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Thermal decomposition of rhodopsin, photoregenerated rhodopsin and P470
Won Res.
Vol.8,
pp. 1467-1469.
PergamonPress1968. Printedin GrertBritain.
THERMAL DECOMPOSITION OF RHODOPSIN, PHOTOREGENERATED RHODOPSIN AND P4701 BARBARA N. BAKER
and THEODOREP. WILLIAMS
Department of Biological Sciences and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306 (Received 8 August 1968)
HUBBARD
(1958) has shown that opsin derives much of its stability from interaction with its prosthetic group. She showed that rhodopsin, the 1l-k, pigment was more stable than isorhodopsin, the 9-cis pigment, which in turn was more stable than opsin, the protein moiety with no prosthetic group. In the accompanying paper (WILLIAMS, 1968), it was found that the formation of rhodopsin from meta II requires some time for the opsin to conform to the 1I-& geometry. It was suggested that the steric requirements of 11-cis retinal compel the meta II protein configuration to adjust and to become a “rhodopsin contiguration”. On the other hand, the production of P470 from meta II was found to be instantaneous, and it was proposed that the chromophore of this pigment (13-k?) finds opsin of meta II in a conformation that is suitable for a fairly good fit. It was proposed, therefore, that the opsin of P470 is essentially the opsin of meta II, the shift in absorption from 380 mn to 470 nm being due to increased chromophore-protein interaction. If Hubbard’s idea is correct, this increased interaction should cause P470 to be more stable than meta II. But since freshly extracted rhodopsin and photoregenerated (from meta II) rhodopsin appear identical in spectra and optical rotatory dispersion (WILLIAMS, 1966), it might be expected that these two substances should have similar stabilities. In order to test these hypotheses, the thermal stabilities of photoregenerated rhodopsin, freshly-extracted rhodopsin, P470 and meta II were compared. Cattle rhodopsin solutions (2% digitonin, pH 6.5) were prepared and divided into -two equal parts. One portion was reserved and run as “freshly extracted” rhodopsin. The remainder was subjected to a double-flash procedure : First, a “white” electronic flash was given which caused all the molecules to absorb at least one quantum, and then, after a one-second delay, a U.V.flash was given. The second flash photolyzes meta II and produces rhodopsin (some im) and P470. If the thermal decay of photoregenerated rhodopsin was to be studied, NHzOH (final concentration O*lM) was added. This rapidly destroyed the P470 pigment (and other unstable intermediates), leaving only photoregenerated rhodopsin and a small amount of iso-rhodopsin. The decay of this rhodopsin along with the freshly extracted material was followed in a conventional way (cf. WIUUMS and MILBY, 1968). When studying the thermal stability of P470, no NH20H was added to the doubly1 This work was supported by USPHS Grant NB-07140. 1467
1468
BARBARA N. BAKER AND THEODOREP. WILLIAMS
flashed solutions. Since P470 is quite labile, it was possible to follow its decomposition at temperatures at which rhodopsin is stable. Difference spectra obtained from P470 decomposition runs showed that it was a pigment of Ai?,, 465-470 nm, i.e. one produced by the U.V.flash, that was being destroyed. This was checked to prevent confusion with acid NRO (440 nm) or other possible species. The data for the decomposition of fresh and photoregenerated rhodopsins are shown as circles in Fig. 1. The open circles are the rate constants for the decomposition of the fresh
i
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lo-s1 ’ 3.00
I
I
3.20
I
I
3.40
10001-f FIG. 1. Arrhenius plots of rates of decomposition of native (open circles) and photoregenerated rhodopsin (filled circles), P470 (triangles), and metarhodopsin II (dashed line). The respective activation energies are 50 kcal/mole, 48 kcal/mole and 20 kc&/mole.
rhodopsin pigment and the filled circles are those for the photoregenerated material. No major differences in the rates are obvious. What appears to be a trend toward less stability in the photoregenerated pigment is reversed in one case. Furthermore, repeated measurements of the rate at selected temperatures did not show that either kind of rhodopsin was consistently more stable than the other. The rate constants for decomposition of P470 (triangles) are cu. 500 times greater than
those of rhodopsin. However, they are smaller than those (dashed line) calculated from the data of HUBBARD et al. (1965) for the decay of meta II. In summary, the conclusions are: (a) Photoregenerated rhodopsin is as stable toward heat bleaching as freshly extracted rhodopsin; and (b) P470, while much more labile than rhodopsm, is more stable than the meta II from which it was produced.
