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Light scattering from albumins
Volume
24, number 4
15 February
CHEMICAL PHYSICS LETTERS
LIGHT SCATTERING
1974
FROM ALBUMINS
J.P. BISCAR and N. KOLLIAS Department
of Physics and .4strottomy. the University Larantie, Wyoming S2070, USA Received
31
of Wyoming,
July 1973
In light scattering data of albumins. one distinguishes a large broad band from the normal Raman lines. That broad band in ovuline albumin is shown to have a pseudo-Raman behavior.
There is growing interest in the Raman investigation of long chain polymers, some of them being of biological origin. invariably in the spectra of such molecules there is a large broad band [l] which at times renders the recording of the Raman peaks very difficult. On top of a medium intensity broad band one can still detect the individual Raman lines as it has been done for bovine serum albumin [Z] (BSA) and human serum albumin [3] (HSA). These Raman lines and the broad band are two distinct phenomena. To investigate the latter, one has to separate the broad band data from the Raman peaks. The investigator can find himself confronted with three cases: (a) the intensity of the broad band is so large that the Raman peaks cannot be detected and thus they do not disturb;(b) the broad band is of medium intensity and the Raman peaks can be resolved. One can minimize their contribution to the profile of the broad band by decreasing the resolution of the monk chromator to about 200 cm-’ which will average the sharp Raman peaks. A better solution would be to resolve the whole spectrum and draw the base line at the bottom of the Raman peaks; (c) if the broad band is small, one has to resolve the complete spectrum and draw the base line of the Rarnan peaks separating the broad band for further study. When one is interested only in the conventional Raman peaks, one can destroy the broad band by long exposure of the molecules (not without molecular changes) to high laser intensity [4] _This is the tech- .’ nique recommended by the Spex Speaker: “Despite ‘.
the absence of an adequate explanation of the phenomenon, this drench-quench technique has proved so universally applicable that it shou!d be tried on every sample exhibiting high levels of broad band” [5]. Our point is that by doing so the Raman spectroscopists destroy an important phenomenon which is not fluorescence and deserves the most careful investigation. The proper investigation of a broad band is not as easy as obtaining the conventional Raman lines. The present technology provides argon laser lines of several watts’enhancing the Raman output and at the same time destroying the broad band. To carefully investigate the latter, one has to use laser input powers in the range of 1 mW to l/l00 mW in order to minimize the molecular perturbations. This implies that in order to compensate for the low intensity one has to use wide angle monochromators, and photon-counting systems. A Spectra Physics argon laser (16503) provided the exciting lines (with plasma fluorescence suppressor): The sample was irradiated for only one second every 30 seconds to allow recuperation time for the molecules. The scattered light was directed into an F/3, F matched monochromator preceded by filters attenuating the laser line by six ordersof magnitude with less attenuation than with.a second monochromatorThe intensity measurements were carried out using a GaAsP photocathode photomultiplier. followed by SSR, :I 129 amplifier discriminator and an 11 lo photon-counter. -. Data of the broad band obtained by this techniquein ovulirie albumin (GA) are clearly non-fhrorescenti The~erystallized samples . were obtained from Schwarz’ . ,-
-I_:_
. . ‘2
,56i.’
..,
‘.
1.
:.
.’ ‘:
.“._:
_:.:.
Volume
24, number 4
I
2
CHEMICAL PHYSICS LETTERS
3
WAVENUMBER
4
5
6x1000
hi’)
Fig-l. Pseudo-Raman broad band of ovuline albumin obtained at four exciting lines: 4579,4658,4880 and 5i45.4. The corresponding spectra are in decreasing intensity order. Laser power: 1 mW.
