Characterization and conformational analysis by Raman spectroscopy of human airway lysozyme

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  • 488 Biochimica et Biophysica Acta 870 (1986) 488-494 Elsevier

    BBA 32493

    Characterization and conformational analysis by Raman spectroscopy of human airway lysozyme

    J. Marx a, j. Jacquot b, M. Berjot a, E. Puchelle b and A.J.P. Alix a a Groupe de Spectroscopie Vibrationnelle des Systbmes Bio-Molbculaires, GSV-SBM, Laboratoire de Recherches Optiques,

    UER Sciences, BP 347, 51062 Reims Cedex, and b Groupe d'Etude des Systbmes Mucociliaires, GEMUC, Laboratoire d'Histologie, UER Mbdecine, 51095 Reims Cedex (France)

    (Received July 26th, 1985) (Revised manuscript received December 6th, 1985)

    Key words: Lysozyme secondary structure; Raman spectroscopy; Conformational analysis; Human airway; (Hen egg-white)

    Human airway iysozyme, purified from pathological bronchial secretions, is characterized by a specific activity 3-fold higher than that of hen egg-white lysozyme. The amino acid composition of human airway lysozyme is identical to that of other human lysozymes. The laser Raman spectra of human airway lysozyme and hen egg-white lysozyme in phosphate buffer solution (pH 7.2) are recorded in the range 300-1900 cm-t at 488 nm. Drastic intensity differences are observed between the spectra analyzed in the ranges characteris- tic of the peptide backbone (e.g., l-sheet; Ca-C, Ca-N), and of the aromatic side-chain vibrations (tyrosine, tryptophan). The deconvolution of the Raman amide I band gives secondary structures of 38% and 39% a-helix, 25% and 20% fl-sheet, and 37% and 41% undefined structure for the human and hen lysozymes, respectively.

    Introduction

    Although human lysozyme has been isolated, purified and characterized from various sources such as tears, saliva, milk and urine [1-3], human airway lysozyme has received little attention, ex- cept for its immunocytochemical localization and quantitation in pathological conditions [4-5]. Hu- man airway secretions contain a variety of locally produced proteins, including secretory IgA, lacto- ferrin and human airway lysozyme, which are potentially important in the lung microbial de- fense. Human airway lysozyme is an antibacterial enzyme secreted in large quantities (10-20 mg of lysozyme cleared from airways daily) essentially by submucosal tracheal glands, surface epithelium and pulmonary alveolar macrophages [6]. Investi- gations on the conformational and three-dimen- sional structures of lysozyme have been carried

    out mostly with enzyme of non-human origin (es- sentially from hen egg-white) by circular dichro- ism [7], nuclear magnetic resonance [8], X-ray crystallographic analysis [9] and Raman spec- troscopy [10]. Only human lysozymes purified from urine of leukemic patients have been studied in crystallized forms by the X-ray diffraction tech- nique [11-12]. The usefulness of Raman spec- troscopy applied to the investigation of biological molecules lies in its capability to study the material in any phase (solid state, crystal; powder, lyophi- lized; aqueous solution) and under various experi- mental conditions (pH, temperature, concentra- tion). The X-ray technique is only applicable to crystallized substances, although practically all proteins in biological systems are in aqueous solu- tion. In addition, the question always arises of whether the conformational structure of biological proteins is really the same in crystals and in the

    0167-4838/86/$03.50 1986 Elsevier Science Publishers B.V. (Biomedical Division)

  • natural state. In contrast, Raman spectroscopy can be applied to the conformational analysis of proteins in aqueous solution and, moreover, gives information on free and/or bound-molecules (molecular interactions).

    Therefore, this technique is a powerful tool for studying the conformational structures (sec- ondary, tertiary) of a protein, as it provides infor- mation on the peptide backbone structure, the geometries of the disulfide S-S and/or the C-S bonds and the environment of some side chains such as those of tyrosine and tryptophan [13].

    The purpose of the present study is first to characterize the bacteriolytic activity and molecu- lar properties of human airway lysozyme, and second to analyze the Raman spectrum of the lysozyme in solution, within the same experimen- tal conditions. In addition, the human airway lysozyme Raman spectrum is compared to that of hen egg-white lysozyme and the secondary struc- tures of both lysozymes are determined from the deconvolution of the Raman amide I band (Refer- ence Intensity Profiles Method [14]).