Thermal Decomposition of Rhodopsin, Photoregenerated Rhodopsin and P470
1469
These results provide further evidence that photoregenerated rhodopsin is identical to freshly extracted rhodopsin. However, in this study, as in an earlier one (WILLUMS, 1966), it is not possible to ascertain the nature of photoregenerated opsin in the region of protein absorption (u.v.). (Compare WILLIAMS, 1966, for a discussion of the difficulties attendant to making optical rotatory dispersion measurements directly in such regions.) The comparison of P470 and meta II should be qualified: Many of the known intermediates of bleaching are more stable than their precursors. For example, huni- is more stable than prelumi-; meta I is more stable than lumi-, etc. Therefore, it might be expected that P470 would be more stable than the meta II from which it is produced. However, the difference is that in this case the means of producing P470 is photic, not thermal as it is in the “normal” sequence. Difference spectra showed that cu. 15% iso-rhodopsin was mixed with the photoregenerated rhodopsin. Since it comprises such a small fraction of the total pigment and does not decay at a rate grossly different from rhodopsin (HUBBARD, 1958), it did not distort the kinetic measurements. REFERENCES HUBBARD, R. (1958). The thermal stability of rhodopsin and opsin. J. gen. PhysioZ. 42,259-280. HUBBARD,R., Bows, D. and Yosxuz~w~, T. (1965). The chemistry of visual photoreception. Cold Spring Harb. Symp. quant. Biol. 30, 301-315. WILL.UM$T. P. (1966). Induced asymmetry in the prosthetic group of rhodopsin. Vision Res. 6, 293-300. WILLUMS,T. P. and MILBY,S. (1968). The thermal decomposition of some visual pigments. Vision Res. 8,359-367. WILUAMS,T. P. (1968). Vision Res. 8. Abstract-The thermal stabilities of rhodopsin and photoregenerated rhodopsin in the temperature range 4O-6O”Care determined and found to be the same. The thermal decomposition of P470, produced by photolysis of rnetarhodopsin II, is measured in the temperature range 23-4O”C. The rates of decomposition of P470 are compared with those observed (by other workers) for metarhodopsin II. All rates are found to be first-order. Arrhenuis activation energies are given for all pigments studied. R&am&-Gn determine la stabilite thermique de la rhodopsine et de la rhodopsine photor&&n&& darts l’intervalle de temperature 4O-6O”C: on lea trouves identiquea. Gn mesure aussi dans l’intervalle 23-4O”C la decomposition thermique du pigment P470, produit par photolyse de la m&arhodopsine II. Gn compare lea vitessea de decomposition de P470 a celles que d’autres auteurs ont observees pour la metarhopsine II. On trouve que toutes les vitesses sont du premier ordre. On donne pour tous lea pigments &udiC les energies d’activation d’Arrhenius. Zusamrnenfassung-Die therm&hen Stabilititen von Rbodopsin und photoregeneriertem Rhodopsin im Temperaturbereich von 4O-6O”C wurden bestimmt und erwiesen sich als gleich. Der thermische &fall von P470, das durch Photolyse von Metarhodopsin II entsteht, wurde im Bereich von 23-4o”C gemessen. Die Zerfallsgeachwindigkeiten von P470 werden mit jenen vergleichen, die (von anderen Autoren) bei Metarhodopsin II beobachtet wurden. Alle Geschwindigkeiten waren von erster Grdnung. Die Arrheniusschen Axmegungsenergien werden ti alle untersuchten Sehstoffe angegeben.
Pe3lome -
OnpexeJIriJxacb ~phKKTa6EJIbHoCTb po~ortcr5ra H ~OToreriepripyeMoro pononcziua npri TeMnepaType rtopma 4O-6O”C H rra&reuo, 910 orra omiria~o~a. TepMaJtbHoe pasnoxemie P470, Bbr3bmaeM0e (PoToJni3~c0M Merapo~oncrina II, u3Mepeso rrprr TeMnepaType nopgmta 234°C. C~opocn, pa3Jroxcerrna P470 cpaenuBanacb c TaKOBOtixe rra6mo~errrrofi (ApyrEiMEEUxIenOBaTeJUIMX)J~J’~T MeTapo~oJIcE?ra II. Ha.@cerro, VT0 Bee 3TSiCKOpocTuCOOTB~TCI~~I~T m ncpu0ro nopea. 3rieprmi arcrrmamiri no Appermycy Aat% AJIg BCeXrfs~errr3bxX mirMeEtTOB.