and used as received under temperature and moisture controlled conditions. Since only the profile of the broad band was desired, the monochromator was set for a resolution of approximately 200 cm-l_ Scattering data obtained that way, with exciting lines of 4579, 4658,488O and 5145 A can be plotted (after corrections for filters, grating blaze and photocathode quantum efficiency) versus monochromator wavelength setting. The display does not visualize a fluorescence phenomenon. The photon counting rate goes to zero at the proximity of each exciting line. This could not be the case if the latter was located inside, and especially near the peak, of a real fluorescence band. Another unusual feature is that the peak of the broad band shifts with. the exciting line and in the same direction. This is a clue for a Raman-like behavior_
IS February
1974
If one calculates the frequency difference (in cm-‘) between the exciting line and each data point the latter can be plotted in a Raman-like scale as in fig.]. The superposition of the broad bands of ovuline albumin obtained at four different exciting lines gives credence to the pseudo-Raman nature of this phenomenon. In the same figure one can also note a Raman-like susceptibility decrease with increasing exciting wavelength. For further confirmation, the existence of an antiStokes band was verified at 4579A. Its shape was, however, modified from the one displayed here on the Stokes side. This is very likely due to the fact that intermolecular couplings were modified by higher input laser power needed for the anti-Stokes recording. I In conclusion, albumins investigated do exhibit, in addition to the normal Raman lines an intense broad band which is not a fluorescence, and is very sensitive to !aser exposure (its investigation requiring low intensities). The data of ovuline albumin displayed in fig. 1 show that this broad band is a new pseudo-Raman phenomenon. By a special treatment of the sample we have resolved this broad band [6] in a series of peaks which make up the asymmetrical shape of this band. Similar results have been also obtained with other albumins and other chain molecules (to be reported).
References [ 11 W.B. Rippon, J.L. Koenig and A.G. Walton, I. Agr. Food Chem. 19 (1971) 692. [Z] J.P. Biscar. P. DhaII and J. Pennison, Chem. Phys. Letters 14 (1972) 569. [3] J-P. Biscar, P.K. Dhall and J.L. Pennison, Phys. Letters 39A (1972) 111. [4] J.L. Koenigand B. Fushour, Biopolymers 11 (1972) 1871. [Sj The Spex Speaker, Vol. 14, Sept. (1970). [6 1 J-P. Biscar and N_ KoIIias, to be published.
24, number 4
15 February
CHEMICAL PHYSICS LETTERS
LIGHT SCATTERING
1974
FROM ALBUMINS
J.P. BISCAR and N. KOLLIAS Department
of Physics and .4strottomy. the University Larantie, Wyoming S2070, USA Received
31
of Wyoming,
July 1973
In light scattering data of albumins. one distinguishes a large broad band from the normal Raman lines. That broad band in ovuline albumin is shown to have a pseudo-Raman behavior.
There is growing interest in the Raman investigation of long chain polymers, some of them being of biological origin. invariably in the spectra of such molecules there is a large broad band [l] which at times renders the recording of the Raman peaks very difficult. On top of a medium intensity broad band one can still detect the individual Raman lines as it has been done for bovine serum albumin [Z] (BSA) and human serum albumin [3] (HSA). These Raman lines and the broad band are two distinct phenomena. To investigate the latter, one has to separate the broad band data from the Raman peaks. The investigator can find himself confronted with three cases: (a) the intensity of the broad band is so large that the Raman peaks cannot be detected and thus they do not disturb;(b) the broad band is of medium intensity and the Raman peaks can be resolved. One can minimize their contribution to the profile of the broad band by decreasing the resolution of the monk chromator to about 200 cm-’ which will average the sharp Raman peaks. A better solution would be to resolve the whole spectrum and draw the base line at the bottom of the Raman peaks; (c) if the broad band is small, one has to resolve the complete spectrum and draw the base line of the Rarnan peaks separating the broad band for further study. When one is interested only in the conventional Raman peaks, one can destroy the broad band by long exposure of the molecules (not without molecular changes) to high laser intensity [4] _This is the tech- .’ nique recommended by the Spex Speaker: “Despite ‘.
