Sodium trichloroacetate-induced helical conformation of poly(l -lysine)

Biochimica et Biophysica Acta, 439 (1976) 302 309

{)i Elsevier Scientific Publishing Company, Amsterdam

Printed in The Netherlands

BBA 37409 SODIUM TRICHLOROACETATE-INDUCED HELICAL CONFORMATION OF POLY(L-LYSINE)

OSAMU TAKENAKA', AKIKO TAKENAKA'" and YUJI INADA Laboratory of Biological Chemistry, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo (Japan)

(Received January 22nd, 1976)

SUMMARY Sodium trichloroacetate which has been reported previously to be an effective denaturation reagent for proteins was applied to poly(L-lysine) and poly(x~-glutamic acid) to see its effects on a coil-to-helix transition and on the chemical reactivities of the e-amino group of poly(l.-lysine). Addition of sodium trichloroacetate to poly(L-lysine) induced a helical conformation even at neutral p H where the e-amino group of the polymer was protonated. On the other hand, little effect was observed on the coil-to-helix transition of poly(e-glutamJc acid). The e-amino group of poly(~-lysine) has an anomalously high reactivity with naphthoquinone 4,6-disulfonate carrying a negative charge. Sodium trichloroacetate inhibited the reaction of the e-amino group with this reagent, while sodium trichloroacetate enhanced slightly the reaction of the e-amino group of poly(L-lysine) with diazonium-l-H-tetrazole carrying a positive charge.

INTRODUCTION In the previous studies [1, 2], trichloroacetic acid known as a precipitant for proteins at strong acidic p H was applied at neutral pH to enzymes, proteins and hemoproteins to see its effects on their structures and activities, and the resulls were compared with those of urea, guanidine. HCI and halogen derivatives of acetate. The enzymatic activities of a-chymotrypsin, ribonuclease A and lysozyme near neutral pH were lost completely by addition of 1.2 M sodium trichloroacetatc (NaCI3Ac). Tyro~.ine residues in a-chymotrypsin, chymotrypsinogen, ribonuclease A and lysozyme which do not ionize at p H 12.0 without reagent ionized on addition of 1.5 M NaCI3Ac at the same pH. These proteins when treated with NaC13Ac at pH 7.0 underwent spectral shifts due to exposure of tyrosine and/or tryptophan Abbreviations: NaCI3Ac, sodium trichloroacetate; poly(Lys), poly(L-lysine); poly(Glu), poly(L-glutamicacid); Lys(Ac), N~-acetyl-L-lysine;NQDS, sodium naphthoquinone 4,6-disulfonate; N2HT, diazonium-l-H-tetrazole. * To whom correspondence should be addressed at the Department of Biochemistry, Primate Research Institute, Kyoto University, Kanrin, Inuyama, Aichi, Japan. "~ Present address : Departnaent of Physiology, Primate Research Institute, Kyoto University.

303 residues from the interior of protein molecules. The helical content of insulin and ¢~-chymotrypsin decreased on addition of NaCIaAc, whereas the helical content of ribonuclease A and lysozyme increased with the ~ame treatment. This result indicates that the structural changes of proteins by NaCI~Ac are not limited to the destruction of the tertiary structure alone. NaCl3Ac was applied at neutral pH to oxy-, deoxy-, met- and cyanometderivatives of hemoglobin and myoglobin. The absorption spectra of these hemoproteins were changed by addition of NaC13Ac above a certain concentration. Difference in the concentration of NaCI3Ac giving spectral change of hemoproteins suggested that the structure of hemoglobin changes to a greater extent than does the structure of myoglobin when they bind ligands. The present study was undertaken to examine the effects of NaCI3Ac on a helix-to-coil transition of poly(L-lysine) (poly(Lys)) and poly(L-glutamic acid) (poly(Glu)) and the effects on the reactivities of e-amino groups of poly(Lys) toward naphthoquino~e 4,6-disulfonic acid (NQDS) and diazonium-l-H-tetrazole (N2HT). MATERIALS AND METHODS