Vol.8,
pp. 1467-1469.
PergamonPress1968. Printedin GrertBritain.
THERMAL DECOMPOSITION OF RHODOPSIN, PHOTOREGENERATED RHODOPSIN AND P4701 BARBARA N. BAKER
and THEODOREP. WILLIAMS
Department of Biological Sciences and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306 (Received 8 August 1968)
HUBBARD
(1958) has shown that opsin derives much of its stability from interaction with its prosthetic group. She showed that rhodopsin, the 1l-k, pigment was more stable than isorhodopsin, the 9-cis pigment, which in turn was more stable than opsin, the protein moiety with no prosthetic group. In the accompanying paper (WILLIAMS, 1968), it was found that the formation of rhodopsin from meta II requires some time for the opsin to conform to the 1I-& geometry. It was suggested that the steric requirements of 11-cis retinal compel the meta II protein configuration to adjust and to become a “rhodopsin contiguration”. On the other hand, the production of P470 from meta II was found to be instantaneous, and it was proposed that the chromophore of this pigment (13-k?) finds opsin of meta II in a conformation that is suitable for a fairly good fit. It was proposed, therefore, that the opsin of P470 is essentially the opsin of meta II, the shift in absorption from 380 mn to 470 nm being due to increased chromophore-protein interaction. If Hubbard’s idea is correct, this increased interaction should cause P470 to be more stable than meta II. But since freshly extracted rhodopsin and photoregenerated (from meta II) rhodopsin appear identical in spectra and optical rotatory dispersion (WILLIAMS, 1966), it might be expected that these two substances should have similar stabilities. In order to test these hypotheses, the thermal stabilities of photoregenerated rhodopsin, freshly-extracted rhodopsin, P470 and meta II were compared. Cattle rhodopsin solutions (2% digitonin, pH 6.5) were prepared and divided into -two equal parts. One portion was reserved and run as “freshly extracted” rhodopsin. The remainder was subjected to a double-flash procedure : First, a “white” electronic flash was given which caused all the molecules to absorb at least one quantum, and then, after a one-second delay, a U.V.flash was given. The second flash photolyzes meta II and produces rhodopsin (some im) and P470. If the thermal decay of photoregenerated rhodopsin was to be studied, NHzOH (final concentration O*lM) was added. This rapidly destroyed the P470 pigment (and other unstable intermediates), leaving only photoregenerated rhodopsin and a small amount of iso-rhodopsin. The decay of this rhodopsin along with the freshly extracted material was followed in a conventional way (cf. WIUUMS and MILBY, 1968). When studying the thermal stability of P470, no NH20H was added to the doubly1 This work was supported by USPHS Grant NB-07140. 1467
1468
BARBARA N. BAKER AND THEODOREP. WILLIAMS
flashed solutions. Since P470 is quite labile, it was possible to follow its decomposition at temperatures at which rhodopsin is stable. Difference spectra obtained from P470 decomposition runs showed that it was a pigment of Ai?,, 465-470 nm, i.e. one produced by the U.V.flash, that was being destroyed. This was checked to prevent confusion with acid NRO (440 nm) or other possible species. The data for the decomposition of fresh and photoregenerated rhodopsins are shown as circles in Fig. 1. The open circles are the rate constants for the decomposition of the fresh
i
!
I o-2
lo-s1 ’ 3.00
I
I
3.20
I
I
3.40
10001-f FIG. 1. Arrhenius plots of rates of decomposition of native (open circles) and photoregenerated rhodopsin (filled circles), P470 (triangles), and metarhodopsin II (dashed line). The respective activation energies are 50 kcal/mole, 48 kcal/mole and 20 kc&/mole.
rhodopsin pigment and the filled circles are those for the photoregenerated material. No major differences in the rates are obvious. What appears to be a trend toward less stability in the photoregenerated pigment is reversed in one case. Furthermore, repeated measurements of the rate at selected temperatures did not show that either kind of rhodopsin was consistently more stable than the other. The rate constants for decomposition of P470 (triangles) are cu. 500 times greater than
those of rhodopsin. However, they are smaller than those (dashed line) calculated from the data of HUBBARD et al. (1965) for the decay of meta II. In summary, the conclusions are: (a) Photoregenerated rhodopsin is as stable toward heat bleaching as freshly extracted rhodopsin; and (b) P470, while much more labile than rhodopsm, is more stable than the meta II from which it was produced.
