Association of monoalkylureas with bovine serum albumin: 1H n.m.r. study

Association of monoalkyl ureas with bovine serum albumin: "H n.m.r. study Anna Michnik and Barbara Lubas Department of Biophysical Chemistry, Faculty of Pharmacy, Medical University School of Silesia, 41-200 Sosnowiec, Jaaieliofiska 4, Poland

(Received 17 July 1985; revised 7 March 1986) The mechanism of interaction of five monoalkylureas with bovine serum albumin (BS A ) was studied by t H n.m.r. spectroscopy in D20 solutions. With BSA concentration increasing flora 0 to 10% (w/v) marked and selective broadening of the n.m.r, lines of apolar fragments of alkylureas was observed. The relative broadening of the lines was higher for long-chained primary alkylureas, but distinctly lower for branched t-butylurea. The broadening of the n.m.r, lines can be interpreted in terms of formation of low affinity complexes between alkyl fraaments of aikylurea molecules and BSA protein. Selectively higher broadening of n.m.r, lines of the ~methylenes adjacent to the amide nitrogens suggests their preferential immobilization at binding to BSA. Therefore, the formed hydrophobic complexes seem to be stabilized by hydrogen bonds involving the amide nitrogen of aikylureas and the peptide linkages of a protein. Keywords: Alkylureas;bovineserum albumin; tH n.m.r, spectroscopy

Introduction The denaturing action of alkylureas on various proteins leads to their unfolding and dissociation into subunits t-s. These effects are generally attributed to the direct interaction of the denaturant molecules with proteins, involving formation of intermolecular complexes, i.e. binding of the alkylurea molecules to the polypeptide chains. The o.r.d, changes observed for ~-chymotrypsinogen in the presence of alkylureas 7 seem to suggest that their denaturing action is predominantly hydrophobic in character. On the other hand, studies of aqueous solutions of alkylureas by various techniques9 -~ 7 provided evidence for the rearrangements of water molecules by ureas, for the interaction between their hydrated molecules and for their self-associations. These effects which seem to be strongly related to formation by ureas of the hydrogen bonds are therefore also taken into account for explanation of the association of the ureas with proteins. The binding constants for the ureas and the average amino acid sites were evaluated from model calculations l"z as increasing in the order 0.042, 0.061, 0.108 and 0.232M - t for methyl-, ethyl-, propyl- and butylureas, respectively. In these calculations group additivity of the hydrophobic and polar contribution to the binding constant was assumed. However, so far no direct experimental evidence has been presented for the existence of complexes formed by alkylureas with the polypeptide chains of proteins. Also, the structure of these complexes remains obscure. We studied the mode of association of alkylureas with a protein in solutions by IH n.m.r, spectroscopy, a method well suited for detection of low-affinity intermolecular complexes which might be formed. The experimental design was essentially the same as previously 0141-8130/86/050289-06503.00 ~ 1986Butterworth& Co. (Publishers)Ltd

applied in our laboratory for aliphatic alcohols t s, i.e. it was based on searching for selective changes in the n.m.r. spectra of alkylureas due to their interaction with protein. Bovine serum albumin (BSA) which shows remarkable affinity for many ligands of widely diverse chemical properties, was chosen as a model protein for binding studies. The approach based on observation of selective broadening or shifting of the n.m.r, peaks attributable to different fragments of small molecules in the presence of protein was introduced by Jardetzky t9. In those particular cases of the weak ligand-protein complexes, where there is a fast exchange (F ~ B) between free (F) and bound (B) ligand molecules, increases in the linewidth are the only spectral changes observed. Selective broadening of particular lines allowed identification of the primary and secondary binding sites of the ligand. The spectral line width at half-height (Art/2) proportional to the relaxation rate can then, in the simplest case, be expressed by the relation Avl/2,o = BAy1/2,B+ (i - B)Avl/2.F

(1)

where Avt/z.o, Art~2. B and Avl/2. ~ are the observed, bound and free linewidths, respectively and 'B' is the fraction of the ligand molecules bound. Since the pioneering experiments directed by Jardetzky on sulphonamides z° and penicillins 2~ bound to BSA in several systems examined by this approach, especially in low-affinity complexes, the binding sites have been satisfactorily identified (for review see Ref. 22). In the systems in which spectral changes are more complex, the influences of the different terms to the relaxation rates can be resolved by extrapolation procedures tg'zL22. Justification of the use of the n.m.r. method as a measure of weak binding of the denaturants

Int. J. Biol. Macromoi., 1986, Vol 8, October

289

t H n.m.r, study of monoalkylureas association with BSA: A. Michnik and B. Lubas Table 1 Ranges of alkylurea/BSA molar ratio and positions of the n.m.r, lines analysed for individual alkylureas in the systems alkylurea/BSA/D20 Positions ( + 0.02 ppm)" of the individual signals b for linewidth analysis Monoalkylurea

CH3

CH2

Methylurea Ethylurea 1-Propylurea

2.62 (S) 1.02 (T) 0.94 (T)

1-Butylurea t-Butylurea

0.85 (T) 1.23 (S)

3.01 (Q) 1.37 (M) 3.12 (T) 3.08 (T) -

Range of an alkylurea/BSA molar ratio 560-I 70ff 340-1700 d 270--1350a 50(04080 ~ 215-102(}r

Chemical shifts in ppm from external TMS, pH = 5.5 + 0.5 b From singlets (S), triplets (T), quartets (Q) and multiplets (M), respectively c In BSA range 2-6~ow/v, methylurea0.5 M d In BSA range 2-10~ow/v, ethylurea 0.5 M, propylurea 0.4 M In BSA range 0.5-4~ w/v, butylurea 0.3 M f In BSA range 0.4-4~ow/v, t-butylurea 0.06M to protein in model systems is discussed for aliphatic alcohols in the first paper of the present series ~a and in two other recent papers 23'24.