Materials Trichloroacetic acid obtained commercially was purified by distillation at 98 °C/20 mm Hg. NaC13Ac was prepared by neutralization of trichloroacetic acid solution in an ice bath with solid NaOH and then with a concentrated NaOH solution until the pH of the solution became 7.0. NaCI3Ac solutions at various concentrations were prepared by dilution of this solution, e-Benzyloxycarbonyl-i_-Iysine and poly(I~ys) hydrochloride were purchased from the Protein Research Foundation, Osaka University and were used without further purification. The molecular weight of poly(l~ys) was examined with thin-layer gel filtration using Sephadex G-200 [3] and was greater than 2" 104. The content of the benzyloxycarbonyl group, the e-aminoprotecting group during polymerization, was estimated spectrophotometrically and was found to be 1.4 ~ of lysine residues. The molecular extinction coefficient of the benzyloxycarbonyl group was assumed to be 115 at 257 nm, which is obtained by e-benzyloxycarbonyl-~_-lysine. Poly(Glu) was kindly donated by Kyowa Hakko Kogyo Co. and was purified as follows. Poly(Glu) dissolved in a small amount of water (100 mg/ml) was treated with active charcoal followed by ethanol precipitation. The resultant precipitate was dissolved in a small amount of 0.1 M NaC1 (50 mg/ml) and subjected to gel filtration using Sephadex G-75 super fine (4 × 30 cm) equilibrated with 0.1 M NaC1. The fractions of the molecular weight over 2. l04 were collected and lyophilized after exhaustive dialysis against water. The concentrations of poly(Eys) and of poly(Gtu) were determined by acid-base titration. Acetyl-L-lysine (Eys(Ac)) was synthesized by the method of Neuberger and Sanger [4]. NQDS and 5-amino-l-H-tetrazole~-were purchased from Seikagaku Kogyo Co. and Tokyo Kasei~Kogyo Co., respectively.

Modification of poly(Lys) Modification of poly(Eys) and of Eys(Ac) with NQDS was carried out by the method of Matsushima et al. [5]. Eys(Ac) or poly(Eys) with and without 2.5 M

3~24 NaClaAc in 0.1 M phosphate buffer (pH 8.3) was incubated with NQDS of various concentrations for 2 h. A sample mixture with NaCl3Ac (2 ml) was acidified with 2 ml of 1 M acetic acid and a sample mixture without NaCl3Ac (2 ml) with 2 ml of 1 M acetic acid containing 2.5 M NaClaAc before photometry. Reaction of e-amino group with the reagent was determined spectrophotometrically assuming a molecular extinction coefficient of 4010. Modification of poly(Lys) and Lys(Ac) with NQDS was followed by NaOH consumption measured with a pH-stat at pH 8.3 as well as photometry. NzHT was synthesized as described previously [6] and modification of poly(Lys) with NzHT was carried out as follows. Lys (Ac) or poly (Lys) with and without 2.5 M NaClaAc in 0.1 M phosphate buffer (pH 8.3) was incubated for 1 h with N i H T of various concentrations. A sample mixture (2 ml) with NaCl3Ac was diluted with 4 ml of 0.2 M phosphate buffer (pH 8.3) and a sample mixture without NaClaAc (2 ml) with 4 ml of 1.25 M NaCl3Ac in 0.2 M phosphate buffer (pH 8.3). Reaction of e-amino group was followed by measuring the absorbance value at 326 nm.

Measurements Optical rotatory dispersion of poly(Lys) and of poly(Glu) with and without 2.5 M NaClaAc was measured with a Jasco ORD/UV-5, using a 10.0 cm cell, The paramater, b0, was determined by use of the Moffit-Yang equation. The refractive index of 2.5 M NaCl~Ac was 1.388 and the value of 20 was assumed to be 212 rim. The mean residue weight was assumed to be 128 for poly(Lys) and 129 for poly(Glu). The acid-base titration was carried out using a Metrohm automatic titrator model 15 258. The pH-Stat used was a Toa 151ectric model HS-2A. Spectrophotometric measurements were made with a Shimadzu MPS-50 spectrophotometer using 1.0-cm cells. RESULTS