Thermal Decomposition of Rhodopsin, Photoregenerated Rhodopsin and P470
1469
These results provide further evidence that photoregenerated rhodopsin is identical to freshly extracted rhodopsin. However, in this study, as in an earlier one (WILLUMS, 1966), it is not possible to ascertain the nature of photoregenerated opsin in the region of protein absorption (u.v.). (Compare WILLIAMS, 1966, for a discussion of the difficulties attendant to making optical rotatory dispersion measurements directly in such regions.) The comparison of P470 and meta II should be qualified: Many of the known intermediates of bleaching are more stable than their precursors. For example, huni- is more stable than prelumi-; meta I is more stable than lumi-, etc. Therefore, it might be expected that P470 would be more stable than the meta II from which it is produced. However, the difference is that in this case the means of producing P470 is photic, not thermal as it is in the “normal” sequence. Difference spectra showed that cu. 15% iso-rhodopsin was mixed with the photoregenerated rhodopsin. Since it comprises such a small fraction of the total pigment and does not decay at a rate grossly different from rhodopsin (HUBBARD, 1958), it did not distort the kinetic measurements. REFERENCES HUBBARD, R. (1958). The thermal stability of rhodopsin and opsin. J. gen. PhysioZ. 42,259-280. HUBBARD,R., Bows, D. and Yosxuz~w~, T. (1965). The chemistry of visual photoreception. Cold Spring Harb. Symp. quant. Biol. 30, 301-315. WILL.UM$T. P. (1966). Induced asymmetry in the prosthetic group of rhodopsin. Vision Res. 6, 293-300. WILLUMS,T. P. and MILBY,S. (1968). The thermal decomposition of some visual pigments. Vision Res. 8,359-367. WILUAMS,T. P. (1968). Vision Res. 8. Abstract-The thermal stabilities of rhodopsin and photoregenerated rhodopsin in the temperature range 4O-6O”Care determined and found to be the same. The thermal decomposition of P470, produced by photolysis of rnetarhodopsin II, is measured in the temperature range 23-4O”C. The rates of decomposition of P470 are compared with those observed (by other workers) for metarhodopsin II. All rates are found to be first-order. Arrhenuis activation energies are given for all pigments studied. R&am&-Gn determine la stabilite thermique de la rhodopsine et de la rhodopsine photor&&n&& darts l’intervalle de temperature 4O-6O”C: on lea trouves identiquea. Gn mesure aussi dans l’intervalle 23-4O”C la decomposition thermique du pigment P470, produit par photolyse de la m&arhodopsine II. Gn compare lea vitessea de decomposition de P470 a celles que d’autres auteurs ont observees pour la metarhopsine II. On trouve que toutes les vitesses sont du premier ordre. On donne pour tous lea pigments &udiC les energies d’activation d’Arrhenius. Zusamrnenfassung-Die therm&hen Stabilititen von Rbodopsin und photoregeneriertem Rhodopsin im Temperaturbereich von 4O-6O”C wurden bestimmt und erwiesen sich als gleich. Der thermische &fall von P470, das durch Photolyse von Metarhodopsin II entsteht, wurde im Bereich von 23-4o”C gemessen. Die Zerfallsgeachwindigkeiten von P470 werden mit jenen vergleichen, die (von anderen Autoren) bei Metarhodopsin II beobachtet wurden. Alle Geschwindigkeiten waren von erster Grdnung. Die Arrheniusschen Axmegungsenergien werden ti alle untersuchten Sehstoffe angegeben.
Pe3lome -
OnpexeJIriJxacb ~phKKTa6EJIbHoCTb po~ortcr5ra H ~OToreriepripyeMoro pononcziua npri TeMnepaType rtopma 4O-6O”C H rra&reuo, 910 orra omiria~o~a. TepMaJtbHoe pasnoxemie P470, Bbr3bmaeM0e (PoToJni3~c0M Merapo~oncrina II, u3Mepeso rrprr TeMnepaType nopgmta 234°C. C~opocn, pa3Jroxcerrna P470 cpaenuBanacb c TaKOBOtixe rra6mo~errrrofi (ApyrEiMEEUxIenOBaTeJUIMX)J~J’~T MeTapo~oJIcE?ra II. Ha.@cerro, VT0 Bee 3TSiCKOpocTuCOOTB~TCI~~I~T m ncpu0ro nopea. 3rieprmi arcrrmamiri no Appermycy Aat% AJIg BCeXrfs~errr3bxX mirMeEtTOB.