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Turbidity correction for absorption spectra of colored solutions
584
SHORT COMMUNICATIONS
BBA 43 O7T Turbidity correction for absorption spectra of colored solutions Absorption spectra of heme proteins in aqueous extracts of fresh muscle tissue are often inaccurate because of light scattering caused b y turbidity, which is difficult to remove b y centrifugation or filtration. Even where filtration through a fine filter paper yields a clear solution, quantitative estimation of heme protein concentration b y absorbance measurement is subject to serious error owing to adsorption of some of the protein on the filter paper. One method of correcting for turbidity depends upon reading the absorbance due only to turbidity at a series of wavelengths (2) where the chromophore does not absorb light (the "baseline"). It has long been known that the intensity of scattered light produced b y a given amount of turbidity varies inversely with 2, being higher at the blue end than at the red end of the visible spectrum l& Accordingly, from these readings, a correction for turbidity, at a wavelength where the chromophore absorbs strongly, is calculated by extrapolation. This value is then subtracted from the apparent absorbance of the chromophore at that wavelength, in order to obtain the corrected absorbance ~,4. In another method, a piece of opal glass is placed after both sample and reference cells in the light paths of the spectrophotometer 5. The improved spectra resulting from this method can be read directly, but still do not give zero baselines. PERSON AND FINE have modified this procedure for microvolumes by applying the sample and reference solvent to small squares of filter paper 6. BARER achieved considerable reduction of the effect of light scattering by turbidity, by reducing to zero the difference between the refractive index of the suspension and that of the surrounding mediumL However, light scattering was found not to be completely eliminated in the intense Sorer absorption region of whole red blood cells; anomalous dispersion was thought to be likely to occur where there are sharp changes of refractive index. The best method would be one which not only corrects directly for turbidity in the instrument, but also requires the minimum of theoretical assumptions. In the sample cell we have solvent, colored solute plus turbidity: in the reference cell we want only the solvent ph~s a turbidity identical in light-scattering properties with that in the sample. We have obtained excellent results with turbid myoglobin and hemoglobin solutions by using, as a reference, very dilute solutions of ordinary milk, which very closely approaches the ideal requirement. Fig. I shows a comparison of the absorption spectra of two solutions of chicken oxyhemoglobin of equal colorimetric concentration at their Soret maxima, one being clear and the other including a fixed amount of turbidity. The small divergence of Curve c from a smooth curve between 41o and 45o m/z is attributable to dilution errors, which will be obvious in a region where the absorbance changes greatly with wavelength. The value of absorbance on this curve corresponding to the Soret m a x i m u m is exactly equal to Curve b mit~us Curve a. Similar spectra with Soret maxima from 0.3 to 1.8 in clear solution (up to 2.5 with added turbidity) have shown that, at any given wavelength between 400 and 700 m/,, the light scattering due to turbidity is independent of the absorption of the chromophore. The difference spectra were equal to the turbid mi~us the clear Biochim. Biophys. Acta, 112 (1966) 584-586
SHORT COMMUNICATIONS
585
spectra within a maximum error of o.o8 absorbance, in a range up to o.3 at 7oo m/~ (where almost no light is absorbed by the chromophore) corresponding to 0.8 at 4o0 m~. They were also identical with spectra of equivalent concentrations of turbidity in distilled water. All recordings were made with the instrument slit width set at the minimum for maximum resolution.
h5 ~ ~ . b
1,5
1,0 !
1.0
il°
0.5
0.5
0.5
0.3
-
0.2
0.1 0 4(?0
500 Wavelength
•, . L _
600 (mju)
T
700
400
I
500 Wavelength
]
600 (m/J)
7QO
Fig. I. A b s o r p t i o n spectra of clear a n d t u r b i d solutions of c h i c k e n o x y h e m o g l o b i n . R e c o r d i n g s m a d e in a Cary Model II s p e c t r o p h o t o m e t e r , I - c m light path. (a) I o m l of a clear s o l u t i o n of o x y h e m o g l o b i n d i l u t e d w i t h IO m l distilled water. (b) IO m l of t h e s a m e o x y h e m o g l o b i n s o l u t i o n d i l u t e d w i t h IO m l distilled w a t e r w h i c h includes 4 drops of m i l k diluted I : I . (c) D i f f e r e n c e s p e c t r u m b e t w e e n a and b. F i g . 2. A b s o r p t i o n spectra of dilute m i l k s o l u t i o n s w i t h absorbances at 7oo m # of o.o5, o . I o , o.15, o.20, 0.25, 0.30, 0.40, a n d 0.50. F o r use in t u r b i d i t y correction, see text.
