Eur. J. Biochem. 177. 383-394 (1988) 0 FEBS 1988

Investigation of protein structure by means of 19F-NMR A study of hen egg-white lysozyme

Peter ADRIAENSENS, Marie Elise BOX, Henri .I. MARTENS, Erik ONKELINX, Jos PUT and Jan GELAN

Department SBM, Limburgs Universitair Centrum, Diepenbeek

(Received January 11/April29. 1988) - EJB 88 0028

A '"F-labeled derivative of hen egg-white lysozyme, in which the six &-amino groups are trifluoroacetylated (LF,), was prepared by reaction of lysozyme with S-ethyltrifluorothioacetate. The reaction mixture was fractionat- ed by cation-exchange chromatography at pH 7.3.

A comparison of the circular dichroic spectra and the activity towards Micrococcus lysodeikticus of both LF6 and native lysozyme reveals that the labeling causes no major conformational changes of the polypeptide backbone.

Assignment of the six resonances present in the 19F-NMR spectrum of LF6 was accomplished by using a variety of techniques : specific chemical modifications, the effect of the inhibitor (G~cNAc)~, "F-shift/pH information and relaxation parameters.

Hen egg-white lysozyme, a protein composed of 129 amino acid residues, has been intensively investigated using a variety of experimental techniques [I], including nuclear magnetic resonance spectroscopy. The use of nuclei as probes for the study of structural and conformational properties of proteins in solution requires the allocation of resonances to specific nuclei in the molecule. Due to the large number of resonances, 'H and 13C-NMR spectra of large proteins are extremely complex. For 'H-NMR. the main problems are the small range of chemical shifts and the spectral splittings arising from homonuclear scalar coupling, giving rise to relatively unresolved spectra.

For 3C-NMR, the range of chemical shifts is much larger than for protons and splittings caused by heteronuclear cou- pling to protons can be eliminated by proton decoupling. The main problems are the low natural abundance (1.1 %) and the low sensitivity of the 13C nucleus.

In spite of these difficulties, resonances of more than 120 of the 129 amino acid residues have been assigned in the 'H spectrum of lysozyme by using various techniques [2 - 41 (and references therein; C. M. Dobson and C. Redfield personal communication). In the I3C spectrum, a number of aromatic and aliphatic resonances are assigned [6].

Unfortunately, molecular masses of around 20 kDa are near the practical limit for two-dimensional (2D) proton- proton NMR techniques [7]. For this reason we made a com- plementary 19F-NMR study of fluorine-labelled lysozyme, a

Correspondence to J . Gelan, Department SBM, Limburgs Universitair Centrum, Campus Diepenbeek, B-3610 Diepenbeek, Belgium

Abbreviations. LF., trifluoroacetyl derivative of lysozyme, n refers to the number of trifluoroacetylated amino groups; F3Ac, tri- fluoroacetyl-; CM, carboxymethyl-, (Tyr-NO,),,, ovtho-nitrated tyro- sine residues, n refers to the number of tyrosine residues nitrated; COSY, correlation spectroscopy; NOESY, nuclcar Overhauser en- hancement spectroscopy; ( G I c N A ~ ) ~ , tris(N-acetylglucosamine).

Enzymes. Carboxypeptidase A (EC 3.4.17.1); hen egg-white lysozyme (EC 3.2.1.17).

well known protein which can serve as a good model com- pound for the development of NMR methodologies for in- vestigating larger proteins. For the introduction of the fluor- ine probes, we have chosen trifluoroacetylation of the lysine c-amino groups because (a) the F3Ac probe is small and contains three equivalent fluorine atoms, giving rise to rela- tively intense signals and (b) except for the 'H- and 13C-NMR studies of methylated amino groups by Bradbury and Brown [8] and Gerken et al. [9], respectively, little is known about the side-chain environment of the amino groups for lysozyme. This technique makes it possible to study biochemical pro- cesses (e. g. denaturation, inhibitor binding, etc) very quickly and at concentrations of less than 0.1 mM, the latter being very interesting because at these low concentrations it is probably always possible to study the monomeric species. Although we certainly do not want to compete with 'H-NMR, we believe that the technique is also useful for studying lower- molecular-mass proteins where the labels can be used for (a) the assignment of lysine spin systems (which are sometimes difficult to assign, even with 2D-NMR techniques) by using, for instance, heteronuclear relayed COSY experiments; (b) the assignment of other proton resonances by using heteronuclear NOE(SY) experiments and (c) the study of the behaviour of the lysine environments in biochemical processes at very low concentrations because, although I9F-NMR spectra contain less information than 'H-NMR spectra, these I9F-NMR spectra contain well resolved resonances which are acquired very rapidly.

In general, we believe that "F-labeling is of interest for obtaining relevant information about biochemical processes, rather than for carrying out complete structure determination.

In the first part of this paper, we present the preparation and purification of a F3Ac derivative of lysozyme in which the six lysine &-amino groups are labeled (LF,). Since the introduction of labels in a protein can have an effect on the structure and properties, it is important to compare these derivatives with the native protein.

The second part deals with the assignment of the six reso- nances present in the I9F-NMR spectrum of LF,.

384

l b io 30 40 45 f ract ion number

10 20 30 40 f ract ion number

Fig. 1. Elution profile (CM-Sepharose CL-6B column) of the soluble ,fruction of the reaction mixture after trifluoracetylution. (a) CF3COSEt elution buffer: 0-0.1 M NaCI in 0.05 M TrisiHCl buffer, pH 7.3; (b) CF3COOPh-pN02, elution buffer: 0-0.5 M NaCl in 0.05 M Tris/ HC1 buffer, pH 8.0

MATERIALS AND METHODS

Materials

Hen egg-white lysozyme was obtained from Serva and was used without further purification. p-Nitrophenyltrifluoro- acetate was a product from Janssen Chimica (Beerse, Belgium). Acrylamide and N,N'-methylene-bisacrylamide were products of Bio-Rad. S-Ethyltrifluorothioacetate, tetra- nitromethane and iodoacetic acid were obtained from Aldrich Chemical Co. CM-Sepharose CL-6B and Sephadex G-100 were obtained from Pharmacia. Micrococcus lysodeikticus and the inhibitor tris(N-acetylglucosainine), (GICNAC)~, were products of Sigma. Carboxypeptidase A was obtained from Worthington. These reagents were used without further purifi- cation.