Optical rotatory dispersion of poly(Lys) and poly(Glu) The helical content of poly(Glu) and poly(Lys) was determined from O R D measurements, since the trichloroacetate anion has a strong absorption band in the ultraviolet region (below 270 nm). The Moffitt-Yang plot of poly(Glu) and poly(Lys) with and without NaCI3Ac gave straight lines and the --bo value obtained was plotted against p H and shown in Fig. 1. Curve A is the pH dependence of the -b0 value obtained for poly(Glu). The value was 500 at acidic pH and decreased with increasing p H. The apparent pK of this sigmoidal curve was 5.3. The presence of 2.5 M NaCI3Ac (Curve B) did not affect the pH dependence of the bo value, but the values were slightly higher than those without NaClaAc. However, the meaning of this result was not clear, since the acid-base titration curve of poly(Glu) in the presence of 2.5 M NaC13Ac agreed with that of poly(Glu) without NaCI~Ac, and the apparent pK was 5.1. The pH dependence of the --b0 value of poly(Lys) (Curve C) showed a sigmoidal curve and its apparent p K was 9.8. On the other hand, the - -bo value obtained for poly(Lys) in the presence of 2.5 M NaCI~Ac (Curve D) did not change in the pH range 8-12. The Moffitt-Yang plot of poly(Lys) with 2.5 M NaCI~Ac at various pH values gave straight lines, as seen in Fig. 2. This enabled us to obtain the -b0 value

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Fig. 1. The pH dependence of the b0 value of poly(Glu) and poly(Lys): Curve A, poly(Glu) and Curve B, poly(Glu) in the presence of 2.5 M NaC13Ac. The concentration of v-carboxyl groups was 2.19 mM. Curve C, poly(Lys) and Curve D, poly(Lys) in the presence of 2.5 M NaCI3Ac. The concentration of e-amino groups was 2.32 mM. Buffers used were acetic acid/NaOH in the pH range 4.2-5.6, KHzPO4/Na2HPO4 in the pH range 5.8-6.0 (ionic strength - 0.2), NaHCO3/Na2CO3 in the pH range 8.2-11.4 and KC1/NaOH in the pH range 12.0 12.5 (ionic strength 0.1).

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x~/(>#- xD Fig. 2. Moffitt-Yang plots obtained for poly(Lys) in the presence of 2.5 M NaCI3Ac. (0) pH 12.0, ((:.) pH 11.5, (A) pH 10.2 ( ~ ) pH 9.0. Buffers as in Fig. 1.

and the value was 480 in this pH range. The poly(Lys) molecule in the presence of 2.5 M NaCI3Ac may be in a helical conformation even at neutral pH. The value of ao, the intercept value of the plot, is ascribed to the specific rotation of amino acid residues and is thought to be influenced easily by the environment of the molecule. The decrease seen in Fig. 2, therefore, is not well defined. Acid-base titration of poly(Lys) and Lys(Ac) was carried out in the presence of various concentrations of NaC13Ac. Poly(Lys) with NaCI~Ac (0.2-1.5 M) precipitated upon addition of N a O H in the pH range 10-12. The titration curves, however,

3{36 were not disturbed appreciably by the precipitation. Poly(Lys) without NaCI3Ac and with NaCl3Ac above 2.0 M did not precipitate. The apparent pK values were obtained from the titration curves and were plotted against the concentration of NaCI3Ac in Fig. 3. The value obtained for poly(Lys) in the absence of NaCl3Ac was 10. I and agreed with the apparent pK value, 9.8, obtained from the pH dependence of - b0 (Fig. 1). The apparent pK value of poly(Lys) (Curve A) increased with increasing NaCI~Ac concentration and approached a constant level, 11.0, above 1.0 M NaCI3Ac. The e-amino group of poly(Lys) was, therefore, expected to be protonated at neutral pH in the presence of 2.5 M NaCI3Ac. The apparent pK value of Lys(Ac) increased slightly with increasing NaCI3Ac concentration (Curve B). r

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[~oC~A~](~) Fig. 3. The apparent pK value of poly(Lys) and Lys(Ac) plotted against NaCI3Ac concentration. Curve A, poly(Lys) and Curve B, Lys(Ac). The concentrations of e-amino groups of poly(Lys) and of Lys(Ac) were 12.4 m M and 20.0 raM, respectively.