the absence of an adequate explanation of the phenomenon, this drench-quench technique has proved so universally applicable that it shou!d be tried on every sample exhibiting high levels of broad band” [5]. Our point is that by doing so the Raman spectroscopists destroy an important phenomenon which is not fluorescence and deserves the most careful investigation. The proper investigation of a broad band is not as easy as obtaining the conventional Raman lines. The present technology provides argon laser lines of several watts’enhancing the Raman output and at the same time destroying the broad band. To carefully investigate the latter, one has to use laser input powers in the range of 1 mW to l/l00 mW in order to minimize the molecular perturbations. This implies that in order to compensate for the low intensity one has to use wide angle monochromators, and photon-counting systems. A Spectra Physics argon laser (16503) provided the exciting lines (with plasma fluorescence suppressor): The sample was irradiated for only one second every 30 seconds to allow recuperation time for the molecules. The scattered light was directed into an F/3, F matched monochromator preceded by filters attenuating the laser line by six ordersof magnitude with less attenuation than with.a second monochromatorThe intensity measurements were carried out using a GaAsP photocathode photomultiplier. followed by SSR, :I 129 amplifier discriminator and an 11 lo photon-counter. -. Data of the broad band obtained by this techniquein ovulirie albumin (GA) are clearly non-fhrorescenti The~erystallized samples . were obtained from Schwarz’ . ,-
-I_:_
. . ‘2
,56i.’
..,
‘.
1.
:.
.’ ‘:
.“._:
_:.:.
Volume
24, number 4
I
2
CHEMICAL PHYSICS LETTERS
3
WAVENUMBER
4
5
6x1000
hi’)
Fig-l. Pseudo-Raman broad band of ovuline albumin obtained at four exciting lines: 4579,4658,4880 and 5i45.4. The corresponding spectra are in decreasing intensity order. Laser power: 1 mW.
and used as received under temperature and moisture controlled conditions. Since only the profile of the broad band was desired, the monochromator was set for a resolution of approximately 200 cm-l_ Scattering data obtained that way, with exciting lines of 4579, 4658,488O and 5145 A can be plotted (after corrections for filters, grating blaze and photocathode quantum efficiency) versus monochromator wavelength setting. The display does not visualize a fluorescence phenomenon. The photon counting rate goes to zero at the proximity of each exciting line. This could not be the case if the latter was located inside, and especially near the peak, of a real fluorescence band. Another unusual feature is that the peak of the broad band shifts with. the exciting line and in the same direction. This is a clue for a Raman-like behavior_
IS February
1974
If one calculates the frequency difference (in cm-‘) between the exciting line and each data point the latter can be plotted in a Raman-like scale as in fig.]. The superposition of the broad bands of ovuline albumin obtained at four different exciting lines gives credence to the pseudo-Raman nature of this phenomenon. In the same figure one can also note a Raman-like susceptibility decrease with increasing exciting wavelength. For further confirmation, the existence of an antiStokes band was verified at 4579A. Its shape was, however, modified from the one displayed here on the Stokes side. This is very likely due to the fact that intermolecular couplings were modified by higher input laser power needed for the anti-Stokes recording. I In conclusion, albumins investigated do exhibit, in addition to the normal Raman lines an intense broad band which is not a fluorescence, and is very sensitive to !aser exposure (its investigation requiring low intensities). The data of ovuline albumin displayed in fig. 1 show that this broad band is a new pseudo-Raman phenomenon. By a special treatment of the sample we have resolved this broad band [6] in a series of peaks which make up the asymmetrical shape of this band. Similar results have been also obtained with other albumins and other chain molecules (to be reported).
References [ 11 W.B. Rippon, J.L. Koenig and A.G. Walton, I. Agr. Food Chem. 19 (1971) 692. [Z] J.P. Biscar. P. DhaII and J. Pennison, Chem. Phys. Letters 14 (1972) 569. [3] J-P. Biscar, P.K. Dhall and J.L. Pennison, Phys. Letters 39A (1972) 111. [4] J.L. Koenigand B. Fushour, Biopolymers 11 (1972) 1871. [Sj The Spex Speaker, Vol. 14, Sept. (1970). [6 1 J-P. Biscar and N_ KoIIias, to be published.