Analytical methods

The concentration of lysozyme derivatives was determined spectrophotometrically using a molar absorption coefficient of 39000 M-' cm-' at 280 nm [lo]. A Radiometer Copen- hagen PHM82 was used for the pH measurements. Glycine/ HCl buffer, formate buffer and acetate buffer, all of ionic strength 0.1 M, were used for the pH ranges 1.8 - 2.9, pH 3 - 4 and pH 4.1 - 5.7 respectively, unless otherwise stated.

Cation-exchange chromatography was performed on a CM-Sepharose CL-6B column (1.6 x 20 cm), eluted with a linear NaCl gradient in 0.05 M Tris/HCl buffer pH 7.3 or pH 8 (first fractionations).

Pre-column dialysis was performed in two steps: first against water and then against the fractionation buffer (0.05 M Tris/HCl pH 7.3 or pH 8).

For polyacrylamide gel electrophoresis (PAGE), all samples were dissolved in water after freeze-drying and loaded on 10% polyacrylamide gels of 2 or 3 mm thickness. Staining was accomplished with Coomassie brilliant blue R-250 [I I]. Separations were carried out at pH 7.3 (Tris/HCl buffer) or pH 8.9 (Tris/glycine buffer).

Titration with the inhibitor, (G~cNAc)~, was performed by addition of 1.66 mM ( G ~ C N A C ) ~ solution to 0.5 ml 0.1645 mM LF, solution.

Circular dichroic spectra were recorded on a Cary 61 CD spectropolarimeter at 24 "C. Samples with a concentration of 0.02 - 0.036 mM were dissolved in 0.05 M phosphate buffer, pH 5.7. For calculations, the average residue mass, 112.4 Da, of lysozyme was used.

Nuclear magnetic resonance. The solutions of lysozyme derivatives were prepared in the appropriate buffers, ionic strength 0.1 M. "F-NMR spectra were recorded on a Varian XL-200 spectrometer a t 188.18 MHz, using sample D 2 0 (10%) as internal lock. A Motorola 68000 computer was used to accumulate the free induction decays and to perform the Fourier transformations. The ppm scale was set to trifluoroacetone as internal reference. The "F-spectra were recorded at 30.0 0.1 "C. Spin-lattice relaxation times ( T I ) and spin-spin relaxation times (T,) were measured using the inversion recovery and the Carr-Purcell-Meiboom-Gill se- quence, respectively.

A non-linear least-square fit of the experimental "F-shift/ pH dependence was carried out using an iterative computer program. From the observed shifts (dabs) at different pH values, the program determines Ka, the ionization constant of the neighbouring ionizing group, dAH and dA, the chemical shifts corresponding to the protonated and non-protonated states of the neighbouring titrating group, according to:

Preparative methods

Preparation of LF6 and LF , using CF3COSEt. Trifluoro- acetylation of the six lysine amino acid residues was performed according to Fanger and Harbury [ 121. S-Ethyltrifluorothio- acetate (226 mol reagent/mol lysozyme) was added to a 0.69 mM lysozyme solution in water at 20°C. Higher tempera- tures result in a lower yield. The pH was maintained at 9.5- 10 by addition of 1 M NaOH. After 2 h base consumption ceased. The reaction mixture was brought to pH 7 with 0.1 M HC1, dialyzed and centrifuged. The pellet was dialyzed against water and freeze-dried. The supernatant was fractionated on a CM-Sepharose/column pH 7.3 or pH 8 (200 ml; 0-0.1 M NaC1) (Fig. 1 a). The fractions (a) (LF,) and (b) (LF,) were collected and dialyzed. Each fraction was rechromatographed on the same column and eluted with the same gradient. The fractions LF6 and LF, were collected, dialyzed against water and freeze-dried. The reaction products (pellet and super- natant) were characterized by gel filtration on Sephadex G- 100 in 0.05 M Tris/HCl buffer pH 8 containing 8 M urea (see Results). The purity of the derivatives LF, and LF5 was checked by PAGE (Fig. 2A).

Preparation of L F , and LF6 using CF3COOPh-pN02. Tri- fluoroacetylation of the six eamino groups and the a-amino group was performed with p-nitrophenyltrifluoro- acetate. To a 0.345 mM solution of lysozyme in water, 456 mol reagent/mol lysozyme was added at 20°C. The pH was maintained at 9.5- 10 with 1 M NaOH. After 2 h, the base consumption ceased and the pH was brought to 7 with 0.1 M HC1. After the reaction mixture was dialyzed and centrifuged, the supernatant was fractionated on a CM-Sepharose column, pH 8 (200 ml; 0-0.5 M NaCI) (Fig. 1 b). The fractions (a) (LF,) and (b) (LF,) were collected, dialyzed and re- chromatographed using a gradient of NaCl (200 ml; 0- 0.1 M). The fractions LF7 and LF6 were collected, dialyzed against water and freeze-dried. The purity of the derivatives was checked by PAGE (Fig. 2B).

385

Fig. 2. PAGE of Iy.sozvmr trifluoroacc~tyl derivutives. The cathode is at the top and the origin is marked with an arrow. (A) LF6 (1); LF5 (2) and the soluble fraction of the reaction mixture obtained with CF3COSEt (3) in Tris/HCI buffer, pH 7.3. (B) LF, ( 1 ) ; LF, (2); CM-LF, (3); CM-LFs (4); [(Tyr-N02)2]LF6 (5); [ ( T Y ~ - N O ~ ) ~ ] L F ~ (6); [(Tyr-NO,),]LF, (7) and [(Tyr-N02)1]LFS (8) in Tris/HCI buffer, pH 7.3. (C) LFo ( 1 ) ; nativc lysozyme (2); CM-L (3); [(Tyr-NO,),]L (4) and [(TY~-NO,)~]L (5) in Tris/HCI buffer, pH 7.3. (D) Asp-lOl- modified LF6 (1) and niixturc of LF6 and LFS (2) in Tris-glycine buffer, pH 8.9. Exccpt for LF,, Asp-101-modified LF6 and Tyr-nitrat- ed derivatives, the fractionation of thcse dcrivatives on a CM- Sepharose column was accomplished at pH 7.3

Preparation of des-Leu-LF,. LF6 (see earlier for prep- aration) was treated with carboxypeptidase A for 52 h [13]. The removal of the C-terminal amino acid residue (Leu-129) was followed by thin-layer chromatography on Silica gel G in butanol/acetic acid/water (12: 3 : 5 ) and detected with ninhydrin. After the reaction, the mixture was fractionated from carboxypeptidase A by chromatography on Sephadex G-100 in 1% acetic acid. des-Leu-LF6 was collected, dialyzed against water and freeze-dried.

