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Absence of effect of hydroxylamine upon production rates of some rhodopsin photo intermediates
VisionRes. Vol. 10, pp. 897-900.
PergamonPress 1970. Printedin Great Britain.
RESEARCH NOTE ABSENCE OF EFFECT OF HYDROXYLAMINE UPON PRODUCTION RATES OF SOME RHODOPSIN PHOTO INTERMEDIATES RAYMOND
Department
H.
JOHNSON*
of Biological Sciences and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, U.S.A. (Received 17 March 1970)
HYDROXYLAMINE has Iong been used in the spectral examination of visual pigments. In this laboratory, its function has been presumed to be twofold: (1) hydroxylamine reacts with free retinal to form an oxime product having a blue-shifted X,,, and a narrower absorption envelope compared to other intermediates present during light bleaching, and (2) hydroxylamine hastens the decomposition of these bleaching intermediates. The purpose of this note is to show that hydroxylamine does not change the rates of formation of photo intermediates up to and including meta II and, furthermore, does not change the position of photoequilibrium between rhodopsin and meta I achieved during the time course of intense light flashes. These conclusions are developed for rhodopsin solubilized in three different solvent systems and exposed to flashes from two different light sources. METHODS
AND
MATERIALS
Cattle rod segments were isolated by the usual sucrose flotation procedures. Rhodopsin was solubilized by 2% (w/v) digitonin, cetyltrimethylammonium bromide (CTAB) or Triton X-100 solutions made up in 0.15 M phosphate buffer, pH 6.6. A O-5 M hydroxylamine stock solution was prepared by neutralizing the hydrochloride salt with KOH to a pH of 6.6 and diluting to volume with buffer solution. The stock solution was stored in a refrigerator until needed; tests showed the pH of the stock solution was unchanged over the duration of the experiments. The iinal concentration of hydroxyl~ine in all the experiments reported here was O-1 M. Results from two kinds of experiments are reported: measurements of the fraction of rhodopsin bleached by intense light flashes and rates of production of meta II. Determinations of the fraction bleached were made by accurately measuring @20 ml of rhodopsin solution into each of two cuvettes and adding 0.05 ml of the stock hydroxylamine solution to one of the cuvettes. The mixture was stirred and the OD at 500 nm of each cuvette measured on a Gilford spectrophotometer. Both cuvettes were then &shed separately in the apparatus described below, 0.05 ml of hydroxyiamine added to the second cuvette and the OD of each cuvette again measured. FinaS OD me~urements were made after the contents of each cuvette had been completely bleached by the house lights. Temperature control was maintained by circulating water of the appropriate temperature through an aluminium block designed to fit snugly around the cuvette (10 mm pathlength) that contained the sample. Short, intense light flashes were provided by either a Honeywell 65-C Aash gun (flash duration of 2 msec) or Sylvania 26 Ep flash bulbs (flash duration of 60 msec). The flasher was positioned so that light was delivered vertically upward through the bottom of the cuvette and its contents. Ultraviolet light was blocked from the sample by a Coming 3-72 filter. Rates of production of meta If were measured on the kinetics apparatus previously described by WELLUS (1968). Meta II concentrations were determined by monitoring OD changes at 380 nm. Ultraviolet light from the 2 msec flash source was prevented from reaching the sample. Initial sample preparation was identical to that described above for the fraction bleached measurements. *This work supported
by USPHS Grant EY 00479-03 and PHS Traineeship 897
Grant
lTOl-MH-1121801.
