/ . Biochem., 81, 1769-1779 (1977)
I.
Studies by Circular Dichroism and Viscosity Measurements Taro IZUMI and Hideo INOUE1 Shionogi Research Laboratory, Shionogi & Co., Ltd., Fukushima-ku, Osaka, Osaka 553 Received for publication, November 1, 1976
We have measured the circular dichroism (CD) spectrum of lysozyme [EC 3.2.1.17] at 25 and 30°C and the viscosity at 25°C in water-dioxane mixtures within the range of apparent pH 2.0-2.4. Above 20% dioxane, three positive bands at 294, 289, and 283 nm and a negative band at 262 nm in the near-ultraviolet region gradually decreased in ellipticity until they were replaced by an overall negative broad spectrum above 30% dioxane. On the other hand, negative CD bands in the far-ultraviolet region with a shoulder at 222 nm and an extremum at 208 nm first decreased slightly in ellipticity at 20-30% dioxane, and then gradually increased up to 40 % dioxane, above which the ellipticity of these bands was still further enhanced. Such profiles were common to the two temperatures studied, but at 30°C a higher resolution of the far-ultraviolet CD bands was observed for lysozyme in 60 and 72% dioxane, suggesting the partial disruption of /9-structure. These results indicate that the tertiary structure reflected by the near-ultraviolet CD bands was initially disrupted at 20-30% dioxane with possible slight disorganization of the ordered secondary structure, and then a marked increase in helical structure was induced at high dioxane concentrations. The helical content at 72% dioxane calculated from the ellipticity values at 222 nm was about twice that of lysozyme in the absence of dioxane at both temperatures. The gross structural changes above 20% dioxane were confirmed by the intrinsic viscosities observed at 0, 20, 40, and 60 % dioxane. The ordered structure enhanced by dioxane in the lysozyme molecule was found to be more discernible when the difference CD spectrum was constructed from the observed spectra, taking the CD spectrum of lysozyme in the absence of dioxane as a reference. This procedure was also applied to investigate the effect of pH on the conformation of lysozyme at 60% dioxane. The appearance of ^-structure at apparent pH 5.6 and of aggregated helices at apparent pH 7.5 was apparently induced by 60% dioxane.
Since organic solvents usually affect the native structure of proteins, it is of interest to see how proteins in water-organic solvent mixtures interact 1
Present address: Institute for Plant Virus Research, Ministry of Agriculture and Forestry, Yatabe-cho, Tsukuba-gun, Ibaraki 300-21. Vol. 81, No. 6, 1977
1769
with solvent components when they undergo conformational changes, especially in denaturing solvents that induce more ordered structures. From this point of view, proteins such as /9-lactoglobulin, lysozyme, bovine serum albumin, and insulin (1-3) and a water-soluble nonionic homopolypeptide, poly(N'-(3-hydroxypropyl)-L-gluta-
Downloaded from https://academic.oup.com/jb/article-abstract/81/6/1769/2185726 by Stockholm University Library user on 19 January 2019
Effect of Dioxane on the Conformation of Lysozyme at Low pH
1770
T. IZUMI and H. INOUE
In their earlier work, Hamaguchi and Kurono (5) reported the effects of dioxane on the conformation of lysozyme at an apparent pH of about 5.6 by optical rotatory dispersion (ORD), difference spectroscopy, and viscosity measurements. They found that the lysozyme molecule did not unfold below 50% (v/v) dioxane; above this level the protein underwent conformational changes, suggestive of the appearance of /3-structure. Unlike these observations near neutral pH, the present study gives data showing that lysozyme at pH 2 begins to denature at a dioxane concentration of about 20% (v/v) and assumes a more helical conformation above 40% (v/v) dioxane. Preliminary results of CD measurements concerning the effects of higher pH on the ordered structure of lysozyme produced by dioxane are also presented.