Fig. 2 shows a series of recordings of the turbidity of dilute milk solutions. A trace of these on translucent paper placed over the absorption spectrum of a turbid myoglobin solution permits a rapid and simple correction, which is only slightly less accurate than if a reference solution of milk turbidity is made up to match exactly that of the sample at 700 m~. KOCHs has shown that for suspensions of biological particles which are large with respect to 2, the absorbance A ~ 1/22. A plot of log absorbance v s . log A from our curves gives parallel straight lines with a negative slope of about 2. Similar plots made from turbidity curves obtained in a Cary Model 15, a Bausch and Lomb Spectronic 505, and a Zeiss PMQ II all give parallel straight lines with very nearly the same slope as those obtained from the Cary Model II. Additional confirmation of the efficacy of the method has been achieved where, in one case, we succeeded in filtering clear a turbid myoglobin solution, minimizing adsorption losses with a very small fine filter paper: the corrected absorbance of the turbid solution agreed exactly with that of the filtrate. I3iochim.
Biophys. Acta,
112 (1966) 5 8 4 - 5 8 6
586
SHORT COMMUNICATIONS
This work was supported in part by grants from the U.S. Public Health Service (GM-o9899 ) and from the Muscular Dystrophy Association.
Institute of Marine Resources, Department of Nutritional Sciences, University of California, Berkeley, Calif. (U.S.A.)
D. E. GOLDBLOOM W. DUANE BROWN
I J. W . STRUT* (LORD I:{.AYLEIGH/,Phil. Mag., 41 (1871) lO 7. 2 I{. T. BALCH, Ind. Eng. Chem. Anal. Ed., 3 (1931) 124. 3 0 . H. LOWRY AND A. t3. HASTINGS, J. Biol. Chem., 143 (1942) 257. 4 G. W E B E R AND F. ~V. J. TEALE, i n H. NEURATH, The Proteins, Vol. 3, N e w Y o r k , 1965, p. 493. 5 t~[. SHIBATA,J. Biochem. Tokyo, 45 (1958) 599. 6 P. PERSON AND A. FINB, Biochim. Biophys. ,4eta, 90 (1964) 168. 7 R. BARER, Science, 121 (1955) 709 . 8 A. L. KOCH, Biochim. Biophys. Acta, 51 (1961) 429 .
Academic
Press,
Received October I8th, 1965 Biochim. Biophys. Acta, i z 2 (1966) 5 8 4 - 5 8 6
BBA
43 072
An electron microscopical study of the sheath covering collagen fibres The occurrence of a sheath-like material surrounding collagen fibres has been well established for a long time, even though its exact nature is a subject of controversy. JORDAN-LLoYD AND MARRIOTT1 attributed the heterogeneous swelling of collagen-fibre bundles to the reticular tissues encircling them. SHARPEY-SCHAFER2 indicated that such restricted swelling was caused by biologically functional entities such as elastic fibres, which tied the collagen bundles together. That this sheath m a y have a chemical composition different from that of collagen is seen from the fact that it is quite stable in formic acid while collagen is dissolved. Also, the sheath has about 1% sulphur incorporated into it 3. Experiments were undertaken recently to characterize the sheath covering kangaroo-tail tendon fibres. Optical and electron microscopical studies are reported in this paper; the biochemical studies will be reported elsewhere. While teasing out kangaroo-tail tendon fibres, it was possible to isolate a membranous structure covering the tendons. Its thickness was in the range lOO-2OO/,. It was white as normal collagen fibres and was sheet-like in appearance. Sections, about 5o/* thick, were cut in a freezing microtome. These revealed a fibrous network, the components running in all directions. A small portion of the sheath was disintegrated in deionized distilled water using a hand homogenizer made of glass. Drops of the cloudy suspension were deposited over formvar films floating on distilled water and thus dialyzed overnight. The samples were shadowcast using a palladium-gold alloy (4o:6o, w/w) at an angle of 14 °. Alternatively, the specimens were stained with a i % solution of uranyl acetate in 5o % ethanol. The samples were examined in a Siemens Elmiskop I. No uniformity could be observed in the nature of the structures obtained from Biochim. Biophys. Aeta, I I 2 (1966) 5 8 6 - 5 8 9
SHORT COMMUNICATIONS
