Conformational transitions of immunoglobulin fragment Fc(t) incited by alkyl sulfates of various hydrophobic chain lengths

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<ul><li><p>BIOCHIMICA ET BIOPHYSICA ACTA 631 </p><p>BBA 35783 </p><p>CONFORMATIONAL TRANSITIONS OF IMMUNOGLOBULIN FRAGMENT </p><p>Fc(t) INCITED BY ALKYL SULFATES OF VARIOUS HYDROPHOBIC CHAIN LENGTHS </p><p>PAK KAI JANE LEE AND BRUNO JIRGENSONS* </p><p>The Umvers,ty of Texas, M. D. Anderson Hospital and Tumor Instztute at Houston, Department of Biochem,stry, Houston, Texas 77o25 (U.S.A.) </p><p>(Received November 9th, 197 o) </p><p>SUMMARY </p><p>The effect of alkyl sulfates of various chain lengths on immunoglobulin frag- ment Fc(t) was studied. Circular dichroism measurements indicated that the non- helical polypeptide chains of Fc(t) were ordered by anionic detergents to a certain degree. Resolution of the eUipticity curves into Gaussian peaks showed that the detergents incited the formation of negative bands centered at 206 and at 222 nm and a positive band at 19o-192 nm. This partial transition to a-helical structure was enhanced by increasing amount of detergents until the critical micelie concentrations of the detergents were reached, when no further change was observed with additional detergents. The effect of ionic strength seemed to be on the critical micelle concen- tration. These conformational changes were also influenced by pH and the hydro- phobic chain length of the detergents. In acid solution, anionic detergents appeared to both promote a-helix formation and reduce the amount of disordered structure present in Fc(t). The order of effectiveness of the detergents as helix-forming agents was tetradecyl &gt; dodecyl &gt; decyl &gt; octyl sodium sulfate, indicating the importance of hydrophobic bonding in Fc(t). In near ultraviolet zone, the circular dichroic bands were weakened by the detergents, although the Cotton effects were not completely eliminated. </p><p>INTRODUCTION </p><p>The interaction of proteins with detergents has been extensively studied and several reviews are available (e.g. refs. I, 2). Depending on the nature of protein </p><p>Abbreviations: IgG, Fab and Fc recommended by the World Health Organization 15" Fab(t) and Fc(t) used for the tryptic digestion fragments similar to the papain digestion fragments Fab and Fc; CD, circular dichroism. </p><p>* Postal Address: The University of Texas M. D. Anderson Hospital and Tumor Institute, Department of Biochemistry, 6723 Bertner Avenue, Houston, Texas 77o25, U.S.A. </p><p>Biochim. Bwphys. Acta, 229 (1971) 631-641 </p></li><li><p>632 P .K . J . LEE, B. JIRGEI~SONS </p><p>under investigation and study of such conditions as temperature, pH and ionic strength, detergents can cause dissociation 3, association 4, precipitation 5, complex formation and denaturationS, v. </p><p>The conformational changes of the protein molecules induced by detergents have been investigated by methods of optical rotatory dispersion (see refs. 8-12), ultraviolet absorption difference spectrum (e.g. refs. 12, I3), and recently, by circular dichroism (CD) 14. </p><p>The present work offers data on the conformational transitions of IgG fragment Fc(t) incited by alkyl sulfates of various chain lengths as shown by CD spectra, The dependence of the conformation on detergent concentrations and on the hydro- phobic chain length of detergents are emphasized. </p><p>MATERIALS AND METHODS </p><p>Normal human IgG was obtained from Pentex Inc. (Kankakee, Ill.). Highly purified sodium octyl, decyl, dodecyl and tetradecyl sulfates were obtained from Mann Research Laboratories (New York, N.Y.). All other reagents used were of analytical grade. </p><p>Preparation of normal human Fc(t). Partial reduction and alkylation of the interchain disulfide bonds of normal human IgG were done according to EDELMAN et al. le. The tryptic digestion fragment Fc(t) was obtained by the