ANALYTICAL
BIOCHEMISTRY
Scattering
65, 596-599
( t 975)
of Light -A
Risk in Circular
Serious
Dichroism
in the Far Ultraviolet
Potential
Measurements Region
In our present spectropolarimetric studies on the effect of small molecules on the secondary and tertiary structures of proteins, some peculiar results were obtained. When the far-ultraviolet circular dichroism (far-uv CD) spectra of several proteins were recorded, the ellipticity of these proteins apparently was not directly proportional to the concentration at the wavelengths around 208 nm and below where the negative Cotton effect of cu-helices ascribed to the rr - n*-transition appears. As these observations may be of practical importance in other CD studies in the far-UV region, the results of our investigation to find the causes of the anomalies are reported here. Lysozyme, myoglobin and human serum albumin were of normal commercial standard and were used without further purification. Circular dichroism spectra were recorded in a JASCO J-20 spectropolarimeter, Japan Spectroscopic Co., Tokyo. This instrument has a large cell compartment of 25 cm length. As standard, a fixed, circular diaphragm is placed in the cell compartment just in front of the cellholder. The rectangular cells with pathlengths 60.5 mm are placed at the far end of the thermostated universal cell-holder. Before scanning, each sample was filtered through a Millipore filter in order to eliminate particles larger than 0.3 pm. No light scattering effect was detectable in a recording spectrophotometer, when tested according to Beaven and Holiday (1). For other technical details, such as calibration, see Ref. (2). Figure 1 shows the CD spectra of lysozyme at different concentrations (l-10 mglml), recorded with a 0. I8 mm rectangular cell 3 cm from the opening to the detector unit. As is evident, the apparent molar ellipticity decreases at the lower wavelengths with increasing concentration. Similar results were obtained with myoglobin and human serum albumin. However, when a diluted sample of human serum albumin was studied in cells with increasing pathlength, the same CD spectra on a molar basis were obtained from all the cells in the wavelength region studied. Thus, the changed ellipticities noted were not related to changes of the photomultiplier dynode voltage or decreased signal/noise ratio. The first tentative conclusion from the results obtained above was that the CD spectra did deviate from Lambert-Beer’s law already at modera596 Copyright Q 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
0
7 Y E v .
200
r
210
597
COMMUNICATIONS 220
230
240
-5
-5
-10
-10
“5 . 7‘F 0 ; zf
-15
I 200
I 210
I 220 Wavelength,
I 230
I240
-15
nm
1. Circular dichroism spectra of lysozyme at pH 7.5, 25” and varying tions. All measurements were made in 0.18 mm rectangular cells. Lysozyme tions (mglml): A. 1.O; B. 3.0: C. 5.0; D, 10.0 with the cell close to the detector; with the cell far from the detector and a diaphragm placed after the cell. FIG.
concentraconcentraand E, I .O
tely high concentrations. However, when the CD spectra were checked in a JASCO J-40 and a Cary 6001 spectropolarimeter, these instruments gave correct results also at the highest protein concentration ( 10 mglml). Thus, the anomalous results could be related to the specific experimental set-up in the JASCO J-20 instrument. After several tests with different cells, cell-holders and cell positions, it was found that correct results could be obtained with the JASCO J-20 instrument provided that the cell was placed far from the detector or that a diaphragm was inserted between the cell and the detector. Evidently, the CD instrument is very sensitive to some kind of anomalous light, which is eliminated by the circular diaphragm inserted qfter the sample. Moreover, the disturbance was dependent on the wavelength, the concentration and the distance from the detector. These findings indicate that the erroneous spectra obtained at the higher concentrations might be due to a scattering effect of the sample having a deleterious effect on the optical purity of the transmitted light reaching the detector. The intensity of scattered light (i) in relation to the incident radiation of linearly polarized light (I,,) is given by the following equation (3):
(1) where CY~denotes molar polarizability: c, concentration; 4, the angle normal to the scattered light beam; r, the distance from the sample; and A, the wavelength.