Reaction of poly(Lys) with NQDS Fig. 4 shows the reaction of poly(Lys) with NQDS in the presence and absence of 2.5 M NaCI3Ac. The absorbance value at 480 nm is plotted against NQDS concentration. The curve obtained for poly(Lys) without NaCI3Ac (Curve A) rises steeply at low concentration of NQDS and reached a constant level above 1 mM, This level means that half of the e-amino group of the poly(Lys) molecule reacted with the reagent. The curve obtained for poly(Lys) with NaCI3Ac (Curve B) increases gradually in the low concentration range of NQDS but rises over that for poly(Lys) at higher NQDS concentration. NaC13Ac did not affect appreciably the reaction of the e-amino group of Lys(Ac) (Curves C and D). The reaction of an amino group with one NQDS molecule produces one proton [7]. The reaction of poly(Lys) with NQDS at pH 8.3 was followed by measuring the consumption of NaOH, and the results are shown in Fig. 5. Curves A - F are the time course of the NaOH consumption after the addition of NQDS of 0.05, 0.1, 0.2, 0.4, 0.6 and 1.0 mM, respectively, to poly(Lys) (e-amino group = 0.35 mM) without NaCI3Ac. When the concentration of NQDS is high (Curves D-F), NaOH consumption occt~rred rapidly and ceased in 2 min. Some precipitates were formed at the points indicated by arrows, and the bar, 500/0, indicates the level where a half

307

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Fig. 4. Reaction of poly(Lys) and Lys(Ac) with NODS in the presence and absence of NaCI3Ac. Absorbance value at 480 nm plotted against NQDS concentration. Curve A, poly(Lys), Curve B, poly(Lys) in the presence of 2.5 M NaC13Ac, Curve C, Lys(Ac) and Curve D, Lys(Ac) in the presence of 2.5 M NaC13Ac. The concentration of e-amino group of poly(Lys) and of Lys(Ac) were 0.35 and 0.40 raM, respectively, in the reaction mixture.

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Fig. 5. Time course of NaOH consumption of poly(Lys) and Lys(Ac) after addition of NQDS. Curves A - F were obtained for poly(Lys) (e-amino group -- 0.35 mM) without NaC13Ac by addition of NQDS of 0.05, 0.1,0.2, 0.4, 0.6 and 1.0 m M, respectively. Curve G for poly(Lys) (e-amino group 0.35 mM) in the presence of 2.5 M NaCI3Ac by addition of NQDS of 1.0 mM. Curves H and I were obtained for Lys(Ac) (0.40 mM) in the absence and presence of 2.5 M NaCI3Ac, respectively, by addition of 1.0 mM NQDS.

of the e-amino groups in the poly(Lys) molecule react with N Q D S . The reaction was terminated probably due to the formation of precipitates. N a O H consumption curves (Curves A, B and C) approached the levels where 0.4, 0.9 and 1.7 ffmol of N a O H , respectively, were consumed. Therefore, 8 0 - 9 0 ~ of the N Q D S added reacts with the amino group in a few minutes, since a sample mixture indicated by Curves A, B and C contains 0.5, 1.0 and 2.0 ffmol of N Q D S , respectively. Curve G in the same figure is the time course of the N a O H consumption of poly(Eys) with 2.5 M NaCI~Ac

308 by addition of NQDS of 1.0 raM. The reaction was inhibited by NaCI3Ac as compared w,th Curve F. Curves H and 1 are obtained for 0.40 mM Lys(Ac) by addition of 1.0 mM NQDS in the absence and presence of 2.5 M NaCI3Ac. The presence of NaCI3Ac slightly enhanced the reaction of Lys(Ac) with NQDS and the results are compatible with those measured spectrophotometrically (Fig. 4j.

Reaction qf poly(Lys) with N2HT Fig. 6 shows the reaction of poly(Lys) with N2HT, where the absorbance value at 326 nm was plotted against NzHT concentration. Curves A and B were obtained for poly(Lys) without and with 2.5 M NaC13Ac, respectively. In contrast to the results with NQDS (Figs. 4 and 5), the presence of NaCI3Ac enhanced the reaction of poly(Lys) with N2HT. As seen from Curves C and D, the reactivity of ~-amino group of Lys(Ac) was not affected by the presence of NaCI3Ac.