Selective modification of Asp-101 in LF,. Asp-101 in LF, was selectively modified under slightly modified conditions of Yamada et al. [14]. LF, (0.35 mM) and ethylenediamine (0.5 M) were dissolved in water and the pH of the solution adjusted to 5.0 with HC1. l-Ethyl-3-[3-(dimethylamino)- propyllcarbodiimide HCl(3.5 mM) was added to the solution, whilst stirring at room temperature, to initiate the reaction, followed by the addition of water to 1.8 ml. The pH was maintained at 5.0 with HCI during the reaction. After 2 h (pH change ceased), the solution was dialyzed, centrifuged and fractionated on a CM-Sepharose column pH 8 (200 ml; 0- 0.2 M NaCl). The purity of this derivative was checked by PAGE (Fig. 2D).

Carhoxymetliylation 9fHi.s-1.5 in LF, and LF,. His-15 in lysozyme was carboxymethylated under slightly modified con- ditions of Kravchenko et al. [15]. To an aqueous solution of 0.0276 mmol lysozyme, pH 4.6, an aqueous solution of 4.30 mmol iodoacetic acid (pH 4.6 with 0.1 M NaOH) was added. The pH of the reaction mixture was brought to 5.5

with 0.1 M NaOH and the volume was brought to 100ml. The mixture was kept at 40°C for 6 h in the dark, followed by dialysis against water (pH 3 ) in order to stop the reaction. After centrifugation, the mixture was fractionated on a CM- Sepharose column pH 7.3 (200 ml; 0.1 -0.3 M NaC1, followed by 0.3 M NaC1) and the fraction which eluted before lysozyme was collected, dialyzed and rechromatographed on the same column. After dialysis against water, carboxymethylated lysozyme (CM-L) was freeze-dried. CM-L was then trifluoro- acetylated with CF,COSEt as described earlier. After frac- tionation (200 ml; 0-0.15 M NaCl), the first (CM-LF6) and the second (CM-LF,) fractions were dialyzed against water and freeze-dried. The purity of CM-L, CL-LF5 and CM-LF6 was checked on PAGE (Fig. 2 B, C).

Modification of' Tyr-23 (and Tyr-20) in LF, and LF,. Ni- tration of lysozyme was accomplished under the conditions of Atassi and Habeeb [16] with tetranitromethane'. To a solution of lysozyme (0.022 mmol) in 22 mlO.05 M Tris/HCl, 1 M NaCl buffer pH 8, tetranitromethane (0.22 mmol) was added as a10% solution in 95% ethanol. The reaction mixture was stirred for 2 h at room temperature. After dialysis against water and 0.05 M Tris/HCl buffer pH 8, the mixture was centrifuged and fractionated on a CM-Sepharose column (400 ml, 0.1 -0.25 M NaCl in the same buffer). Fractionation of Tyr-nitrated derivatives was performed at pH 8 (the pK, value ofTyr changes from 10.1 to 7.2 upon nitration [19]). The first [(Tyr-NO,),]L and the second [(Tyr-NOz),]L fractions eluting before lysozyme were collected, dialyzed (as above) and rechromatographed on the same column. The molar amount of o-nitrotyrosine relative to protein was determined spectrophotometrically by measuring the total protein con- centration and the o-nitrotyrosine concentration at 280 nm and 381 nm, respectively (isosbestic point; E = 2200 M-' cm-' [20]). After dialysis, the two derivatives were dialyzed against water and freeze-dried. [(Tyr-NO,),]L was trifluoro- acetylated with CF,COSEt and after fractionation (200 ml; 0 - 0.025 M NaCl), [(Tyr-NO,),]LF, (first fraction) and [(Tyr- N02),]LF5 (second fraction) were collected, dialyzed against water and freeze-dried. [(Tyr-NO,),]L was also trifluoro- acetylated with CF,COSEt and after fractionation (200 ml; 0 -0.1 M NaCl), [(Tyr-N02)1]LFr, and [(Tyr-NO,),]LF, were collected, dialyzed against water and freeze-dried. The purity of all these derivatives was checked on PAGE (Fig. 2B, C).

Preparation of c-N-trifluoroacetyl-lysine. This was prep- ared as described by Schallenberg and Calvin [21]. TLC on silica gel (3-25 with BuOH/HAc/H,O (12:3:5) as eluent, yields RF values of 0.15 and 0.55 for lysine and F,Ac-Lys, respectively. In agreement with Schallenberg and Calvin, a melting point of 228 -230°C was found. The 19F-NMR spec- trum (Fig. 8) shows a single resonance at pH values 5 or higher, as reported by Huestis and Raftery [22], but at lower pH values a side resonance at higher field is observed.

RESULTS AND DISCUSSION

Structure and activity of LF, compared to native lysozyme

The effect of labeling the lysine c-amino groups on the conformation and activity of native lysozyme is investigated

Under these conditions, only Tyr-23 and Tyr-20 are nitrated bccausc both are exposed and located in nonhehcal regions [17]. The third tyrosyl, Tyr-53 (with a pK, = 12.6 [7]) is inaccessible to reaction with tetranitromethane [16] or to iodination'[lX], probably due to its location in the short antiparallcl pleatcd-shect structure comprising residues 41 - 54 [17] and its cngagcmcnt in hydrogen-bonding.

386

i +2500

by a comparison of the circular dichroic spectra and activity measurements towards Mic.rococcus Iysodeikticus.

CD sprctrci of' L F , cind ncitivr 1.y.sozymc. Fig. 3 shows the C D spectra of LF6 and native lysozyme. In agreement with Ikeda et al. [23], the C D spectrum of native lysozyme has positive maxima at 295 nm, 290 nm and 284 nm and a broad negative band at about 260 nm, in addition to the double minima at 222 nin and 208 nm which reflect the conformation of the polypeptide backbone. Comparison of the spectra of LF, and native lysozyme shows that trifluoroacetylation of the six r:-amino groups has only a slight effect on the ellipticity in the 200-250-nm region ([O],,, is - 7280 and - 7640 deg . cm2 dmol-' for native lysozyme and LF6, respectively) and a decrease in the intensity of the bands in the aromatic region. These effects were also noticed by Nakea et al. [24] in their study of acetylated lysozyme. In an analogous study of neurotoxin I1 of Nqjo nc/jci osicinri, Tsctlin et al. [25] pointed out that the trifluoroacetylation of the amino groups has only a minor effect on the resonances in the 'H-NMR spectrum, although there is a considerable effect on the bands in the C D spectrum. 'However, they concluded that the trifluoroacetyla- tion of the six amino groups of neurotoxin I1 has, in general, no conformational effect and that the conclusions from the 19F-NMR study on the spatial structure of the F,Ac deriva- tive are applicable to the native neurotoxin.