598
SHORT
COMMUNICATIONS
The spectral effects noted when the distance between the sample and the detector, r, was changed and with different concentrations, c, suggest at least two reasons for the anomalies; first effects outside that part of the light beam, which reaches the detector, secondly effects inside the light beam. When the cell is placed close to the detector, light scattered from any object outside the beam will increase the total amount of light entering the detector unit. When the cell is removed from the detector, a decreasing fraction of the scattered light will hit the detector, as the light is scattered in all directions from the scattering object. When the concentration of the sample is increased, an increasing portion of the radiation in the light beam will be used to polarize the molecules in the sample. The radiation is then emitted as lirzearly polarized light. As the light incident to the sample is circularify polarized, the scattering effect will counteract the polarization in the Pockel cell. In spectropolarimeters, when CD is measured, the instrument recording is based on an electronic comparison between the different absorptions, AL and Aa, experienced by the light ray oscillating between left and right polarization, and a direct current due to the total radiation reaching the photomultiplier. The latter current is by a servo technique compensated to a constant value, via the photomultiplier voltage, so that the deflection on the recorder depends on the rippling current only. It is thus easy to see, that if the polarization is partly lost due to some kind of scattering process in the light beam, the intensity difference due to the CD of the sample will be seriously decreased. The same effect will also be seen, if the light is scattered from objects outside the beam into the photomultiplier or for some reason reaches the photomultiplier without passing the sample. The distortion of the CD spectra seen is similar to those often noticed in ORD, CD and absorption spectra of certain membranes and macromolecules (4,5). However, some controversy still exists as to the theoretical explanation for these optical artifacts, but the side effects can in some cases be related to the particle size according to the classical Mie theory (6). In the present study, the particle size cannot be the only reason for the artifacts as correct results were obtained with the same samples in an alternative arrangement of the cell and cell-holder. It is thus reasonable to assume that reflections in the cell-holder and/or cell may also contribute to the anomalies. Circular dichroism instruments are often modified to be used with special cells or cell-holders. From our experience it is evident that each experimental arrangement has to be carefully investigated to reveal any unwanted effects on the polarization of the radiation, especially when CD spectra are studied in high absorbing solutions in the far uv-region. The instrument can be easily checked by running CD spectra with two different concentrations.
599
SHORT COMMUNICATIONS
ACKNOWLEDGMENT We thank Dr. Bengt NordCn, Lund, Sweden, for valuable discussions and for the tests on the JASCO J-40 and Cary 60 spectropolarimeters.
REFERENCES 1. Beaven, G. H., and Holiday, E. R. (1952) Advan. Protein Chem. 7, 3 19. 2. SjGholm. I.. and Sjodin, T. (1972) Biochem. Pl~armacol. 21, 3041-3052. 3. van Holde, K. E. (1971) in Physical Biochemists, p. 182, Prentice Hall. Englewood Cliffs, NJ. 4. Ottaway. C. A., and Wetlaufer, D. B. (1970) Arch. Biochem. Biophys. 139, 257-264. 5. Urry, D. W. (1972) Biochim. Biophys. Acta 265, 115-168. 5. Gordon, D. J. (1972) Biochemistry 11, 413-420. INGVARSJ~HOLM
Bo EKMAN Department of Pharmaceutical Biochemistry Faculty of Fharmucy Biomedical Center, Box 578 Uppsala, Sweden Received June 10, 1974: accepted December 13, 1974
BIOCHEMISTRY
Scattering
65, 596-599
( t 975)
of Light -A
Risk in Circular
Serious
Dichroism
in the Far Ultraviolet
Potential
Measurements Region
In our present spectropolarimetric studies on the effect of small molecules on the secondary and tertiary structures of proteins, some peculiar results were obtained. When the far-ultraviolet circular dichroism (far-uv CD) spectra of several proteins were recorded, the ellipticity of these proteins apparently was not directly proportional to the concentration at the wavelengths around 208 nm and below where the negative Cotton effect of cu-helices ascribed to the rr - n*-transition appears. As these observations may be of practical importance in other CD studies in the far-UV region, the results of our investigation to find the causes of the anomalies are reported here. Lysozyme, myoglobin and human serum albumin were of normal commercial standard and were used without further purification. Circular dichroism spectra were recorded in a JASCO J-20 spectropolarimeter, Japan Spectroscopic Co., Tokyo. This instrument has a large cell compartment of 25 cm length. As standard, a fixed, circular diaphragm is placed in the cell compartment just in front of the cellholder. The rectangular cells with pathlengths 60.5 mm are placed at the far end of the thermostated universal cell-holder. Before scanning, each sample was filtered through a Millipore filter in order to eliminate particles larger than 0.3 pm. No light scattering effect was detectable in a recording spectrophotometer, when tested according to Beaven and Holiday (1). For other technical details, such as calibration, see Ref. (2). Figure 1 shows the CD spectra of lysozyme at different concentrations (l-10 mglml), recorded with a 0. I8 mm rectangular cell 3 cm from the opening to the detector unit. As is evident, the apparent molar ellipticity decreases at the lower wavelengths with increasing concentration. Similar results were obtained with myoglobin and human serum albumin. However, when a diluted sample of human serum albumin was studied in cells with increasing pathlength, the same CD spectra on a molar basis were obtained from all the cells in the wavelength region studied. Thus, the changed ellipticities noted were not related to changes of the photomultiplier dynode voltage or decreased signal/noise ratio. The first tentative conclusion from the results obtained above was that the CD spectra did deviate from Lambert-Beer’s law already at modera596 Copyright Q 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
0
7 Y E v .
200
r
210
597
COMMUNICATIONS 220
230
240
-5
-5
-10
-10
“5 . 7‘F 0 ; zf
-15
I 200
I 210
I 220 Wavelength,
I 230
I240
-15
nm
1. Circular dichroism spectra of lysozyme at pH 7.5, 25” and varying tions. All measurements were made in 0.18 mm rectangular cells. Lysozyme tions (mglml): A. 1.O; B. 3.0: C. 5.0; D, 10.0 with the cell close to the detector; with the cell far from the detector and a diaphragm placed after the cell. FIG.