898
&SEAXC?I
Norm
RESULTS AND DISCUSSION Figure la shows the fraction of rhodopsin bleached by a 2 msec flash in each of the three solvents, as a function of temperature. Although, at a given temperature, the fraction bleached differs from solvent to solvent, there is no apparent dependence of this result upon the presence of hydroxylamine during the course of the flash. Figure lb is a similar plot of results obtained by using 60 msec flash bulbs as the quanta1 source. Again, the presence of hydroxylamine exercises no apparent effect upon the outcome of the experiment. It may
be noted that, in a given solvent at the same temperature, the fraction bleach& is greater for the 60 resee flash than for the 2 msec flash, Note, however, that irrespective of the #ash source used, the fraction bleached tends toward an upper limit, that is less than 1.0, as thetem~ra~~ is increased. This trend is more evident in CTAB and Triton than in digitonin; it would have become manifest also in digitonin at higher temperatures than those used in the present experiments. WIUIAMS (1970) has pr@ that moderately short, intense flashes establish a photoequilibrium between rhodopsin and meta I in digitonin solution and, by analogy, in solutions of other surf&ants. The amount of rhodopsin bleached at the end of a f&h is one measure of the ~rn~a~ depe&nee of the rate at which meta I, during the flash, is converted to intermediatea further along in the bleaching pathway. Thus, the results plotted in Fig. 1 illustrate the following points: (1) hydroxylamine does not affect the position of photoequilibrium between rhodopsin and meta I in any of the three solvents, and (2) it does not affect the rate (on a msec time scale) at which meta I is converted to subsequent bleaching intermediates. To corroborate the seoond conolusion above, rn~~erne~~ made of the actual production of meta II during the lifaime of the 2 msec flash are plotted in Fig. 2. A temperature (16”) was chosen such that meta II was still being produced by the end of the fiat& in each of the three solvents, i.e. there was a thermal component to meta II formation as well as a photic
RESEARCH
899
Nm
component (WILLIAMS and BREIL, 1968). There are, therefore, only two ways by which the presence of hydroxylamine could affect the rate of meta II formation: either by modifying the position of some kind of photoequilibrium or by changing the rate at which meta II is thermally produced from meta I. Because meta II itself was not allowed to absorb light, there was no complication that a photoequilibrium might be established between it and any other intermediate or rhodopsin itself. Therefore, if hydroxylamine does affect the rate of meta II production, its influence becomes apparent solely by its effect upon this thermal component. As the data in Fig. 2 illustrate, hydroxylamine exerts no control upon the thermal rate of meta II formation.
O-6
s
Ed .
/-
1’ 0.6
n
Time,
set
FIG. 2. Production of meta II in CTAB (curve A), Triton X-100 (B) and digitonin (C) during the lifetime of a 2 msec flash, at 16” and pH of 6.6. R. is the initial concentration of rhodopsin in each of the three solutions. The size of the symbols is proportional to the magnitude of the experimental differences resulting from the presence and absence of 0.1 M NH,OH during the flash. There was no observable trend in the distribution of points, i.e. the values of points obtained from solutions containing hydroxylamine were never consistently higher or lower than those obtained from solutions not containing the reagent. (- - - -) Integrated flash output (units on right vertical axis).
Thus, the results reported in this note appear to indicate that, in the three different solutions examined, hydroxylamine is an inert ingredient throughout many of the initial bleaching stages. Only after meta II has been produced does the possibility exist that the final steps, leading ultimately to the oxime, may be accelerated by the presence of this reagent. It has been proposed (POINCELOT,MILLAR, KIMBEL and ABRAHAMSON, 1969) that a transfer of the retinal chromophore from a lipid portion of opsin to a protein portion of opsin occurs at some moment in time between the meta I and meta II stages of the bleaching sequence. Given the existence of this transfer, it might be expected that the rate or extent of meta II production would be somewhat different in solutions containing hydroxylamine compared to those without this reagent. One might reason that at some stage during the transfer, the chromophoric aldehyde must be more or less free, since the actual transfer reaction presumably involves the rupture of one imine bond between the chromophore lipid and formation of a second imine bond between the chromophore and a nearby lysine
RESEARCH NOTE
900
residue. The reaction between hydroxylamine and retinal also leads to formation of an imine bond and it would appear that any appreciable concentration of hydroxylamine at the transfer site could quite effectively compete with the protein for the chromophore. As mentioned, the results reported in this note show no distinction in rates or extent of meta II production. This fact does not in itself preclude the possibility of a lipid to protein transfer of the chromophore, but it does seem to indicate that the transfer site is sterically or in some other way masked from hydroxylamine in the bulk of the solution. As a consequence of these results, our own procedures used in obtaining flash photolysis data have been considerably simplified. Heretofore we have always had to apply a dilution factor when measuring OD’s of rhodopsin solutions, since hydroxylamine was added after a flash was given to the sample. Now necessary pH adjustments, etc. can be made and hydroxylamine added before a determination of initial concentration, with confidence that rates of meta II production and the photoequilibrium between rhodopsin and meta I will not be changed during the time course of those reactions presently of interest to us. AcknowMpnevm-The author thanks Dr. T. P. WULLUU for numerous constructive suggestions and discussions and Mrs. M. M~DDUIWNand Mr. B. WILLIAMS for their assistance in preparing this note.
REFERENCES E. W. (1969). Lipid to protein chromoPUNCELJX,R. P., M~LLAR,P. G., KIMEEL,R. L. and ,4mummo~, phom transfer in the photolysis of visual pigments. Ncrlurc, Land. Z&256-257. Ww+xms, T. P. (1968). Photolysis of metarhodop& II: Rates of production of P470 and rhodopsiu Vision Res. 8, 1457-1466. WILLLU&T. P. and B~EIL,S. J. (1968). Kinetic measurements on rhodopsin solutions during intense k&es. Vision Res. 8,777-786.
WILLUMS,T. P. (1970). An isochromic change in the bkachhq of rhodopsin. lb&n Res. 10,525-533.