Activit?; meLiSuretiietit.s of' LF, cind native lysozyme. The activity of lysozyme toward M . Iysodeikticus cells is affected by trifluoroacetylation of the r:-amino groups [26]. A reduction in the number of positive charges on lysozyme displaces the activity maximum to lower pH values at constant ionic strength. This seeins logical since the first step in the enzymatic reaction is the binding of lysozyme to the negatively charged cell wall. Because trifluoroacetylation results in a loss of posi- tive charge, the electrostatic interaction will be weaker, re- sulting in a lower activity for LF6 when compared to native lysozyme.

Davies and Neuberger [27] studied the activity of a lysozyme derivative with all Lys residues and 1.4 Tyr residues acetylated. They found, in agreement with our results, a re- duction of activity and a lower pH optimum for this derivative compared to native lysozyme. If the activity of lysozyme and

Fig. 4. Clir.or?icrtogrNpli~~ on S~~p1iude.r G- 100 in 0.05 M Tri.71 HCI h u f i r p H 8.0, 8 M u r w . Native lyso7yme (-), the supernatant of the reaction mixture obtained with CF,COSEt (......) and the precipitate of this reaction (LF,,),,, (- -. - -)

acetyl-lysozyme derivatives is measured towards ( G I C N A ~ ) ~ [27] or towards glycol chitin [lo], no influence of acetylation on the activity is observed.

Prewrvation of' the ncitivr disulfide bonds in LF6. During the trifluoroacetylation of lysozyrne with both CF,COSEt and CF3COOPh-pN02, 25- 30% of the protein is irreversibly precipitated. In the reaction with CF,COSEt, a large amount of ethane thiol/thiolate (pK, = 10.5) is formed as a leaving group in the reaction, due to the hydrolysis of the reagent in an aqueous environment at high pH. This could lead to a considerable disruption and rearrangement of the disulfide bonds in the protein. Therefore, some authors [25, 281 have performed the trifluoroacetylation in N,N-dimethylforma- mide to avoid the reaction on the disulfide bonds. However, lysozyme is denatured in N,N-dimethylformamide [29,30] and reaction under these conditions might prevent the enzyme from regaining its native conformation. Because there is also a precipitate formed with CF3COOPh-pN02 and the I9F- N M R spectra of LF, obtained with the two reagents are the same (Fig. 6a , b), we believe that the precipitation is caused by aggregation of the protein at this high pH. To strengthen this conclusion, the composition of the reaction mixture was investigated by gel filtration on Sephadex G-100 in 0.05 M Tris/HCl buffer pH 8.0, 8 M urea (Fig. 4). This figure shows that the soluble products of the reaction mixture are eluted at the same position as native lysozyme whereas the precipitate (LF,,),?, contains compounds with a higher molecular mass. This means that LF,, which is the major product used in the further study, is a F3Ac derivative of the lysozyme monomer and that precipitation is caused by aggregation of the protein. Therefore, we performed the labeling in an aqueous environ- ment.

Summarizing, since (a) the 200-250-nm region of the C D spectra of LF6 and native lysozyme show no significant difference, (b) there is activity left towards M . lysodeikticus (50-60% at low pH and ionic strength), (c) the Lys residues are situated on the outside of the molecule and (d) no disrup- tion of disulfide bonds occurs when CF,COSEt is used, we conclude that trifluoroacetylation of lysozyme causes only local conformational changes of some side chains but no conformational change of the polypeptide backbone.

387

Fig. 5. PAGE of' trifliioroocrtyl derivritive.s in Trislglycine huffir p H 8 . Y . (A) LFS (1) and LF6 (2) both fractionated a t pH 8.0. (B) Mixture of LFS and LF4 dissolved in buffer, pH 3.1 (1); pH 6 (2); pH 7 (3); pH 8 (4); pH 8.9 ( 5 ) and mixture of LF6 and LFS in buffer PH 6 (6 )

19F-NMR spctrri c)fI;,Ac,-derivative.s

Hydrolysis of' thr F3Ac group. At first, fractionations (on CM-Sepharose) and PAGE of F,Ac-lysozyme derivatives were performed at pH 8 and pH 8.9, respectively, instead of pH 7.3. When the purity of derivatives obtained in this way was checked on PAGE, traces of derivatives substituted in one position less were always found (Fig. 5A). Since the chromatographic separations seemed alright, hydrolysis dur- ing the last chromatography step, during the time between chromatography and dialysis (separations overnight), during dialysis or during PAGE could be responsible for these im- purities. Therefore, several samples of a freeze-dried mixture of LFs and LF4 at different pH values were prepared. After 24 h, these samples were electrophoresed (Fig. 5B). From Fig. 5B, it can be concluded that hydrolysis occurs at pH values of 8 or higher. As a consequence of hydrolysis, the amount of trifluoroacetate was dependent on the length of the last dialysis before freeze drying. For this reason, the I9F-NMR spectra of derivatives chromatographed at pH 8 contain varying (and sometimes large) amounts of tri- fluoroacetate (labeled *). All later fractionations (except for the fractionations of Tyr-nitrated derivatives) and PAGE were performed at pH 7.3.

Fig. 6 shows some "F-NMR spectra of LF6 at different pH values. These spectra contain six signals of almost equal intensity, corresponding to the six F3Ac groups bound to the E-amino groups of lysozyme. The fluorine resonances are sensitive to changes in pH (see later) but they are separately resolved, reflecting the difference in environment of the F3Ac groups in the enzyme.

Fig. 7 shows some '"F-NMR spectra of LFS. They also show six signals but of varying intensities, reflecting the differ- ent amounts of the six possible kinds of LFS. Depending on which F3Ac group is absent, the resonance position of some of the six signals can be slightly shifted (Fig. 7a-d). Compari- son of Fig. 6a, c and Fig. 7a, b shows that hydrolysis of the F3Ac group responsible for signal 5 (decreased intensity rela- tive to the other resonances) has no influence on the resonance position of the other signals. A comparison of Fig. 6a, c and Fig. 7c, d shows the partial hydrolysis of the F3Ac groups corresponding to resonances 6 and 2. Hydrolysis of the re- sonance-6 F3Ac group has an affect on the resonance position of the signals 1 and 2 at pH 3.1 and on the position of signal 1 at pH 5.5 (this effect can be explained only after assignment of the resonances).