BBA 43 O7T Turbidity correction for absorption spectra of colored solutions Absorption spectra of heme proteins in aqueous extracts of fresh muscle tissue are often inaccurate because of light scattering caused b y turbidity, which is difficult to remove b y centrifugation or filtration. Even where filtration through a fine filter paper yields a clear solution, quantitative estimation of heme protein concentration b y absorbance measurement is subject to serious error owing to adsorption of some of the protein on the filter paper. One method of correcting for turbidity depends upon reading the absorbance due only to turbidity at a series of wavelengths (2) where the chromophore does not absorb light (the "baseline"). It has long been known that the intensity of scattered light produced b y a given amount of turbidity varies inversely with 2, being higher at the blue end than at the red end of the visible spectrum l& Accordingly, from these readings, a correction for turbidity, at a wavelength where the chromophore absorbs strongly, is calculated by extrapolation. This value is then subtracted from the apparent absorbance of the chromophore at that wavelength, in order to obtain the corrected absorbance ~,4. In another method, a piece of opal glass is placed after both sample and reference cells in the light paths of the spectrophotometer 5. The improved spectra resulting from this method can be read directly, but still do not give zero baselines. PERSON AND FINE have modified this procedure for microvolumes by applying the sample and reference solvent to small squares of filter paper 6. BARER achieved considerable reduction of the effect of light scattering by turbidity, by reducing to zero the difference between the refractive index of the suspension and that of the surrounding mediumL However, light scattering was found not to be completely eliminated in the intense Sorer absorption region of whole red blood cells; anomalous dispersion was thought to be likely to occur where there are sharp changes of refractive index. The best method would be one which not only corrects directly for turbidity in the instrument, but also requires the minimum of theoretical assumptions. In the sample cell we have solvent, colored solute plus turbidity: in the reference cell we want only the solvent ph~s a turbidity identical in light-scattering properties with that in the sample. We have obtained excellent results with turbid myoglobin and hemoglobin solutions by using, as a reference, very dilute solutions of ordinary milk, which very closely approaches the ideal requirement. Fig. I shows a comparison of the absorption spectra of two solutions of chicken oxyhemoglobin of equal colorimetric concentration at their Soret maxima, one being clear and the other including a fixed amount of turbidity. The small divergence of Curve c from a smooth curve between 41o and 45o m/z is attributable to dilution errors, which will be obvious in a region where the absorbance changes greatly with wavelength. The value of absorbance on this curve corresponding to the Soret m a x i m u m is exactly equal to Curve b mit~us Curve a. Similar spectra with Soret maxima from 0.3 to 1.8 in clear solution (up to 2.5 with added turbidity) have shown that, at any given wavelength between 400 and 700 m/,, the light scattering due to turbidity is independent of the absorption of the chromophore. The difference spectra were equal to the turbid mi~us the clear Biochim. Biophys. Acta, 112 (1966) 584-586
SHORT COMMUNICATIONS
585
spectra within a maximum error of o.o8 absorbance, in a range up to o.3 at 7oo m/~ (where almost no light is absorbed by the chromophore) corresponding to 0.8 at 4o0 m~. They were also identical with spectra of equivalent concentrations of turbidity in distilled water. All recordings were made with the instrument slit width set at the minimum for maximum resolution.
h5 ~ ~ . b
1,5
1,0 !
1.0
il°
0.5
0.5
0.5
0.3
-
0.2
0.1 0 4(?0
500 Wavelength
•, . L _
600 (mju)
T
700
400
I
500 Wavelength
]
600 (m/J)
7QO
Fig. I. A b s o r p t i o n spectra of clear a n d t u r b i d solutions of c h i c k e n o x y h e m o g l o b i n . R e c o r d i n g s m a d e in a Cary Model II s p e c t r o p h o t o m e t e r , I - c m light path. (a) I o m l of a clear s o l u t i o n of o x y h e m o g l o b i n d i l u t e d w i t h IO m l distilled water. (b) IO m l of t h e s a m e o x y h e m o g l o b i n s o l u t i o n d i l u t e d w i t h IO m l distilled w a t e r w h i c h includes 4 drops of m i l k diluted I : I . (c) D i f f e r e n c e s p e c t r u m b e t w e e n a and b. F i g . 2. A b s o r p t i o n spectra of dilute m i l k s o l u t i o n s w i t h absorbances at 7oo m # of o.o5, o . I o , o.15, o.20, 0.25, 0.30, 0.40, a n d 0.50. F o r use in t u r b i d i t y correction, see text.