PergamonPress 1970. Printedin Great Britain.
RESEARCH NOTE ABSENCE OF EFFECT OF HYDROXYLAMINE UPON PRODUCTION RATES OF SOME RHODOPSIN PHOTO INTERMEDIATES RAYMOND
Department
H.
JOHNSON*
of Biological Sciences and Institute of Molecular Biophysics, Florida State University, Tallahassee, Florida 32306, U.S.A. (Received 17 March 1970)
HYDROXYLAMINE has Iong been used in the spectral examination of visual pigments. In this laboratory, its function has been presumed to be twofold: (1) hydroxylamine reacts with free retinal to form an oxime product having a blue-shifted X,,, and a narrower absorption envelope compared to other intermediates present during light bleaching, and (2) hydroxylamine hastens the decomposition of these bleaching intermediates. The purpose of this note is to show that hydroxylamine does not change the rates of formation of photo intermediates up to and including meta II and, furthermore, does not change the position of photoequilibrium between rhodopsin and meta I achieved during the time course of intense light flashes. These conclusions are developed for rhodopsin solubilized in three different solvent systems and exposed to flashes from two different light sources. METHODS
AND
MATERIALS
Cattle rod segments were isolated by the usual sucrose flotation procedures. Rhodopsin was solubilized by 2% (w/v) digitonin, cetyltrimethylammonium bromide (CTAB) or Triton X-100 solutions made up in 0.15 M phosphate buffer, pH 6.6. A O-5 M hydroxylamine stock solution was prepared by neutralizing the hydrochloride salt with KOH to a pH of 6.6 and diluting to volume with buffer solution. The stock solution was stored in a refrigerator until needed; tests showed the pH of the stock solution was unchanged over the duration of the experiments. The iinal concentration of hydroxyl~ine in all the experiments reported here was O-1 M. Results from two kinds of experiments are reported: measurements of the fraction of rhodopsin bleached by intense light flashes and rates of production of meta II. Determinations of the fraction bleached were made by accurately measuring @20 ml of rhodopsin solution into each of two cuvettes and adding 0.05 ml of the stock hydroxylamine solution to one of the cuvettes. The mixture was stirred and the OD at 500 nm of each cuvette measured on a Gilford spectrophotometer. Both cuvettes were then &shed separately in the apparatus described below, 0.05 ml of hydroxyiamine added to the second cuvette and the OD of each cuvette again measured. FinaS OD me~urements were made after the contents of each cuvette had been completely bleached by the house lights. Temperature control was maintained by circulating water of the appropriate temperature through an aluminium block designed to fit snugly around the cuvette (10 mm pathlength) that contained the sample. Short, intense light flashes were provided by either a Honeywell 65-C Aash gun (flash duration of 2 msec) or Sylvania 26 Ep flash bulbs (flash duration of 60 msec). The flasher was positioned so that light was delivered vertically upward through the bottom of the cuvette and its contents. Ultraviolet light was blocked from the sample by a Coming 3-72 filter. Rates of production of meta If were measured on the kinetics apparatus previously described by WELLUS (1968). Meta II concentrations were determined by monitoring OD changes at 380 nm. Ultraviolet light from the 2 msec flash source was prevented from reaching the sample. Initial sample preparation was identical to that described above for the fraction bleached measurements. *This work supported
by USPHS Grant EY 00479-03 and PHS Traineeship 897
Grant
lTOl-MH-1121801.
898
&SEAXC?I
Norm
RESULTS AND DISCUSSION Figure la shows the fraction of rhodopsin bleached by a 2 msec flash in each of the three solvents, as a function of temperature. Although, at a given temperature, the fraction bleached differs from solvent to solvent, there is no apparent dependence of this result upon the presence of hydroxylamine during the course of the flash. Figure lb is a similar plot of results obtained by using 60 msec flash bulbs as the quanta1 source. Again, the presence of hydroxylamine exercises no apparent effect upon the outcome of the experiment. It may
be noted that, in a given solvent at the same temperature, the fraction bleach& is greater for the 60 resee flash than for the 2 msec flash, Note, however, that irrespective of the #ash source used, the fraction bleached tends toward an upper limit, that is less than 1.0, as thetem~ra~~ is increased. This trend is more evident in CTAB and Triton than in digitonin; it would have become manifest also in digitonin at higher temperatures than those used in the present experiments. WIUIAMS (1970) has pr@ that moderately short, intense flashes establish a photoequilibrium between rhodopsin and meta I in digitonin solution and, by analogy, in solutions of other surf&ants. The amount of rhodopsin bleached at the end of a f&h is one measure of the ~rn~a~ depe&nee of the rate at which meta I, during the flash, is converted to intermediatea further along in the bleaching pathway. Thus, the results plotted in Fig. 1 illustrate the following points: (1) hydroxylamine does not affect the position of photoequilibrium between rhodopsin and meta I in any of the three solvents, and (2) it does not affect the rate (on a msec time scale) at which meta I is converted to subsequent bleaching intermediates. To corroborate the seoond conolusion above, rn~~erne~~ made of the actual production of meta II during the lifaime of the 2 msec flash are plotted in Fig. 2. A temperature (16”) was chosen such that meta II was still being produced by the end of the fiat& in each of the three solvents, i.e. there was a thermal component to meta II formation as well as a photic
RESEARCH
899
Nm
component (WILLIAMS and BREIL, 1968). There are, therefore, only two ways by which the presence of hydroxylamine could affect the rate of meta II formation: either by modifying the position of some kind of photoequilibrium or by changing the rate at which meta II is thermally produced from meta I. Because meta II itself was not allowed to absorb light, there was no complication that a photoequilibrium might be established between it and any other intermediate or rhodopsin itself. Therefore, if hydroxylamine does affect the rate of meta II production, its influence becomes apparent solely by its effect upon this thermal component. As the data in Fig. 2 illustrate, hydroxylamine exerts no control upon the thermal rate of meta II formation.