One can see that, for LF, at pH 3.1 (Fig. 6c, e), the side- resonance at lower field of signals 1 and 2 has disappeared

and that the resolution between the signals 1 and 2 at pH 5.5 (Fig. 6 b, d) is increased. This is due to the fact that there are less LFS impurities when the fractionations were done at pH 7.3 instead of pH 8. The LFS spectra in Fig. 7c, d confirm that, in the '"F-NMR spectra of LF,, the shoulders at lower field of the signals 1 and 2 (at pH 3.1) which move between signal 1 and 2 and behind signal 2 at pH 5.5, respectively, result from specific kinds of LFS impurities.

Side-resonances at higher field. At pH 3.1 (and less than at pH 5.5) the main resonances still show a small shoulder, all at 5 Hz to higher field. To investigate the origin of these side-resonances, a study of c-N-trifluoroacetyl-lysine as a function of temperature and pH was carried out. Since the model compound is a rather small molecule, one should expect an increase of both T1 and T2 (extreme narrowing range) relaxation times when temperature is raised. The spectra on the other hand (not shown) show an increase of T1 (decrease of area, no waiting time) but a pronounced decrease of T2 (increase of band width). This is probably the result of a coalescence phenomenon (with a coalescence temperature above 1OO')C) meaning that there are two different environ- ments for the F3Ac group which are, at 30°C, in slow exchange relative to the NMR time-scale. We interpret the origin of this side-resonance as an enolic form of the - NHCOCF3 function. Fig. 8 shows the effect of pH on the side-resonance at 30°C. This figure clearly demonstrates the side-resonance at pH 3.1, which disappears behind the main resonance (slow exchange) when the pH is raised probably as a consequence of a shift of the equilibrium to the main form.

Assignment qf the six resonances in the LF6 spectrum

Before one can make any qualitative or quantitative mea- surements with LF6 (e.g. use of inhibitors, denaturation, etc) to obtain information about which sites of the molecule are influenced, it is necessary to assign the six fluorine resonance signals in the LF6 spectrum to the respective lysine F3Ac groups using the following items.

Specijically modijl'ed L F , derivatives. LF,, lysozyme with the six E - and the a-amino group trifluoroacetylated with p-nitrophenyltrifluoroacetate; des-Leu-LF6, LF6 in which the C-terminal Leu-I 29 is removed by carboxypeptides A; Asp- 101-modified LF6, LF6 in which Asp-I01 is modified with ethylenediamine in the presence of carbodiimide; His-1 5- modified LF,, LF, in which His-I 5 is carboxymethylated with iodoacetic acid; Tyr-23 (and Tyr-20) modified LF,: LF, with one or two Tyr residues nitrated with tetranitromethane.

pH measurements. A comparison of the '"F-NMR spectra of LF6 at pH 3.1 and pH 5.5 (Fig. 6d, e) shows that the fluo- rine resonances are sensitive to changes in pH. The pH de- pendence of the '"F chemical shifts is shown in Fig. 9a. Sig- nal 2 experiences the largest influence in the pH range 2- 5.6. Table 1 gives the chemical shifts S A H , S,,, A 6 = B A H - S A and the pK,, values of the ionic groups involved, determined from the titration curves according to Eqn (1). Since lysozyme undergoes no major conformational change in the pH range investigated [I], the observed pH dependence of the chemical shifts may be rationalized in terms of titrating ionic groups in the proximity of the F,Ac groups.

E&t qf'inhihitor-binding. Fig. 10 shows the effect of the inhibitor (GIcNAc)~ on the 19F-NMR spectrum of LFs. The major effects are the upfield shift of signal 6 and a decrease of the intensity of signal I , while an increase of intensity at 11.235 ppm (position of signal 2) is observed. This means that the binding of (GlcNAc), a t the binding sites A-B-C of the

388

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Fig. 6. "F-NMR spectra of liF6 (obtained with CF,COSEt or CF3COOPh-pN02) in buiyer, I = 0.1 M , at 30 'C. (a) pH 5.5, CF3COSEt, fractionated at pH 8.0; (b) pH 5.5, CF3COOPh-pN02, fractionated at pH 8.0; (c) pH 3.1, CF3COOPh-pN02, fractionated at pH 8.0; (d) pH 5.5, CF,COSEt, fractionated at pH 7.3; (e) pH 3.1, CF3COSEt, fractionated at pH 7.3. Concentrations used were in the range 0.15- 0.25 mM. *, CF3COO- from hydrolysis

' I " ' ~ ' " ' 1 ' ' " " ' " I ' ' ~ ' l ' ' ' ' 1 ~ ' ' ' I ' " 1 " ' 1 ' " ' I ' ' , ' ( I 1 " " ,

11 i 11 3 11 2 11 1 11 0 10 9 m a u p m

Fig. 7. I9F-NMR spectra q j LF5 (fractionated at p H 7.3) in huger, I = 0.1 M , at 30°C. The LF5 derivative used for the spectra (a) (pH 5.5) and (b) (pH 3.1) was obtained from another synthesis than the one used for the spectra (c) (pH 5.5) and (d) (pH 3.1). Concentrations used were in the range 0.15-0.25 m M

pH 3 10 JL " " ' I " 1 , ' ' l r ' ' ' I e ' I ' s ' t ' ' L ' I ' ' ' 1 ' I r I r c . "

10 9 10 a '3 ? 10 5 : 3 5 i p m

Fig. 8. "F-NMR spectra of c-N-trifuoroacetyl-I~sine as a,function of p H at 30°C. The concentration used was 0.7 mM

active site 11, 311 produces a different effect on signals 1 and 6. The F3Ac group corresponding to signal 1 gives two resonances: one for LF6 not bound with (GlcNAc)3 (at 11.178 ppm) and another for LF6 with (GlcNAc)3 bound (at 11.235 ppm). This resonance shows a slow exchange rate with respect to the exchange rate at the coalescence-temperature. For signal 6 the observed chemical shift is a weighted average of the chemical shifts of both environments (fast exchange) based on the respective mole fractions. A more detailed study of the binding of (GlcNAc)3 as a function of temperature is in progress.

