aluminum block which was mounted on the flash unit of a kinetic spectroscopic apparatus. The rhodopsin solution was brought to 5” and then irradiated from above with the orange light. The irradiation was of 3 min duration. At the end of the irradiation the photoreversin8 tlash was delivered and the scope triggered. The density changes occurring during the flash and for several seconds after the flash were monitored between 575 nm and 380 nm as previously described by W~LLMM(1968). A new sample was utilized at each wavelength. When investigating the extent of meta I photoreversed, several additional measurements were made. The initial rhodopsin concentration of the sample (RJ was determined by measurement of the density at 500 nm on model 2000 Gilford Spectrophotometer. The amount of meta IL (MII) produced during the 3 min orange

Photolysis of Metarhodopsin I

451

irradiation was evahaated by monitoring the increase in densityat 380 mn with the kinetic spectroscopic apparatus. After addition of 2Ohof 1 M NHIOH to prevent photoproduct absorption at 500 nm, the density of the rhodopsin mmaining after orange irradiation (RnWtntsl) was measured. The meta I concentration at the time the photoreversing Sash was delivered was then calculated by difference, i.e.

Anothersample was treated precisely as described above but the photoreversing Bash was delivered immediately following the orange irradiation. The rhodopsin photoregenerated by the flash was evaluated by determhmtion of density at 500 rm~of the sample after flashii. Due to the extreme overlap of the spectra of rhodopsin and meta I, some rhodopsm is bleached by the photoreversing fIash. Therefore, the concentration of rhodopsin in the Sashed sample had to be corrected for this. In order for the above analysis to be considered correct it was nv to show that the only substances present after orange irradiation were rhodopsin, meta I and meta II. Therefore, the following experiments were performed. Samples at pH 4,6-S and 8 were maintained at 53 irradiated with orange light as usual, and scam& using a Cary Model 14 S~ophoto~~. These spectra when corrected for rhodopsin and isorhodopsin remaining, indicated only the presence of meta I and meta IL Furthermore, it is reasonable that no other substances would exist since at this temperature, reactions beyond meta II are very slow while at the same time, precursors of meta I are very short-lived. Finally, the ratio of meta I to meta II was found to change significantly as the pH was varied, which, qualitatively, agrees with the effect found by M~rrrraws et of. (1963). The equilibrium was displaced toward meta II acid solution and favored meta I in the abtahne condition. Therefore, what we call meta 1 and meta 11seem to be those compounds descrii in earlier work by other investigators. RESULTS &te

of reaction

The fact that the only change occurring during the flash is the conversion of metarhodopsin I to rhodopsin is illustrated in Fig. 2. The data points are the experimentally determined density changes at the end of the 2-msec Bash, the solid curve is the difference 1

I

t

I

400 410 430

I

450

I

I

I

470 490 510 Wovetcngth. nm

I

1

I

550

550

570

Fro. 2. Density changes occur&g durhrg the 2 msec Sash in the photo&&s of metarhodopsin I at 5’. (-1 Known difference spectrum of rhodopsin and metarhodospin f.

452

BARBARA N. BAKERANDTHEODQR~ P. WILLIAMS

spectrum for metarhodopsin I and rhodopsin as measured by HUBBARDand KROPP (1958). The points follow the curve well enough to justify the conclusion that no other reaction is occurring during the time that Iight is entering the sample. Although not shown in the figure, slow changes in density take place in the period between 2 msec and 5 sec. These involve the re-establishment of the metarhodopsin I-metarhodopsin II equilibrium. There is no reaction of metarhodopsin I to rhodopsin after the flash. The extent to which a thermal component may exist in the photolysis of metarhodopsin I to rhodopsin is shown in Fig. 3. The bars indicate the range of values obtained in the four determinations of the rate of density change at 530 nm. This wavelength is the maximum in

0'

I”

I

05 msec

I

I.0 Time, msec

I

I*5

L 24

FIG. 3. Rate of rhodopsin regeneration from photolysis of metarhodopsin 1. ( I11I) Experimentally determined density changes at 530 nm. (-) Integrated flash output.

the difference spectrum of metarhodopsin I and rhodopsin. The solid line is the integrated flash output. The 50-100 psec displacement of the data points from the integrated fiash curve demonstrate that at 5” any thermal limitation which may exist in the conversion of meta I to rhodopsin cannot exceed 100 psec. This is to say that there is Iittle or no dark component in the conversion of meta I to rhodopsin at 5”. Extent of reaction