O-6
s
Ed .
/-
1’ 0.6
n
Time,
set
FIG. 2. Production of meta II in CTAB (curve A), Triton X-100 (B) and digitonin (C) during the lifetime of a 2 msec flash, at 16” and pH of 6.6. R. is the initial concentration of rhodopsin in each of the three solutions. The size of the symbols is proportional to the magnitude of the experimental differences resulting from the presence and absence of 0.1 M NH,OH during the flash. There was no observable trend in the distribution of points, i.e. the values of points obtained from solutions containing hydroxylamine were never consistently higher or lower than those obtained from solutions not containing the reagent. (- - - -) Integrated flash output (units on right vertical axis).
Thus, the results reported in this note appear to indicate that, in the three different solutions examined, hydroxylamine is an inert ingredient throughout many of the initial bleaching stages. Only after meta II has been produced does the possibility exist that the final steps, leading ultimately to the oxime, may be accelerated by the presence of this reagent. It has been proposed (POINCELOT,MILLAR, KIMBEL and ABRAHAMSON, 1969) that a transfer of the retinal chromophore from a lipid portion of opsin to a protein portion of opsin occurs at some moment in time between the meta I and meta II stages of the bleaching sequence. Given the existence of this transfer, it might be expected that the rate or extent of meta II production would be somewhat different in solutions containing hydroxylamine compared to those without this reagent. One might reason that at some stage during the transfer, the chromophoric aldehyde must be more or less free, since the actual transfer reaction presumably involves the rupture of one imine bond between the chromophore lipid and formation of a second imine bond between the chromophore and a nearby lysine
RESEARCH NOTE
900
residue. The reaction between hydroxylamine and retinal also leads to formation of an imine bond and it would appear that any appreciable concentration of hydroxylamine at the transfer site could quite effectively compete with the protein for the chromophore. As mentioned, the results reported in this note show no distinction in rates or extent of meta II production. This fact does not in itself preclude the possibility of a lipid to protein transfer of the chromophore, but it does seem to indicate that the transfer site is sterically or in some other way masked from hydroxylamine in the bulk of the solution. As a consequence of these results, our own procedures used in obtaining flash photolysis data have been considerably simplified. Heretofore we have always had to apply a dilution factor when measuring OD’s of rhodopsin solutions, since hydroxylamine was added after a flash was given to the sample. Now necessary pH adjustments, etc. can be made and hydroxylamine added before a determination of initial concentration, with confidence that rates of meta II production and the photoequilibrium between rhodopsin and meta I will not be changed during the time course of those reactions presently of interest to us. AcknowMpnevm-The author thanks Dr. T. P. WULLUU for numerous constructive suggestions and discussions and Mrs. M. M~DDUIWNand Mr. B. WILLIAMS for their assistance in preparing this note.
REFERENCES E. W. (1969). Lipid to protein chromoPUNCELJX,R. P., M~LLAR,P. G., KIMEEL,R. L. and ,4mummo~, phom transfer in the photolysis of visual pigments. Ncrlurc, Land. Z&256-257. Ww+xms, T. P. (1968). Photolysis of metarhodop& II: Rates of production of P470 and rhodopsiu Vision Res. 8, 1457-1466. WILLLU&T. P. and B~EIL,S. J. (1968). Kinetic measurements on rhodopsin solutions during intense k&es. Vision Res. 8,777-786.
WILLUMS,T. P. (1970). An isochromic change in the bkachhq of rhodopsin. lb&n Res. 10,525-533.