Relaxation parameters. Table 2 gives the T I and T2 relax- ation times for the fluorine signals. The relaxation times for the six signals are different, indicating the different mobility of the F3Ac groups. Since, for all the signals, the TI value is larger than the T2 value, we can conclude that the correlation

389

11.

- E a /

0

11.1

10.

a LF6

2 3 i 5 OH

b DES-LEU-LF6

2 3 4 5 PH

Fig. 9 .pH dependence of’thc, l y F c h r ~ i c u l shifts for LF6 ( a ) unddes- leu-LF~, ( b ) in buffer, I = 0.1 M , at 30°C. Soiid lines show the pH curves calculated according to the parameters in Table 1 ; dotted lines connect the experimental points. The concentrations used were in the range 0.1 -0.2 mM

Table 1. Purumeters derivedjrom the p H dependence o f the ”F chemical shifts f o r LF6 and de.s-Leu-12Y-LF6, according to Eyn ( I ) A negative sign for A 6 indicates a shift to lower field with increasing pH

Protein Signal PKA 6 A H 6 A A6

PPm

LF.5 1 4.30 k 0.7 11.137 f 0.004 11.178 f0.007 - 0.041 2 3.61 & 0.04 10.969 f 0.005 1 1.233 k 0.004 ~ 0.264 3 3.97 & 0.3 10.937 f 0.006 10.982 0.006 - 0.045

des-Leu-129-LF6 1 4.41 f 0 . 3 11.141 If: 0.003 11 . I 75 0.003 - 0.034

3 3.95 f 0.3 10.929 k 0.009 10.987 0.003 - 0.058

.-

2 3.69 f 0.12 10.974 f 0.013 11.231 t 0 . 0 1 0 - 0.251

Fig. 10. I9F-NMR .spectru of LF6 in the presence qf (GlcNAc)3 in buffer pH 5.5, I = 0.1 M , ut 30°C. The molar ratio of (GIcNAc)~ to LF6 was: 0 (a); 0.1 (b); 0.4 (c) and 1 (d). (NAG)3 = (GIcNAc)~. The concentration of LF6 was 0.1645 mM

3 90

Table 2. Spin-lattice relu.uution time Ti and spin-spin reluxafion time T2 .for LI.b measured in huifer at p H 5.5 and p H 3.1 ionic screngch 0.1 M , at 30°C Values are prescntcd with their standard dcviation. The conccn- trations used were in the range 0.226-0.247 mM

Signal Relaxation times at

pH 3.1 pH 5.5

TI T2 7-1 T2

S

1 0.83 fO.01 0.31 k0.01 0.83 kO.01 0.31 kO.01 2 0.93 f 0.02 0.24 0.03 0.77 f 0.01 0.25 k 0.01 3 0.83 k 0.01 0.23 f 0.01 0.87 f 0.02 0.20 0.02 4 0.82 f 0.01 0.25 f 0.02 0.85 k 0.02 0.22 f 0.01 5 0.86 f 0.01 0.24 k 0.02 0.86 f 0.02 0.22 f 0.01 6 0.97k0.01 0.14f0.01 0.92k0.02 0.10+0.01

4 5

1 1

- 10.8 6,lppm)

Fig. 11. I9F-NMR spectrum c?f Lk76 ( a ) anddes-Leu-LF6 ( b ) In buffer, pH 5.5, I = 0.1 M , at 30 c‘. Thc conccntraiions used were in the range 0.1 -0.2 mM

time for molecular motion of LF6 is situated outside (but close to) the extreme narrowing range at this field strength (188 MHz) [32].

Assignment of the six resonances

Signal 2. Removal of the C-terminal amino acid Leu-129 of LF6 by carboxypeptidase A (des-Leu-LF6), besides having a minor effect on signal 6, gives rise to a new resonance, indicated as signal 2’, and a decrease of the intensity of sig- nal 2 (Fig. 11). The presence of signal 2 in the 19F-NMR spec- trum of des-Leu-LF, indicates that this derivative still con- tains some LF6 molecules in which the C-terminal Leu-129 has not been removed. The pH dependence of signal 2’ is clearly different from that of signal 2, but no drastic change is observed for the remaining fluorine resonances (Fig. 9a, b). Since the removal of Leu-129 has a major effect only on signal 2 and since, in native lysozyme, there is a salt bridge between

the a-carboxyl group of Leu-I29 and the c-amino group of Lys-13 [I, 331 (the other c-amino groups are separated by at least 1.6 nm from the a-carboxyl group, Lys-96), we can assign signal 2 to the Lys-I 3 F3Ac group. The analysis of the chemi- cal shift of signal 2 in the pH-range 2-5.6 yields a pKa of 3.6 for the neighbouring ionizing group (Table 1). The titration curve of signal 2’ (Fig. 9 b) reflects the presence of ionizing groups with a pK, different from that affecting signal 2 (prob- ably the new a-carboxyl group of Arg-128 and the carboxyl group of Asp-18). Therefore, we can conclude that the chemi- cal shift of signal 2 in LF6 is affected by the ionization of the Leu-129 a-carboxyl group with a pK, of 3.6.

Shindo et al. [34] reported, in their 13C-NMR study of native lysozyme, a normal pK, value of 3.1 [35] for the Leu- 129 a-carboxyl group. Since these authors found no change in the pKa value when 4.6 of the lysine residues were converted to homoarginyl residues, they concluded that the salt bridge between the Lys-13 c-amino group and the a-carboxyl group does not exist in solution.

However, in their 13C-NMR study of 13C-labeled dimethylated lysozyme, Gerken et al. [9] found that the pK, value of the dimethylated Lys-13 decreases by 0.5 after re- moval of the C-terminal Leu-129. They concluded that this is consistent with the loss of an ion pair interaction for Lys-13 in the carboxypeptidase-A-treated enzyme. Our results con- firm the existence of an interaction between Lys-13 and Leu- 129: the pK, of 3.1 for the a-carboxyl group in native lysozyme is increased to 3.6 after trifluoroacetylation of the &-amino groups of lysozyme. A possible explanation for the contra- diciting result of Shindo et al. [34] could be that Lys-13 is probably not derivatized under the conditions described by these authors.