Treatment of the system stoichiomet~~lly allows the determination of the extent to which meta I is photoreversible. We have studied this reversibility as a function of both pH and temperature. The effect of pH is shown in Fig. 4. The data are plotted as per cent of meta I photoreversed, i.e. the amount of meta I photoreversed divided by the total meta I present at the time the reversing flash is given. At 5” never more than 33 per cent of the total meta I[ can be photoreversed and this occurs at pH 6.5 and above. At pH values below 6, the per cent of meta I photoreversed falls off sharply, and by pH 4, meta I has essentially no photoreversibility. In view of the similarity of the curve in Fig. 4 to the titration curve reported by MATTHEWS et al. (1963) for the acid-base relationship between meta I and meta II, it was important to establish that the lack of photoreversal of MI observed at acid pH was not simply the result of the decreased concentration of meta I. Therefore, a series of experiments were run in which the initial rhodopsin concentrations were adjusted so as to yield equivalent meta I concentrations at all pH values. Under these conditions the

Photo&& of Metarhodopsin

I

453

FtG, 4. The extent ofmetaI pbotoreversal as a function 0fpI-I at 5”. (0 0 0 0) ~~~ti~~ madeusingthe2msecflash.(~m~O)ResultobtluhsdfromanexperimentwiththeZlas#: Bash when the intensity had been reduced by an O-3 ND. titer, (UUOO) Data given by 60 msec Sylvania FP-26 ~hb~lb at full intensity. (8 I R B Data given by 60 msac Sylvania IV-26 flashbulb with intensity reduced by an &3 N,D. filter.

meta Xin acid media was still less reversible than in base, approac~ng zero per cent reversibility at pH 4. Checks were made to be certain that this extent of photoreversal was not intensitylimited: (1) Bash bulbs whose qua&al output is 30 times greater than the Honewell 2 msec source were used and gave identical results; (2) halving the intensity of both the flash and the flash bulbs had no effect on this limit. iteration of the effect of temperature on the photoreversal of meta I was carried put at pH 6-5. The temperature range employed was from lo to 18”. Above 18” the rate of hydrolysis of MII became significant and the steady state condition did not apply. As the temperature was increased the per cent of the meta I capable of photoregenerating stable pi~~nt decreased. The data are shown in Table 1. This is the same kind of effect seen by

28.6 30% 31.0 30.8 28.6 31.0 31.0 32.7

10.3 12.96 13.1 10-7 10.9 10-g 10-5 9-58

3”84 483 4-33 3.14 2% 253 2.12 l-80

37.3 34.0 33-o 29-4 26.1 23-4 20-l l&8

BARBARA N. BAKERANDTHEO~RB P. WILLIAMS

454

WILLIAMS(1970) in the flash bleaching of rhodopsin and which led to the proposal of the existence of meta I’. Evaluation of the data of this study using his mechanism, MZ7+ MI’ * MZZ + H+, leads to the results shown in Table 2. Meta I concentration was set equal to the TABLE 2. METAI-IWIA

II-MBTA

Ii EQ~~IBRIA

M~]MI

Mff] MI

Temperature (de@

: 5 :9 15 ::

AG

entropy units K -238 -243 -273 -290 -310 -348

1.55 1.56 1.64 1 a67 1*73 1.84 1.86 1.84

+7*33 i-7.31 +7*36 i-7.30 -i-7*30 $7.36 +F32 + 7.32

z

entropy units

callmole

AS

::!i 2.10

-282 -298

2.86 3.35 3.68 6-035 5.04

ZZ -685 -735 -1038 -932

-l-42*4 +42*2 +42*3 +42*2 +42*3 -I-42*2 +42Z! +42*2

K

AS

The values of the equilibrium constantwcm calculated from tbeconumtrstions of each speck at the given temperature. The concentrations were measured 88 described in the text. The fnx energy was calculated from the equation AG - --RTlnK. The AG and the graphically determined values of AH wereemployed to yield the entropy by the equation AG = AH - TAS.

6.

4.

K

2.1

I.(

1

t

340 t/T

I

1

350

360

I

PK-‘! X IO3

FKL 5. Eikt of temperature on the equilibrium constants, K = [MfIl/[MI’] (0 0 0 0) and K = [Ml’flfMfl (0 0 0 0). Data is plotted knn the r8lation8hip dblx/dl/T = -AH/R and From tha slope of the line the cxkthalpy calculated for MI’ + MIX reaction is 1.8 M/mole the MI --c MI’ reaction enthalpy is 11.3 kcal/mol~.