EXPERIMENTAL
Lot 72c-8060) purchased from Sigma Chemical Co. was used as received. Dioxane (spectrograde) was obtained from Merck and was distilled over sodium before use. Water was deionized and redistilled. Reagent grade potassium chloride was a product of Wako Pure Chemicals and was used without further purification. Preparation of Lysozyme Solutions—Solutions for all measurements were usually prepared as described previously (10) by diluting freshly prepared stock solutions of lysozyme in 0.01 M KC1 at pH 2.1 with appropriate aqueous mixtures of dioxane. Dioxane concentrations are expressed as volume per cent throughout this work. To check reversibility, lysozyme solution containing 50% dioxane prepared from the stock solution was diluted to give a solution containing 20 % dioxane, which was used for CD measurements. Lysozyme solutions containing 60% dioxane at apparent pH's of 5.4 and 7.5 were also prepared to examine the effect of pH on the CD spectrum of the protein in 60% dioxane. Lysozyme concentrations were determined spectrophotometrically using E\*m= 26.1 at 282 nm (10). pH Measurements—A Beckman Century SS pH meter equipped with a No. 39030 electrode was used for pH measurements. The apparent pH of lysozyme solutions containing dioxane ranged between 2.0 and 2.4 unless otherwise stated. CD Measurements—CD spectra were recorded on a Jasco ORD/UV/CD-6 spectropolarimeter calibrated with (+)-10-camphorsulfonic acid (11) in a 10-mm cell for the 340-250 nm region and in a 0.1- or 0.2-mm cell for the 250-200 nm region at 25 and 30±l°C. When more precise measurement was necessary, a Jasco J-20 spectropolarimeter was also used. Protein concentrations were 0.6-0.8 mg/ml. The results are represented as residue ellipticity, [6]z, in degree cm1 decimole"1 calculated in the usual way (72), taking a value of 111 (13) as the mean residue weight for lysozyme. Viscometry—Viscosities were measured at 25±0.01°C in a modified Ubbelohd viscometer having flow times of 154.8 and 200.0 s for water and dioxane, respectively, at the same temperature. Relative viscosities rjz and reduced viscosities %p/c for lysozyme in water-dioxane mixtures were obtained at several lysozyme concentrations using the equations (14,15),
Downloaded from https://academic.oup.com/jb/article-abstract/81/6/1769/2185726 by Stockholm University Library user on 19 January 2019
mine) (4) have been studied in water-2-chloroethanol and water-dioxane mixtures. In order to extend such studies to another system, hen egg-white lysozyme [EC 3.2.1.17] in water-dioxane mixtures, it is first necessary to determine the features of conformational changes in the protein in the solvent mixtures. Thus the purpose of this paper is to elucidate the effects of dioxane on the conformation of lysozyme at low pH by circular dichroism (CD) and viscosity measurements. Dioxane was chosen as a denaturant because it can be regarded as a structure-inducing agent, like 2-chloroethanol, for proteins (5, 6) and polypeptides (4, 7, 8) and as a suitable solvent component for NMR studies of solvation of biological macromolecules in aqueous organic cosolvents (4). Lysozyme was selected for investigation because, in addition to extensive accumulated physicochemical and enzymatical findings on lysozyme (9), it is soluble in dioxane-water mixtures at low pH at the highest protein and dioxane concentrations of the three proteins tested, lysozyme, )9-lactoglobulin and bovine serum albumin (Izumi, T., Takayama, Y., & Inoue, H., manuscript in preparation). The high solubility also permitted the adoption of acidic conditions, under which no experiment on the conformational changes in lysozyme brought about by dioxane has previously been carried out.
Materials—Hen egg-white lysozyme (Grade I, J. Biochem.
EFFECT OF DIOXANE ON CONFORMATIONAL CHANGES IN LYSOZYME I
1771
KCI, pH 2.1) at 25°C and 30°C. At low dioxane concentrations (
I.
Studies by Circular Dichroism and Viscosity Measurements Taro IZUMI and Hideo INOUE1 Shionogi Research Laboratory, Shionogi & Co., Ltd., Fukushima-ku, Osaka, Osaka 553 Received for publication, November 1, 1976
We have measured the circular dichroism (CD) spectrum of lysozyme [EC 3.2.1.17] at 25 and 30°C and the viscosity at 25°C in water-dioxane mixtures within the range of apparent pH 2.0-2.4. Above 20% dioxane, three positive bands at 294, 289, and 283 nm and a negative band at 262 nm in the near-ultraviolet region gradually decreased in ellipticity until they were replaced by an overall negative broad spectrum above 30% dioxane. On the other hand, negative CD bands in the far-ultraviolet region with a shoulder at 222 nm and an extremum at 208 nm first decreased slightly in ellipticity at 20-30% dioxane, and then gradually increased up to 40 % dioxane, above which the ellipticity of these bands was still further enhanced. Such profiles were common to the two temperatures studied, but at 30°C a higher resolution of the far-ultraviolet CD bands was observed for lysozyme in 60 and 72% dioxane, suggesting the partial disruption of /9-structure. These results indicate that the tertiary structure reflected by the near-ultraviolet CD bands was initially disrupted at 20-30% dioxane with possible slight disorganization of the ordered secondary structure, and then a marked increase in helical structure was induced at high dioxane concentrations. The helical content at 72% dioxane calculated from the ellipticity values at 222 nm was about twice that of lysozyme in the absence of dioxane at both temperatures. The gross structural changes above 20% dioxane were confirmed by the intrinsic viscosities observed at 0, 20, 40, and 60 % dioxane. The ordered structure enhanced by dioxane in the lysozyme molecule was found to be more discernible when the difference CD spectrum was constructed from the observed spectra, taking the CD spectrum of lysozyme in the absence of dioxane as a reference. This procedure was also applied to investigate the effect of pH on the conformation of lysozyme at 60% dioxane. The appearance of ^-structure at apparent pH 5.6 and of aggregated helices at apparent pH 7.5 was apparently induced by 60% dioxane.