procedure of EDEL- MAN et al. le modified by DoI AND JIRGENSONS 17. Symmetrical chromatographic peaks indicated single components in the Fc(t) preparations. Homogeneity of Fc(t) was determined by cellulose acetate electrophoresis and the antigenic specificity was checked by immunoelectrophoresis using goat antiserum against human Fc. </p><p>Sedimentation. All determinations were made with a Beckman-Spinco Model E ultracentrifuge at a rotor speed of 59 780 rev./min and at 20.0 . Sedimentation coefficients were corrected to the standard conditions. </p><p>Viscosity. Viscosity experiments were performed with a Cannon-Ubelhode dilution viscometer at 28.0 . The data were plotted as ~sp/c against c, where ~sp is the specific viscosity and c is the concentration in g/Ioo ml. The intrinsic kinematic viscosity [*/] was obtained by extrapolation to infinite dilution. </p><p>Molecular weight. Molecular weight of Fc(t) was calculated from the intrinsic viscosity and sedimentation coefficient by using the equation of SCHERAGA AND MANDELKERN ls, in which fi was assumed to be 2.6.IO e (ref. 19) and the partial specific volume was assumed to be 0.73 cm 3 (ref. 16). </p><p>Protein concentration. Protein concentrations were determined using a Beckman DU spectrophotometer. --xEX%cm of 15 at 280 nm was used 17. </p><p>CD measurements and resolutions of ellipticity curves. The new Model CD-SP Durrum-Jasco highly sensitive dichrograph was used. The sensitive scale settings of 2. lO -5, 5" lO-5 or I- lO -4 dichroic absorbance difference (zle) per I cm on the recorder chart was used. The optical path in the near ultraviolet was i.o cm and it was o.I or 0.05 cm in the far ultraviolet spectral region from 250 nm down to a little below 19o nm. The errors in this spectral zone, in terms of mean residual molar ellipticities were between -4- 400 and -- 600 degrees-cm 2 per decimole. They were much smaller at longer wave lengths. The performance of the instrument was checked by using solutions of camphor sulfonic and poly-L-glutamic acids. All recordings were made </p><p>Biochim. Biophys. Acta, 229 (I97I) 631-641 </p></li><li><p>CD OF IgG FRAGMENT Fc(t) WITH ALKYL SULFATES 633 </p><p>at an ambient room temperature of 23-25 . The data were calculated as mean residual molar ellipticities E0]. The mean residue weight of lO 9 was used 17. Reduced mean residual molar ellipticities EO'l, in the dilute salt solution and acid solution were calculated taking the refractive indices of water (see ref. 2o), EO'l = E01" E3/(n2+2)]~2o. The eUipticity curves were constructed from at least three circular dichroism re- cordings by calculating the mean residual molar ellipticities in 2-nm intervals. The ellipticity curves were resolved into Gaussian peaks by means of the DuPont 31o curve resolver. </p><p>pH measurement, pH measurements were carried out on a Coming Model 12 research pH meter with a combination electrode. Standard buffer solutions of pH 4.00 and 7.00 (certified by Fisher Scientific Co., Pittsburgh, Pa.) were used to calibrate the pH meter. </p><p>Interaction of anionic detergents with Fc(t). Stock protein solutions for the near ultraviolet CD measurements were made up of approx. 0.2% Fc(t) in water. For far ultraviolet measurements, I/IO dilutions of the above stock solution were used. The concentrations were checked from absorbance at 280 nm. </p><p>Stock detergent solutions were prepared by dissolving known amounts of sodium octyl, decyl, dodecyl, or tetradecyl sulfates in either water or other solvents, e.g. 0.02 and 0.2 M Na2SO 4, acidified with H2SO 4, whenever necessary to achieve the desired pH. </p><p>All samples were prepared by diluting I.O ml of stock Fc(t) solution with known amounts of detergent and the volume made up to 2.0 ml by addition of appropriate salt solutions. Thus the final solutions consisted of either o.I % or o.oi % Fc(t) and various concentrations of detergent in either water, o.oi