Photolysisof MetarhodopsinI

455

amount of stable pigment produced by the flash; meta I’ concentration was the total meta I minus the amount of stable pigment produced, and the meta II concentration was measured as previously described. Thus the equilibrium constants MI’IMI and MIIIMI’ were calculated. In Fig. 5 the equilibrium constants for the two reactions are plotted as a function of temperature. The lines are obtained from a least squares analysis and the AH obtained for the MI-MI’ equilibrium is +l l-3 kcal/mole, while the MI’-MII equilibrium yields a AH of +1.8 kcal/mole. DISCUSSION When the results of this study are compared with the findings of the earlier investigation of meta II photolysis, two major differences are apparent. First, the reaction of meta II to rhodopsin was shown to involve a substantial dark or thermal component while, in this work, the photolysis of meta I to rhodopsin measured under identical conditions shows little or no thermal limitation. This probably indicates that more extensive protein rearrangement is necessary to yield rhodopsin from meta II than is required to produce rhodopsin from meta I. This is consistent with the accepted view that a large conformational change occurs between meta I and meta II. Second, absorption by meta II was shown to yield predominantly the thermally labile P,,, compound, but in this study no evidence for a P4,,,-like pigment was found. It has been shown that, even though all the meta I molecules absorb during the flash, only 33 per cent, at most, are photoreversed. What is the result of the absorption by the other 67 per cent? While at present no experimental answer to this question exists, an interesting possibility is that these absorptions do produce a new compound but this compound is spectrally identical to the meta I from which it is produced. Some evidence exists in support of this idea. The evidence comes from an experiment by Y~~EIEAWAand WALD (1963) who demonstrated that the conversion of meta I to rhodop sin is blocked at - 195”, and that absorption by meta I at - 195” led to no visible change in its spectrum. It seems unlikely that isomerixation of the chromophore was blocked at - 195” since isomerization of lumirhodopsin is possible at this temperature. Thus, it appears the meta I of Yoshizawa and Wald did undergo isomerixation but it did so without any apparent spectral change. Therefore, in the present work, the absence of a P,,O-analog could possibly be explained if production of a cis isomer, other than the 11 or 9-cis, yields an intermediate of spectral properties so closely resembling those of all-trans meta I that it could not be detected. Both meta I and meta II, when they exist in equilibrium have limited photoreversibility. Why should a given species have “limited” photoreversibility? Why should it not have either zero or full reversibility? There are two possible answers to these questions. First, the quantum efficiency for reversal may be less than unity. In this case, a molecule would simply fall out of the excited state either unchanged (all-trmrP) or into another labile form. Only a limited fraction of absorptions would lead to the 1I-cis configuration. This was, in fact, the explanation given for the limited reversibility of meta II (WILLIAMS, 1968). The second possibility is that what appears spectroscopically to be one species is in reality two substances one of which is photoreversible and the other is not. This is the preferred explanation in this work on meta I because the extent of reversibility is so sensitive to temperature. Quantum e8iciencies are not usually this sensitive to such minor changes in temperature. Our observations are more consistent with the thermal production of a new isochromic species which has lost its ability to be photoreversed to rhodopsin. This is a

456

BARBARA N. BAKERAND THEODORE P. WILLIAMS

molecule with the properties attributed to meta I’ (WILLIAMS, 1970). Therefore, in the experiments carried out here, what appears spectroscopically to be just one intermediate is most probably a composite of meta I and meta I’, the difference between them being their ability to produce stable pigment upon photic absorption. Thus, the limit on the photoreversibility of meta I is determined by the meta I-meta I’ equilibrium constant. On the basis of spectral changes alone, MATTHEWS et al. (1963) determined the enthalpy of the reaction meta I + meta II as +13*1 kcal/mole. They, furthermore, calculated the entropy of this reaction as +46.5 entropy units. In this paper we have presented the equilibrium constants for the successive reactions MI + MI’ -+ MZZ and have found that the major enthalpy and entropy changes occur in the reaction: MI -+ MI’; a reaction which is not accompanied by a spectral change. The Harvard group interpreted the large positive entropy change they observed as evidence of a major conformational change in going from meta I to meta II. By the same reasoning, therefore, we must now assign this conformational change to the meta I -+ meta I’ reaction. The total enthalpy and entropy changes we find for the meta I to meta II reaction are essentially the same as those found by MATTHEWS et al. (1963). The sum of the enthalpy changes for the two reactions as determined by us is exactly the value they found, 13.1 kcal/mole. The sum of the two AS values found in this work is +49*5 entropy units, only 3 entropy units more positive than the earlier measurement. The relatively small AH and AS values we observed for the MI’ -+ MZZreaction may well indicate that little more than the depronation of the Schiff’s base is involved in this step. None of the proposed mechanisms of bleaching can explain the effect of pH on meta I photoreversibility found in our experiments. MATTHEWS et al. (1963) presented evidence for the binding of a proton in the transition from meta I to meta II. WILLIAMS (1970) more recently has shown that at high temperature the rate of meta II production during the flash is increased by basic conditions. He, therefore, proposed that the step which produces meta II involves loss of a proton. The results reported here show that in addition to favoring meta II in the equilibrium situations, high H+ ion concentration produces a meta I with little or no photoreversibility. These three, apparently discordant, facts lead to the conclusion that the role of protons in the meta I-meta II transition is neither simple nor understood. Obviously more work is necessary and such work is now in progress in our laboratory. Acknowledgement-The authors thank Mr. BOB WILLIAMSfor drafting the MIDDLETON for preparing the manuscript.