Since organic solvents usually affect the native structure of proteins, it is of interest to see how proteins in water-organic solvent mixtures interact 1
Present address: Institute for Plant Virus Research, Ministry of Agriculture and Forestry, Yatabe-cho, Tsukuba-gun, Ibaraki 300-21. Vol. 81, No. 6, 1977
1769
with solvent components when they undergo conformational changes, especially in denaturing solvents that induce more ordered structures. From this point of view, proteins such as /9-lactoglobulin, lysozyme, bovine serum albumin, and insulin (1-3) and a water-soluble nonionic homopolypeptide, poly(N'-(3-hydroxypropyl)-L-gluta-
Downloaded from https://academic.oup.com/jb/article-abstract/81/6/1769/2185726 by Stockholm University Library user on 19 January 2019
Effect of Dioxane on the Conformation of Lysozyme at Low pH
1770
T. IZUMI and H. INOUE
In their earlier work, Hamaguchi and Kurono (5) reported the effects of dioxane on the conformation of lysozyme at an apparent pH of about 5.6 by optical rotatory dispersion (ORD), difference spectroscopy, and viscosity measurements. They found that the lysozyme molecule did not unfold below 50% (v/v) dioxane; above this level the protein underwent conformational changes, suggestive of the appearance of /3-structure. Unlike these observations near neutral pH, the present study gives data showing that lysozyme at pH 2 begins to denature at a dioxane concentration of about 20% (v/v) and assumes a more helical conformation above 40% (v/v) dioxane. Preliminary results of CD measurements concerning the effects of higher pH on the ordered structure of lysozyme produced by dioxane are also presented.
EXPERIMENTAL
Lot 72c-8060) purchased from Sigma Chemical Co. was used as received. Dioxane (spectrograde) was obtained from Merck and was distilled over sodium before use. Water was deionized and redistilled. Reagent grade potassium chloride was a product of Wako Pure Chemicals and was used without further purification. Preparation of Lysozyme Solutions—Solutions for all measurements were usually prepared as described previously (10) by diluting freshly prepared stock solutions of lysozyme in 0.01 M KC1 at pH 2.1 with appropriate aqueous mixtures of dioxane. Dioxane concentrations are expressed as volume per cent throughout this work. To check reversibility, lysozyme solution containing 50% dioxane prepared from the stock solution was diluted to give a solution containing 20 % dioxane, which was used for CD measurements. Lysozyme solutions containing 60% dioxane at apparent pH's of 5.4 and 7.5 were also prepared to examine the effect of pH on the CD spectrum of the protein in 60% dioxane. Lysozyme concentrations were determined spectrophotometrically using E\*m= 26.1 at 282 nm (10). pH Measurements—A Beckman Century SS pH meter equipped with a No. 39030 electrode was used for pH measurements. The apparent pH of lysozyme solutions containing dioxane ranged between 2.0 and 2.4 unless otherwise stated. CD Measurements—CD spectra were recorded on a Jasco ORD/UV/CD-6 spectropolarimeter calibrated with (+)-10-camphorsulfonic acid (11) in a 10-mm cell for the 340-250 nm region and in a 0.1- or 0.2-mm cell for the 250-200 nm region at 25 and 30±l°C. When more precise measurement was necessary, a Jasco J-20 spectropolarimeter was also used. Protein concentrations were 0.6-0.8 mg/ml. The results are represented as residue ellipticity, [6]z, in degree cm1 decimole"1 calculated in the usual way (72), taking a value of 111 (13) as the mean residue weight for lysozyme. Viscometry—Viscosities were measured at 25±0.01°C in a modified Ubbelohd viscometer having flow times of 154.8 and 200.0 s for water and dioxane, respectively, at the same temperature. Relative viscosities rjz and reduced viscosities %p/c for lysozyme in water-dioxane mixtures were obtained at several lysozyme concentrations using the equations (14,15),
Downloaded from https://academic.oup.com/jb/article-abstract/81/6/1769/2185726 by Stockholm University Library user on 19 January 2019
mine) (4) have been studied in water-2-chloroethanol and water-dioxane mixtures. In order to extend such studies to another system, hen egg-white lysozyme [EC 3.2.1.17] in water-dioxane mixtures, it is first necessary to determine the features of conformational changes in the protein in the solvent mixtures. Thus the purpose of this paper is to elucidate the effects of dioxane on the conformation of lysozyme at low pH by circular dichroism (CD) and viscosity measurements. Dioxane was chosen as a denaturant because it can be regarded as a structure-inducing agent, like 2-chloroethanol, for proteins (5, 6) and polypeptides (4, 7, 8) and as a suitable solvent component for NMR studies of solvation of biological macromolecules in aqueous organic cosolvents (4). Lysozyme was selected for investigation because, in addition to extensive accumulated physicochemical and enzymatical findings on lysozyme (9), it is soluble in dioxane-water mixtures at low pH at the highest protein and dioxane concentrations of the three proteins tested, lysozyme, )9-lactoglobulin and bovine serum albumin (Izumi, T., Takayama, Y., & Inoue, H., manuscript in preparation). The high solubility also permitted the adoption of acidic conditions, under which no experiment on the conformational changes in lysozyme brought about by dioxane has previously been carried out.
Materials—Hen egg-white lysozyme (Grade I, J. Biochem.
EFFECT OF DIOXANE ON CONFORMATIONAL CHANGES IN LYSOZYME I
1771
KCI, pH 2.1) at 25°C and 30°C. At low dioxane concentrations (