Signal I . A comparison of the 19F-NMR spectra of LF6 and Asp-101-modified LF6 (Fig. 12a-c) shows a major shift of signal 1 to higher field and a small upfield shift of signal 5. The F3Ac group corresponding to signal 1 is in the environ- ment of an ionizing group with a pK, value of 4.3 (Fig. 9a, Table 1). In native lysozyme, Asp-101 has a pK, value of 4.4- 4.5 [36, 37). A downfield shift is observed for signal 1 when ( G ~ c N A c ) ~ is bound. Therefore, the F3Ac group correspond- ing to signal 1 must be in the neighbourhood of the binding sites A, B and C. The c-amino group of Lys-97 is situated at the top of the active cleft, near Asp-I 01 which forms hydrogen bonds with the sugar residues at sites A and B [I].

On the basis of these results, we assign signal 1 to the Lys- 97 F3Ac group. The large T2 relaxation time of signal 1 (Table 2) corresponds to the high solvent accessibility and mobility of the Lys-97 side chain [I]. The low-field position of signal 1, with respect to the other resonances, at low pH values can possibly be explained as a consequence of the ring- current effect of the neighbouring Trp residues 62 and 63.

Signal 3. Comparing the 19F-NMR spectra of LF6 and LF7 (Fig. 12a, b), the spectrum of LF, contains a new reso- nance at low field, a pronounced upfield shift of signal 3 and a minor downfield shift of signal 6. Since the a-amino group (pK, of 7.9 [35]), being less nucleophilic than the &-amino groups (pK, of 10.3 - 10.8 [35]), will be trifluoroacetylated more slowly than the 8-amino groups, we assign the new res- onance to the a-amino F3Ac group. The F3Ac group respon- sible for signal 3 is in the neighbourhood of an ionizing group with a pK, value of 3.97 (Fig. 9a, Table 1). The Lys residues which are in the vicinity of carboxyl groups are Lys-97, Lys- 13 and Lys-1 [l]. Since the signals 1 and 2 are already assigned to the Lys-97 and Lys-13 F3Ac groups, respectively, and the I9F-NMR spectrum of LF, shows a major effect on signal 3

391

' I " ' 1 " 1 ' ' I . ' T 1 ' ' ' 1 ' " ' " , , , I , , , / , . , , , , , , / , I I , , l , . , , " 1 . 1 ' "

1 0 8 p p m 11 5 ;1 L 11 3 11 2 11 1 l i 0 i 0 9

Fig. 12. "F-NMR sprctrurn of' LF, ( a ) , LF, ( h ) and Asp-101-modified LF6 (c) in hufler, p H 5.5, I = 0.1 M , at 30' C. The concentrations used were in the range 0.1 -0.25 mM

L-5

, , , 1 I . , # / / I , . , , , / I , , , z , . < ~ 1 ' ' , . 8 ~ 8 / 8 1 I f 1 8 1 , , 1 8 , I , / / # I 1 1 n r r T 7 1 ( I ' ' ' , i ' ' ' 1 ' 11 L 11 3 1: 2 11 1 11 0 '0 9 :0 3 g ? ~

Fig. 13. ' 9 F - N M R spwtrurn o f [ (Tyr -NO, ) , ]LF , (a ) , [ (Tyr-N02)2]LF6 ( b ) , [ ( T y r - N 0 2 ) J L F 5 ( c ) and [(Tyr-N02),]LF5 (d) in hufer p H 5.5, I = 0.1 M , at 30°C. The conccntrations used were in the range 0.1 -0.29 mM

(the distance between the a- and &-amino group of Lys-I is 0.74nm while the next nearest c-amino group of Lys-33 is 1.5 nm away [9]), signal 3 is assigned to the Lys-1 F3Ac group.

This is in agreement with the results of Gerken et al. [9]. They also observed an effect on the I3C resonance, corre- sponding to the di['3C]methylated a-amino group, when the extent of methylation of the Lys-I c-amino group was differ- ent.

From inspection of the lysozyme model, we can propose that the ionizing group with a pK, value of 3.97 might be Glu-7. In native lysozyme, Shindo et al. [34] determined a pK, of 2.6 for Glu-7. After conversion of 4.6 lysine residues to homoarginyl residues, they found a pK, 3.2 for Glu-7 and these authors concluded that the same interaction between Glu-7 and the c-amino group of Lys-I exists in solution as there is in the crystal [38] (normal pK, values for Glu are 4.1 - 4.4). This was confirmed by a I3C-NMR study of methylated lysozyme [9].

Signals 4, 5 and 6 . Tyr-20 and Tyr-23 are situated in the direct environment of Lys-96 and Lys-I 16, respectively. With respect to these Tyr residues, Lys-33 lies on the other site of

the lysozyme molecule. Figs 13 and 14 show the "F-NMR spectra at pH 5.5 and 3.1 of trifluoroacetylated lysozyme de- rivatives with one or two Tyr residues modified. Comparing the spectra of [ ( T Y ~ - N O ~ ) ~ ] L F ~ with those of LF6, we notice, besides the small downfield shift of signal 2 and 6 at pH 5.5 and some effects on the resonances 1 and 2 at pH 3.1, a major shift of signal 5 to the position of signal 4 at both pH values and no effect on the resonance position of signal 4. Compari- son of the spectra of [(Tyr-NO,),]LF, with those of [(Tyr- NOz),]LF6 shows, besides a minor effect on resonance 1 at pH 3.1, only a shift of resonance 6 to lower field at both pH values. Since the signals 1, 2 and 3 are already assigned and the resonance position of signal 4 stays unaltered, we can assign signal 4 to the Lys-33 F3Ac group. Comparing the 19F- NMR spectra of Tyr-nitrated LF5 derivatives with those of Tyr-nitrated LF6 derivatives at both pH values, only an effect on the signals 1 and 2 is observed due to the absence of resonance 6 (cf. LF5). Since these two resonances are assigned to the Lys-97 and Lys-I 3 F3Ac groups, respectively, and Lys- 96 is the only Lys residue in the neighbourhood of Lys-13 and Lys-97, signal 6 can be assigned to the Lys-96 F3Ac group.