figures and Mrs. MARLENE

REFERENCES HUBBARD,R. and WALD, G. (1952-53). Cis-trans isomers of Vitamin A and retinene in the rhodopsin system. J. gen. Physiol. 38, 269-315. HUBBARD, R. and KROPF,A. (1958). The action of light on rhodopsin. Proc. natl. Acud. Sci., U.S. 44, 130-139. MATTHEWS,R. G., HUBBARD, R., BROWN,P. K. and WALD,G. (1963). Tautomeric forms of metarhodopsin. J. gen. Physiol. 47, 215-240. WILLLAMS,T. P. (1964). Photoreversal of rhodopsin bleaching. J. gen. Physiol. 47,679-689. WILLIAMS. T. P. (1968). Photolvsis of metarhodoosin-II. Rate of production of P,,, and rhodopsin. Vision Res. 8; 1457-1466.. _ WILLIAMS, T. P. (1970). An isochromic change in the bleaching of rhodopsin. Vision Res. 10, 525-533. WALD, G. (1968). The molecular basis of visual excitation. Science, N. I’. 162.230-239. YOSHUAWA,T. and WALD, G. (1963). Pre-lumirhodopsin and the bleaching of visual pigments. Nature, Lo&. 197, 1279-1286.

Photolysis of Metarhodopsin I Abstmet-The photolysis of metarhodopsin I, produced from cattle rhodopsin, has been investigated. The rate of conversion of meta I to rhodopsin has little, if any, “dark” component, i.e. rhodopsin appears nearly as fast as the meta I absorbs light. This differs from what was found in the studies on meta II where an easily measured dark component exists. Also different from the results on meta II was the fact that no product, analogous to PlrO, was noticed when meta I was flashed. The extent of photoreversibility of meta I to rhodopsin was also investigated and was found to depend both on pH and temperature. The reversibility was nearly abolished at pH 4 and reached an upper limit at pH 65 or above. The reversibility was decmased with increasing temperature and these results were interpreted in terms of a mechanism recently proposed by WILLABLE (1970): MI * MI’ z MZZ + H+. Assuming meta I-prime to be photoirreversible, the two equilibrium constants were measured over the range: 1-18”. The thermodynamic parameters were calculated and it was found that AH for the MI-MI’ reaction is 11.3 kcal/mole and for the MI’-MZZ reaction it is 18 kcal/mole. The sum of these, 13.1 kcal/mole, is exactly the value obtained by MATTHEWS et al. (1963) for the overall reaction: MZ-MZZ. RM Ctudie la photolyse de la metarhodopsine I produite g partir de rhodopsine de b&ail. La vitesse de conversion de m&a I en rhodopsine a peu ou pas de composante d’obscurite, ce qui signitie que la rhodopsine apparait presque aussi vite que l’absorption de lurni&e par la m&a I. Ce resultat differe des r&hats obtenus avec la m&a II pour laquelle existe une composante d’obscuritt aidment mesurable. Une autre diff&ence avec la m&a II est l’absence d’un produit analogue a P,,, quand on &claimla m&a I. On etudie aussi la photomversibiliti de la m&a I et de la rbodopsine, et on trouve qu’elle depend a la fois du pH et de la temptrature. LB r&ersibilite est pmsque abolie au pH 4 et atteint sa limite suptrieure vers le pH 6,5 ou plus. LB mversibiliti d&roit quand la temperature augmente, ce qu’on interprete dans le cadre dun mecanisme propose r&emment par WILLUMS(1970): MZ%MZ’%MZZ + H+. Si on admet que la m&a I-prime at photoirreversible, on peut mesurer les deux constantes d’&quilibre dans le domaine l-18”. On calcule Its param&& thermodynamiques et on trouve que pour la reaction MZ-MZ’ la valeur de dH est 11,3 kcal/mole et pour MI’-MZZ 1,8 lad/ mole. LB somme des deux, soit 13,l kcal/mole, est exactement la valeur obtenue par M~~r~mvs et al. (1963) pour la r&action globale MI-MZZ.