    Materials and Methods

    Preparation of lysozymes. Human airway lyso- zyme was prepared from a pool of 2 liters of purulent bronchial secretions collected in patients with chronic bronchitis. The purification proce- dure involved NaC1 extraction, trichloroaceric acid treatment and dialysis followed by cation-ex- change chromatography on CM-Sephadex C-25 resin and gel filtration chromatography on Sep- hadex G-75 Superfine gel (Pharmacia Fine Chem- icals, Uppsala), as previously described [15]. The lysozyme was pure as judged by SDS-polyacryl- amide gel electrophoresis by the method of King and Laemmli [16]. An apparent molecular weight of 15000 was estimated. The lytic activity of the lysozyme was evaluated by measuring the initial rate of lysis of an Micrococcus luteus cell wall suspension (Worthington Biochemicals Corp., Freehold) used as substrate according to a mod- ified procedure of Shugar [17]. A 0.1 ml sample of test enzyme solution was added to 2.9 ml substrate (0.15 mg/ml) in 0.07 M sodium phosphate buffer (pH 7.0). Changes in absorbance (AA) at 450 nm were followed spectrophotometrically for 3 rain at

    489

    25C, and the initial velocity was measured to give AA/min. The specific activity was defined as units of enzyme activity per mg protein determined by the method of Lowry et al. [18], using hen egg- white lysozyme as standard. The amino acid analyses were carried out with a Biotronik LC 6000 analyser after hydrolysis of human airway lysozyme samples for 18, 48 and 96 h in 5.6 M HC1 under vacuum at 110C. Cysteine was de- termined as cysteic acid after performic acid oxidation. Tryptophan was determined spectro- photometrically according to Simpson et al. [19].

    Twice-crystallized hen egg-white lysozyme was purchased from Worthington and was used without further purification.

    Raman spectroscopy. Just before the experi- ment, the enzymes (human and hen) were dis- solved in 0.06 M sodium phosphate buffer, 0.15 M NaC1 (pH 7.20) at a concentration of 10 mg/ml and then centrifuged at 10000 g for 10 min to clarify the lysozyme solution. Raman spectra were recorded on a Coderg PHO spectrometer in the wavelength region 300-1900 cm-1 (HELLMA cell, 10 10 ram). A Coherent Radiation Model 52B Ar + laser at 600 mW power with the 488 nm line was used. In these conditions, no sample degrada- tion was detected by changes in specific lyric activity of the human or hen lysozymes or modifi-, cations in the ultraviolet absorption of either lysozyme, before or after irradiation. The signal pulse obtained by a cool photomultiplier EMI 9558 QB was amplified, counted digitally and then stored in the computer system (ALCYANE, MBC, France), which coordinates the scanning (steps of 2 cm-1 with maximum counting time of 2 s per step) and the spectral data acquisition. Several types of operations may be carried out on the stored spectra [20]. Data accumulation, time aver- aging (16 scans), was used to obtain the spectra and to improve the signal-to-noise ratio. The spec- tra are recorded at room temperature with slit widths of 6 cm-1 or 4 cm-1. The Raman frequen- cies reported here are accurate to + 2 cm-1. The Raman spectral intensities were measured at a particular frequency by their heights above the adjusted baseline. For a broad band, such as the amide I band, the integrated intensity was ob- tained by calculating the band area.

    Amide I band deconoolution. The secondary

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    structure of the enzymes was obtained by applying an original method, the 'Reference Intensity Pro- files Method' [14]. The methods uses the Raman spectral intensities of the backbone amide I vibra- tions of pure classes of secondary structures (a- helix, fl-sheet, undefined conformations) as refer- ence intensity profiles. The reference intensity profiles were determined from the analysis of the Raman amide I bands of a basic set of proteins and polypeptides, the secondary structures of which were estimated from X-ray data. The struc- ture criteria used were identical to those defined by Levitt and Greer [21], except that all 'aperiodi- cal' conformations are put together in a single class called 'undefined' conformation. Using such reference intensity profiles for the quantitative estimation of the secondary structure gave correla- tion coefficients (X-ray versus Raman data) higher than 0.97 for each class of structure. The reference intensity profiles method may be of use to detect characteristic conformational changes when the calculated shifts are larger than 4%. The percent contents are given accurate to ___ 2%. The reference profiles have maximum intensities at 1650, 1673 and 1660 cm -1 for the a-helical, fl-sheet and the undefined conformation, respectively. The 'treated' experimental Raman amide I band of a protein arises from a linear combination of the reference intensities and thus the fitted coeffi- cients give the corresponding amounts of the dif- ferent types of secondary structure. In the case of the study of a protein in solution and in order to take into account the 'solvent' contribution (essen- tially due to the water symmetric bending vibra- tion at 1640 cm-1), the problem of finding a subtraction coefficient working on the overall spectrum is solved together with the secondary structure determination. One introduces the spec- trum of the solvent, in the rang