392

i-: c n

2 3 I \

l ~ " ' 1 " ' I ' ' ' I " , V ' I r r r l r " , l " " " ' i ' I ' ' J " , ' ' I ' ' , , ' , ' I '

11 i l i 3 I 1 2 11 1 $ 1 9 10 9 ' i:9 C'T

Fig 34 "F-NMR ypectrum of [ (Tyr -N02) , ]LF6 ( u ) , [(Tyr-N02),]LF, ( b ) , [ ( T y r - N 0 2 ) J L F ~ l ~ ) and [ ( T Y ~ - N O ~ ) J L F S id) in buffer p H 3 1 , 1 = 0 I M , at 31) C The concentrdtions used were in the range 0 1 -0 25 mM

1 , I " , " ' I ' ' 1 , ' ' / " " I " ' I ' , . , , , . I , I , , , , , , # , , , , 8 , 1 11 L 11 3 11 2 11 1 11 0 :o 9 i C 8 pc;m

Fig. 15. "F-NMR spectra oj His-I5 curboxymethyluted F3Ac derivatives in buffer, I = 0.1 M , ut 30°C. (a) CM-LF6 and (c) CM-LF, in buffer pH 5.5; (b) CM-LF6 and (d) CM-LFs in buffer pH 3 .1 . The concentrations used were in the range 0.13-0.16 mM

Because resonance 5 is also affected by the Tyr modifications, we assign it to the remaining Lys-I 16 F3Ac group which lies in close to Tyr-23.

Since the introduction of a second nitro group affects signal 6 and the 19F-NMR spectra of [(Tyr-NO,),]LF, con- tain only six signals, the product [(Tyr-N02),]LF6 has to be a pure derivative, containing only molecules with Tyr-23 nitrated. This is in agreement with the findings of Hayashi et al. [18] on the iodination of lysozyme and confirms the assignment of resonance 5 to the Lys-116 F3Ac group. Ac- cording to these authors, Tyr-23 is easily iodinated while the reactivity of Tyr-20 is about one-third that of Tyr-23 and Tyr- 53 is not reactive. Probably due to the ionization of His-15 (pK, 5 . Q the introduction of the second nitro group has more influence on signal 6 at pH 5.5 than at pH 3.1. The distance between the Lys-96 c-amino group and the His-15 imidazole ring is about 0.7 nm [9].

From the pH dependence of resonance 6 (Fig. 9a), a pK, of about 5.5 or higher can be predicted for the neighbouring group. The two residues with such a pK, value in native

lysozyme are Glu-35 [34, 351 (the only carboxyl group with such an abnormally high pK, value) and His-15 [7, 351. Glu- 35 is located in the active cleft, acting as a proton-donor in the mechanism of substrate hydrolysis, but the distance between this residue and the nearest Lys residue (Lys-33) is much larger than the distance between His-I5 and Lys-96. Therefore, we conclude that the ionizing group, affecting res- onance 6, is situated on His-15, which is in agreement with the assignment of signal 6 to the Lys-96 F3Ac group.

An upfield shift is observed for signal 6 when (GlcNAc), is bound. Binding of the inhibitor has an effect on the resonance position of the Lys-97 F3Ac group, which probably causes a change in the environment of the Lys-96 F3Ac group. This also confirms the assignment of signal 6 to the Lys-96 F3Ac group.

The influence of carboxymethylation of His-1 5 (mainly on the c2-nitrogen because of steric reasons) on the 19F-NMR spectrum of LF6 at pH 5.5 and 3.2 is shown in Fig. 15. Com- parison of the spectra of CM-LF6 and LF6 at both pH values shows only small effects on signals 1 (Lys-97), 2 (Lys-13) and

Table 3. Assignment of the jhorinc resonance signals of LFb to the appropriate trijluoroucetylated l.ysine wsidues

~

Signal Assignment Reasoning

1 Lys-97 Asp-101 -modified LFb; (GICNAC)~ ; pf l ; TZ

2 Lys-I 3 dCS-LeU-LFb 3 Lys-I LF,; His-15 carboxymethylated LFb 4 Lys-33 LFb with both Tyr-20 and Tyr-23

5 LYS-I 16 Tyr-23-nitratcd LF6 6 LYS-96 LF, with both Tyr-20 and Tyr-23

nitrated

nitrated; (GICNAC)~; His-I 5-modi- lied LF6; pH; His- and Tyr-modi- lied LF, derivatives

4 (Lys-33) but a major downfield shift of the resonances 3 and 6 (which shifts behind the resonance position of the signals 5 and 4 at pH 5.5). Since signal 3 is already assigned to the Lys-1 F3Ac group which is, according to the crystallographic data, orientated towards the His-1 5 carboxymethyl group (the distance between the >:-amino group of Lys-1 and the His-15 imidazole ring is about 1.2 nm) and Lys-96 is the only remain- ing Lys residue in the neighbourhood of the His-15 imidazole ring, the assignment of signals 6 and 3 to the Lys-96 and Lys-1 F3Ac groups, respectively, is strenghtened.

Comparison of the I9F-NMR spectra of CM-LF5 with those of CM-LF6 at both pH values (Fig. 15 a - d) shows that removal of the F3Ac group corresponding to signal 6 again influences the resonance position of the signals 1 and 2, which confirms the assignment of signal 6 to the Lys-96 F3Ac group (see also Tyr modifications).

A survey of the arguments used for the assignment of the six 19F resonances is given in Table 3.

CONCLUSION

This study of trifluoroacetylated lysozyme derivatives shows the ease of labeling proteins with F3Ac probes without causing any drastic conformational changes of the polypep- tide backbone. By labeling specific regions in proteins with I9F probes, specific information can be obtained about the structure and dynamics of these regions.

As a consequence of the main advantages of the I9F- NMR technique (high sensitivity of the 19F nucleus, relatively intense and well-resolved signals, possibility to measure at very low concentrations and the short acquisition times), we believe it is an interesting method especially for investigation of higher-molecular-mass proteins.

Although the I9F-NMR spectra contain less detail com- pared with 'H-NMR spectra, we believe this is a useful method, complementary to 'H-NMR, for studying biochemi- cal processes, even for smaller proteins. The labels can also be used to assign and/or confirm proton resonances (e.g. lysine spin systems).

After the introduction and assignment of the I9F labels, it is our intention to use these labels to study the structure and dynamics of the labeled regions [cf. (GlcNAc), binding]. At this moment, we are working on the binding of (GlcNAc), as a function of temperature, the study of lysozyme as a function of concentration (LF6 with I9F-NMR, as well as native lysozyme with 'H-NMR) and the (selective) denatura- tion effects of temperature and chaotropic agents.

393

We wish to express our gratitude to Prof. G. Prkaux and Dr C. Gielen (Katholieke Universiteit Leuven) for recording the circular dichroism spectra. This work is supported by the NFWO (National Fund of Scientific Research in Belgium).

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