Fig. 2 shows a series of recordings of the turbidity of dilute milk solutions. A trace of these on translucent paper placed over the absorption spectrum of a turbid myoglobin solution permits a rapid and simple correction, which is only slightly less accurate than if a reference solution of milk turbidity is made up to match exactly that of the sample at 700 m~. KOCHs has shown that for suspensions of biological particles which are large with respect to 2, the absorbance A ~ 1/22. A plot of log absorbance v s . log A from our curves gives parallel straight lines with a negative slope of about 2. Similar plots made from turbidity curves obtained in a Cary Model 15, a Bausch and Lomb Spectronic 505, and a Zeiss PMQ II all give parallel straight lines with very nearly the same slope as those obtained from the Cary Model II. Additional confirmation of the efficacy of the method has been achieved where, in one case, we succeeded in filtering clear a turbid myoglobin solution, minimizing adsorption losses with a very small fine filter paper: the corrected absorbance of the turbid solution agreed exactly with that of the filtrate. I3iochim.
Biophys. Acta,
112 (1966) 5 8 4 - 5 8 6
586
SHORT COMMUNICATIONS
This work was supported in part by grants from the U.S. Public Health Service (GM-o9899 ) and from the Muscular Dystrophy Association.
Institute of Marine Resources, Department of Nutritional Sciences, University of California, Berkeley, Calif. (U.S.A.)
D. E. GOLDBLOOM W. DUANE BROWN
I J. W . STRUT* (LORD I:{.AYLEIGH/,Phil. Mag., 41 (1871) lO 7. 2 I{. T. BALCH, Ind. Eng. Chem. Anal. Ed., 3 (1931) 124. 3 0 . H. LOWRY AND A. t3. HASTINGS, J. Biol. Chem., 143 (1942) 257. 4 G. W E B E R AND F. ~V. J. TEALE, i n H. NEURATH, The Proteins, Vol. 3, N e w Y o r k , 1965, p. 493. 5 t~[. SHIBATA,J. Biochem. Tokyo, 45 (1958) 599. 6 P. PERSON AND A. FINB, Biochim. Biophys. ,4eta, 90 (1964) 168. 7 R. BARER, Science, 121 (1955) 709 . 8 A. L. KOCH, Biochim. Biophys. Acta, 51 (1961) 429 .
Academic
Press,
Received October I8th, 1965 Biochim. Biophys. Acta, i z 2 (1966) 5 8 4 - 5 8 6
BBA
43 072
An electron microscopical study of the sheath covering collagen fibres The occurrence of a sheath-like material surrounding collagen fibres has been well established for a long time, even though its exact nature is a subject of controversy. JORDAN-LLoYD AND MARRIOTT1 attributed the heterogeneous swelling of collagen-fibre bundles to the reticular tissues encircling them. SHARPEY-SCHAFER2 indicated that such restricted swelling was caused by biologically functional entities such as elastic fibres, which tied the collagen bundles together. That this sheath m a y have a chemical composition different from that of collagen is seen from the fact that it is quite stable in formic acid while collagen is dissolved. Also, the sheath has about 1% sulphur incorporated into it 3. Experiments were undertaken recently to characterize the sheath covering kangaroo-tail tendon fibres. Optical and electron microscopical studies are reported in this paper; the biochemical studies will be reported elsewhere. While teasing out kangaroo-tail tendon fibres, it was possible to isolate a membranous structure covering the tendons. Its thickness was in the range lOO-2OO/,. It was white as normal collagen fibres and was sheet-like in appearance. Sections, about 5o/* thick, were cut in a freezing microtome. These revealed a fibrous network, the components running in all directions. A small portion of the sheath was disintegrated in deionized distilled water using a hand homogenizer made of glass. Drops of the cloudy suspension were deposited over formvar films floating on distilled water and thus dialyzed overnight. The samples were shadowcast using a palladium-gold alloy (4o:6o, w/w) at an angle of 14 °. Alternatively, the specimens were stained with a i % solution of uranyl acetate in 5o % ethanol. The samples were examined in a Siemens Elmiskop I. No uniformity could be observed in the nature of the structures obtained from Biochim. Biophys. Aeta, I I 2 (1966) 5 8 6 - 5 8 9