concentraconcentraand E, I .O
tely high concentrations. However, when the CD spectra were checked in a JASCO J-40 and a Cary 6001 spectropolarimeter, these instruments gave correct results also at the highest protein concentration ( 10 mglml). Thus, the anomalous results could be related to the specific experimental set-up in the JASCO J-20 instrument. After several tests with different cells, cell-holders and cell positions, it was found that correct results could be obtained with the JASCO J-20 instrument provided that the cell was placed far from the detector or that a diaphragm was inserted between the cell and the detector. Evidently, the CD instrument is very sensitive to some kind of anomalous light, which is eliminated by the circular diaphragm inserted qfter the sample. Moreover, the disturbance was dependent on the wavelength, the concentration and the distance from the detector. These findings indicate that the erroneous spectra obtained at the higher concentrations might be due to a scattering effect of the sample having a deleterious effect on the optical purity of the transmitted light reaching the detector. The intensity of scattered light (i) in relation to the incident radiation of linearly polarized light (I,,) is given by the following equation (3):
(1) where CY~denotes molar polarizability: c, concentration; 4, the angle normal to the scattered light beam; r, the distance from the sample; and A, the wavelength.
598
SHORT
COMMUNICATIONS
The spectral effects noted when the distance between the sample and the detector, r, was changed and with different concentrations, c, suggest at least two reasons for the anomalies; first effects outside that part of the light beam, which reaches the detector, secondly effects inside the light beam. When the cell is placed close to the detector, light scattered from any object outside the beam will increase the total amount of light entering the detector unit. When the cell is removed from the detector, a decreasing fraction of the scattered light will hit the detector, as the light is scattered in all directions from the scattering object. When the concentration of the sample is increased, an increasing portion of the radiation in the light beam will be used to polarize the molecules in the sample. The radiation is then emitted as lirzearly polarized light. As the light incident to the sample is circularify polarized, the scattering effect will counteract the polarization in the Pockel cell. In spectropolarimeters, when CD is measured, the instrument recording is based on an electronic comparison between the different absorptions, AL and Aa, experienced by the light ray oscillating between left and right polarization, and a direct current due to the total radiation reaching the photomultiplier. The latter current is by a servo technique compensated to a constant value, via the photomultiplier voltage, so that the deflection on the recorder depends on the rippling current only. It is thus easy to see, that if the polarization is partly lost due to some kind of scattering process in the light beam, the intensity difference due to the CD of the sample will be seriously decreased. The same effect will also be seen, if the light is scattered from objects outside the beam into the photomultiplier or for some reason reaches the photomultiplier without passing the sample. The distortion of the CD spectra seen is similar to those often noticed in ORD, CD and absorption spectra of certain membranes and macromolecules (4,5). However, some controversy still exists as to the theoretical explanation for these optical artifacts, but the side effects can in some cases be related to the particle size according to the classical Mie theory (6). In the present study, the particle size cannot be the only reason for the artifacts as correct results were obtained with the same samples in an alternative arrangement of the cell and cell-holder. It is thus reasonable to assume that reflections in the cell-holder and/or cell may also contribute to the anomalies. Circular dichroism instruments are often modified to be used with special cells or cell-holders. From our experience it is evident that each experimental arrangement has to be carefully investigated to reveal any unwanted effects on the polarization of the radiation, especially when CD spectra are studied in high absorbing solutions in the far uv-region. The instrument can be easily checked by running CD spectra with two different concentrations.
599
SHORT COMMUNICATIONS
ACKNOWLEDGMENT We thank Dr. Bengt NordCn, Lund, Sweden, for valuable discussions and for the tests on the JASCO J-40 and Cary 60 spectropolarimeters.
REFERENCES 1. Beaven, G. H., and Holiday, E. R. (1952) Advan. Protein Chem. 7, 3 19. 2. SjGholm. I.. and Sjodin, T. (1972) Biochem. Pl~armacol. 21, 3041-3052. 3. van Holde, K. E. (1971) in Physical Biochemists, p. 182, Prentice Hall. Englewood Cliffs, NJ. 4. Ottaway. C. A., and Wetlaufer, D. B. (1970) Arch. Biochem. Biophys. 139, 257-264. 5. Urry, D. W. (1972) Biochim. Biophys. Acta 265, 115-168. 5. Gordon, D. J. (1972) Biochemistry 11, 413-420. INGVARSJ~HOLM
Bo EKMAN Department of Pharmaceutical Biochemistry Faculty of Fharmucy Biomedical Center, Box 578 Uppsala, Sweden Received June 10, 1974: accepted December 13, 1974