K~s~mmenfaswng--Es wurde die Photolyse d*r aus dem Rindemehpurpur gewonnenen Metarhodopsins I untersucht. Die Verwandhmgsgeschwindigkeit des Meta I xum Sehpurpur hat kaum einen Dunkelbestandteil, d.h. der Sehpurpur erscheint beinahe so schnell, wie das Meta I das Licht absorbiert. Dies unterscheidet sich von den Versuchen tiber das Meta II, wo es einen leicht messbaren Dunkelbestandteil gibt. Rim anderer Unterschied besteht darin, dass im Gegensatx xu Meta II Meta I nicht einen Stoff, v&her dem P,rB analog ist, hervorbrachte. Die Lichtwechselverwandlungskraft des Meta I xum Sehpurpur wurde such untersucht: sie h8ngt sowohl vom pH als such von der Temperatur ab. Sie war beinahe Null bei pH 4 und erreichte einen Gipfel bei pH 6,5 und dart&r. Sie venninderte sich bei erhijhter Temperatur und diese Ergebnisse wurden im Sinne eines von WILLMMS(1970) vorgeschlagenen Mechanismus erkl&rt: MZsMZ’%MZZ + H*. Wenn man annimm t, dass das Meta I-Strich lichtunverwandelbar ist, so kann man xwei Gleichgewichtskonstanten im Rereich von 1-18” messen. Die thermodynamischen Parameter wurden berechnet und es wurde gefunden, dass AH f& die MZ-MZ’ Reaktion 11,3 KKal/Mole und fur die MI’-iUZZ Reaktion 1,8 KKal/Mole betrQt. Ihre Summe, niimlich 13,l KKal/Mole, gleicht genau dem von MATT~BWS et al. (1963) erhaltenen Wert fiir die Ganxreaktion des MI xu MZZ.

P~M+~&JI BccnenOBaH @o~o~iu~c ~e~apononc~~a 1, o6pa3o~a~~oro pononcBBo~ gPyWor0 pOIXTOr0CKOW.GICOpOCTB ~B~meBBa MeTa I B pOAOnCBn61.ma Maxa, ecrnr Boo6me xaxo& rnr60 “T~MHoB&‘* xoMlToBeBT,I.e. pOnonclnr nOIlBJUUICx OK0310 Tot-0 MOMenTa,rorna MeTa I nornomam CBeT.310 ommaewa OT TOrO, ¶TO EMeeTMeCTOnpn x3ynemrm hma II, me cymecTByeTBent0 B3Mepneti T~MBOBO~~ xohmoeBT. P~~BBBBB c MeTaHB BmaBamrcB TaK rite B TOM,¶TO Be 6bu1 saMeseA npom arra~orrrxrn& P4,,,, xorna McTa I sacBe¶rBaJtaca BcnBrmxoti. Ftemmrma @oToo6paTBMocrBMeTa I B pOBOncBB 6~ma Tax BeeBccnenoBaBaH 6buro rrB&nerro,BTOoBa sa~rtcrrr xax orpH, -ranH OTnhmepavPH. ‘@OTOO6~~ocTB nO¶TB npexparqanaCanpRpH4~~0~~mana Bepx=ro npcAJnra rtprr pH 6.5 mm xaxe 6onee BLICOKOM PH. QoToo6panrBrocra y~cwmsnacb c YecJrweHHtM V.I. 1l/s-F

451

458

BARBARAN.BAKERAND

THEODOREP.WILLLU+SS

TabaxepaTypH H m-E pe3yJibTRm o6lDKcEaJIE~Ea ocEoBaEmi MCxaEE3Ma npemoxemoro B nocae.mfee BpCMa WILLJAMS(1970): MI* MI’% MIZ + is+. rxpennonarax, ¶I0 McIa l-npm~ He 06~ &m~06pansrmcmo, 6ar~ ICSMC~~ISX me KOH~~ETH paBliOBW&U B npe~max 1-18’. ~LIIE Bhlnp~omraa~we~~ae xxapa~erp~ H 6-0 ~~FIO, PTO AH ~JISIpeaiqn MZ-MI’ pamio 11,3 ranom10pIog aa MOJB, a AJIJIpeanraa MT-MU. 1,8 KRilOBC8JIOpEti HB MOJIL kiX CyMM& 13,1 KEJlOK&lJIOpti/MOJIb, B TOPByWOTBeTCTeT xmmmenonywmoil hhm~~wsn ~p.(1963)arrrrrceti peamsm MI-MZL