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Fig. 6. Reaction of poly(Lys) and Lys(Ac) with NzHT in the presence and absence of NaCI~Ac. Absorbance value at 326 nm plotted against N2HT concentration. Curve A, poly(Lys),Curve B, poly(Lys) in the presence of 2.5 M NaCI3Ac. Solid triangles and open triangles on Curve C are obtained for Lys(Ac) and Lys(Ac) in the presence of 2.5 M NaCI3Ac, respectively. The concentration of ~'amino group of poly(Lys) and that of Lys(Ac) was 0.15 raM, respectively, in the reaction mixture. DISCUSSION The present results indicate that the poly(Lys) molecule has a helical conformation at alkaline pH and agree with the early observation made by Applequist and Doty [8]. On the other hand, the poly(Lys) molecule in the presence of NaC13Ac is probably in a helical conformation even at neutral pH, where e-amino groups of the poly(Lys) molecule are protonated. Trichloroacetate anions bind to the poly(Lys) molecule and may compensate the repulsion among the positive charges of amino groups. Similar conformational transitions of poly(Lys) were observed by Satake and Yang [9]. Recently, they studied the effects of five sodium alkyl sulfates on a coil-to-helix transition of poly(Lys). With octyl sulfate, poly(Lys) has a helical conformation while other alkyl sulfates having longer side chains induce a /3-form. A small anion having a hydrophobic moiety in its molecule may induce a coil-to-helix transition.

309 In a previous paper, we reported that the helical content of ribonuclease A and lysozyme increased on addition of NaCI3Ac. The ribonuclease A and lysozyme molecules may have a region(s) rich in positive charges since these proteins are basic ones. Trichloroacetate anion appears to compensate the repulsion among positively charged groups in such a region(s) and induces a helical conformation. The interaction of the amino groups of poly(Lys) with trichloroacetate anion was examined by chemical modification with NQDS carrying a negative charge and with NzHT carrying a positive charge, e-Amino groups of poly(Lys) without NaCI3Ac have anomalously high reactivity with NQDS as compared with that of Lys(Ac) or poly(Lys) with NaCIsAc (Curves F-I in Fig. 5). When the molar concentration of NQDS was higher than a half of the molar concentration of the e-amino group of poly(Lys), a half of an amino group reacted with NQDS in 2 rain and precipitates were formed. The presence of NaClsAc reduced the reactivity of the e-amino group toward N Q D S to less than 1 ~ . The positive charges of the poly(Lys) molecule may adsorb the negatively charged NQDS and this interaction probably facilitates the reaction. Trichloroacetate anion may bind to poly(Lys) in a competitive fashion with N Q D S and inhibit the reaction of the e-amino group. The reaction introduced the negative charge of naphthoquinone sulfonate group to the poly(Lys) molecule. Electrostatic forces between this negative charge and the positive charge of the unreacted e-amino group may precipitate the modified poly(Lys) molecule. Opposite results were obtained with NzHT carrying a positive charge. The presence of NaCIsAc enhanced the reaction of the amino group with N/HT. Trichloroacetate anion probably lowers the repulsion between positive charges of the amino group and NzHT. The reaction of Lys(Ac) with NQDS or NzHT was not affected appreciably by the presence of NaCI~Ac. Interaction between Lys(Ac) and trichloroacetate anion may not be so effective as to affect the reactivity of the amino group. In the poly(Lys) molecule, the positive charge and the hydrocarbon side chain are accumulated in a spatially limited region. This may be essential for the interaction of trichloroacetate anion with poly(Lys). REFERENCES I Takenaka, A., Takenaka, O., Mizota, T., Shibata, K. and |nada, Y. (1971) J. Biochem. Tokyo 70, 63-73 2 Takenaka, A., Yokoyama, S., Mizota, T., Takenaka, O. and Inada, Y. (1971) Arch. Biochem. Biophys. 146, 348-352 3 Radola, B. J. (1968) J. Chromatogr. 38, 61-77 4 Neuberger, A. and Sanger, F. (1943) Biochem. J. 37, 515-518 5 Matsushima, A., Sakurai, K., Nomoto, M., lnada, Y. and Shibata, K. (1963) J. Biochem. Tokyo 64, 507-514 6 Takenaka, A., Suzuki, T., Takenaka, O., Horinishi, H. and Shibata, K. (1969) Biochim. Biophys. Acta 194, 293-300 7 Frame, E. G., Russell, J. A. and Wilhelmi, A. E. (1943) J. Biol. Chem. 149, 255-270 8 Applequist, J. and Dory, P. (1958) in Abstr. Am. Chem. Soc. 133rd Meet., p. 32Q, San Francisco, Calif. 9 Satake, I. and Yang, J. T. (1973) Biochem. Biophys. Res. Commun. 54, 930-936