M or o.I M Na2SO 4 at certain pH. The samples were kept at room temperature, 23-25 , for 16-24 h or heated in a water bath at 5 for 2 h. In the latter case, CD measurements were performed after complete cooling of the solutions to room temperature. </p><p>RESULTS </p><p>Characterization of Fc(t) The intrinsic viscosity of Fc(t) was o.o512 dl/g and sedimentation coefficient </p><p>S~o,w of 3.75 S was obtained, giving a molecular weight of 51 ooo. These values were similar to those obtained by DoI AND .IIRGENSONS 17 indicating that the preparations were practically the same. The far ultraviolet CD spectrum showed a negative minimum at 217 nm with a definite shoulder at 229 nm and a positive peak at 202 nm (Fig. I). Resolution of this curve gave four Gaussian peaks with a positive peak at 202 nm and negative peaks at 217, 232 and 239-240 nm. The reduced mean residual molar ellipticities at 217 and 202 nm were --173o and + lO5O degrees, cm2/decimole, respectively. </p><p>Effect of different concentrations of anionic detergents on Fc(t) The effect of a series of sodium dodecyl sulfate concentrations on o.o119% </p><p>Fc(t) in o.I M Na~SO 4 (pH 7.1) in the far ultraviolet is shown in Fig. I. At detergent concentrations below o.2 mM sodium dodecyl sulfate no change was observed. When the protein to detergent ratio was increased to I mole Fc(t) to 86 moles sodium dodecyl sulfate, the negative peak at 217 nm increased very slightly while the </p><p>Biochim. Biophys. Acta, 229 (1971) 631-641 </p></li><li><p>634 P .K . J . LEE, B. JIRGENSONS </p><p>6- \ </p><p>4 - """t </p><p>o_ (t) </p><p>-2- 11', ,.8~ .......... , "V Ix ,, ./',-'/ </p><p>-" ! - i ' r~ ' - . . . " / ",,. ..,-" </p><p>\ 1.25B// \ / </p><p>-8 '~T,, / 1 429 .L I1297 </p><p>I I I I I I </p><p>190 2 IO 230 ~.(nm) </p><p>Fig. I. The effect of different concentrat ions of sodium dodecyl sulfate on the far ultraviolet CD spectra of Fc(t) in o.I M NazSO, (pH 7.1). The protein concentrat ion was o.o119,o. Protein to detergent concentrat ions were expressed in molar ratios. </p><p>positive peak was decreased and somewhat shifted towards the lower wave length region. As sodium dodecyl sulfate concentration increased, the positive peak leveled off completely, and shoulders appeared around 208 and 222 rim. Further increase in the detergent to protein ratio resulted in the intensification of the troughs at 222 and 206-208 nm, with corresponding increase of the positive peak at 19o-192 nm until a maximum value was reached. This maximum value was dependent upon the ionic strength and the pH of the solvents used. In this particular case, the ratio was I mole Fc(t) to 429 moles sodium dodecyl sulfate, corresponding to o.o119% Fc(t) and I mM sodium dodecyl sulfate. Increasing the detergent concentration to 50 mM sodium dodecyl sulfate showed no significant difference on the CD spectrum. The curve of the maximum effect of sodium dodecyl sulfate could be resolved into three bands with peaks at 19o, 206 and 222 nm, which are characteristic of a-helix. The mean residual ellipticity of --6000 degrees .cm2/decimole at 222 nm suggested an a-helix content of around 20% using a value of --38 ooo degrees .cm2/decimole for [01222 nm for lOO% a-helix 21 and +4000 degrees.cm2/decimole for lOO% random coil iz. </p><p>Another series was studied using o.oi M NaiSO4 (pH 7.2) as solvent and sodium decyl sulfate as the denaturant. A detergent concentration of I mM sodium decyl sulfate did not induce a change in the protein backbone structure as no change was observed in the far ultraviolet spectrum. When the molar ratio of Fc(t) and detergent reached I to 1972 (corresponding to o.129 % Fc(t) and 5 mM sodium decyl sulfate), the positive peak at 202 nm leveled off completely. The maximum change was reached only when the detergent concentration exceeded 20 mM. It should be noted that in both series, the maximum effect was reached when the detergent </p><p>B~ochim. Blophys. Acta, 229 (1971) 631-641 </p></li><li><p>CD OF IgG FRAGMENT Fc(t) WITH ALKYL SULFATES 635 </p><p>concentrations approached their critical micelle concentrations. At 25 , the critical micelle concentration for sodium dodecyl sulfate in o.I M Na2SO 4 and for sodium decyl sulfate in o.oi M Na2SO 4 are 1.2 and 21 mM, respectively 23. </p><p>In the near ultraviolet region (Fig. 2), sodium dodecyl sulfate is seen to weaken all the bands. Native Fc(t) had only negative bands with major ones located at 262, 274 and 293 nm with a shoulder around 284 nm. At neutral pH, the minimum amount of detergent causing a significant change in the ellipticities was 0. 4 mM sodium dodecyl sulfate, corresponding to a molar ratio of I to 20 (protein concen- tration was O.lO3%). Increase of detergent concentration reduced the intensities of the bands further, until a protein to detergent ratio of I mole Fc(t) to 282 moles sodium dodecyl sulfate was reached. Further increase in detergent concentration, e.g. up to o.I M sodium dodecyl sulfate did not change the effect. However, the Cotton effects were not completely abolished as seen by the persistant ellipticity of --27 degrees, cm~/decimole. </p><p>When the samples were heated at 5 0 for 2 h and then cooled to room temper- ature, protein to sodium dodecyl sulfate concentration of I mole to 15 moles was sufficient in changing the ellipticity at 275 nm from --IOO to --67 degrees.cm~-/deci - mole. Even at a concentration of I mole Fc(t) to 2823 moles of sodium dodecyl sulfate, </p><p>-2 t ' \ t , 12823 </p><p>/ ~i 'k1 :282 </p><p>-4 ~ / L . / " </p><p>-'::' I / - ' ' ..... ! </p><p>- - t e , : ]e~ : , ' --, ',, :00 , - ' ; </p><p>-6 / "~'/"J:i ! H5(50 ) </p><p>1:20/ </p><p>- I0 - </p><p>lalive </p><p>/ tr" "~ </p><p>4- </p><p>! </p><p>-4. \ ~ </p><p>-6 - ~ , ,~, . _ ,~ </p><p>-s . . . . -:. ~-~- - -~ . . . . ~ . . . . ~---_Z.-.."_.-~ . . . . . . . . . . .2 </p><p>2- </p><p>o x 0 - </p><p>-2 </p><p>, , i , t t i </p><p>260 280 300 o out o()~ o'f o~5 ~.(nrn) SODIUM SULFATE CONCN.(M} </p><p>Fig. 2. The effect of different concentrations of sodium dodecyl sulfate on the near ultraviolet CD spectra of Fc(t) in o.oi M Na,SO 4 (pH 7.1). The protein concentration was 0.092%. Protein to detergent concentrations were expressed in molar ratios. </p><p>Fig. 3. The effect of added salts on the ellipticities at 19o, 206 and 222 nm, of sodium dodecyl sulfate-treated Fc(t). The ellipticities at 19o nm were denoted by circles, [6)] , 6 nm by triangles and [O],2z nm by squares. The Fc(t) to sodium dodecyl sulfate molar ratio was i to 396 for the filled symbols .The unfilled ones represented I mole Fc(t) to 472 moles sodium dodecyl sulfate while the half filled ones indicated z mole Fc(t) to 472o moles sodium dodecyl sulfate. </p><p>Biochim. Biophys. Acta, 229 (1971) 631-64t </p></li><li><p>636 P.K.J. LEE, B. JIRGENSONS </p><p>the [0],~5 am remained around --2o degrees-cm*/decimole and could not be reduced further. </p><p>Effect of salt concentration on sodium dodecyl sulfate denaturation of Fc(t) Fc(t) was dissolved in water or Na~SO 4 of various concentrations and the </p><p>effect of sodium dodecyl sulfate was observed (Fig. 3). Since the final pH varied only slightly (from pH 7.2 in water and pH 6. 9 in 0.2 M Na2SO4) no adjustment of pH was made. When a detergent concentration of I mM sodium dodecyl sulfate was used, the [0J,22 nm was increased from --3300 degrees .cm2/decimole in water to --6000 degrees .cm2/decimole in o.I M Na,SO a. No further change was observed when the salt concentration was raised to 0.2 M. </p><p>At higher detergent concentration (o.oi M) which is above the critical micelle concentration of sodium dodecyl sulfate even in water ~3, no salt effects were observed. Moreover, at this high concentration of detergent, the maximum change in CD spectra of Fc(t) a...</p></li></ul>

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