Materials and methods N-Methylurea was a product of the Institute of Organic Chemistry, Polish Academy of Sciences, Warsaw (Poland). Four higher alkylureas were the products of Eastman Kodak Co. (USA). Bovine serum albumin (BSA) used in all experiments, five times crystallized and lyophilized, came from Biomed, Cracov (Poland) and had practically the same characteristics as detailed previously ~s, with dimer fraction not higher than 10~o and fatty acid content below 0.5~. D 2 0 was a product of the Institute of Nuclear Research, Swierk (Poland) and had an isotopic purity of 99.8~. The samples (0.6 ml) were prepared in D 2 0 just before the n.m.r, measurements and were unbuffered but the pH of the solutions was adjusted to 5.5 +0.1. All n.m.r, experiments, linewidth analysis, concentration recalculation and pH adjustment were made as described previously Is. N.m.r. characteristics of the alkylureas both in organic and in aqueous solutions are known 13"25'26. The linewidth was measured for singlets and for the chosen lines of triplets and multiplets (Table 1) corresponding to those previously analysed for alcohols 18. In the chosen range of BSA concentration, the multiplets were not completely fused and the analysed lines could be easily distinguished. The concentrations of the solutions (0.06-0.5 M) chosen according to solubility requirements of particular alkylureas were low enough to avoid conformational destabilization of the protein. The ranges of alkylurea/ BSA molar ratios (Table 1) were similar to those applied for the corresponding alcohol analogues. The reported values of linewidth are the mean results of 25 separate measurements (five independently prepared samples, five spectrum registrations for each).

Results The only spectral changes observed for all n.m.r, signals of

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alkylureas on increasing BSA concentration were the significant gradual increases in their width. Repeated attempts to detect any changes in the chemical shifts of the particular sets of n.m.r, lines have failed. A typical pattern of broadening of the n.m.r, lines of alkylurea observed in the presence of BSA is illustrated for propylurea in Figure I. The average results of segmental linewidth measurements for five analysed ureas are presented in Figure 2a-e respectively. It is clearly seen that broadening of the n.m.r, lines for primary atkylureas increased with increasing BSA concentration in the order methyl < ethyl < propyl < butyl

(2)

Branching of the hydrocarbon portion in t-butylurea caused a decrease of the line broadening (Figure 2e) as compared to the long-chained atkyl derivatives. Also, for each particular alkylurea, the broadening seems to be dependent on the position of the analysed segment in the aliphatic chain. Therefore, for better visualization of the selectivity of this effect, a comparison was made of relative broadening of the lines attributable to protons, appearing in (1) the e-methylenes (next to NH-), (2) the terminal methyls most distant from N H - , (3) the intermediate segments. The relative broadening, as (Av)I/2,aSA/(Av)I/2 where (Av)I/2,aSA and (Av)l/2 are the halfwidths of the respective lines in the presence and absence of BSA, was found to be a function of the ratio of molar concentrations of the denaturant and the protein (solid lines in Figure 3a-c). The broadening effects observed were highest for the lines of the a-methylenes adjacent to the amide nitrogen (Figure 3a), those for terminal methyls most distant from N H were distinctly the lowest (Figure 3b) while for the remaining protons were intermediate (Figure 3c). A direct comparison with our previous results for alcohols obtained at similar molar ratio range (broken lines on Figure 3 as a background for solid lines for alkylureas) indicates that in most cases the broadening effects for alkylureas were relatively higher and in some cases similar to those obtained for alcohols. The highest increase of linewidth observed for ct-methylenes adjacent to the amide nitrogen is separately visualized (Figure 4) for the

I H n.m.r, study of monoalkylureas association with BSA: A. Michnik and B. Lubas (3)

CH3-CHe-CHe-NH-C-NH~ (3) (21" (1)" II " ,,r

(1)

BSA concentration

0

(2)

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4% w/v

0% w/v I

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I

I

3

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Chemical shift (ppm from ext. TMS) Fil~re 1 Spectral pattern illustrating the effect of bovine serum albumin (BSA) on the IH n.m.r, lines of 1-propylurea in D20; 1-propylurea concentration 0.4 M, pH 5.5-1-0.1

alkylurea/BSA molar ratio of 750:1 (i.e. when on average 1.2 alkylurea molecules are attacking one statistical amino acid chain in BSA).

Discussion The absence of chemical shift changes at increased BSA concentration in the 1H n.m.r, spectra of the alkylureas is consistent with the fact that alkylureas upon interaction with proteins, do not form any kind of strong complexes. However, the results of line broadening observed in this situation for all the segments of alkyl chains in alkylurea molecules (Figures 1 and 2a-e) demonstrate that the entire alkyl chains of the ureas are immobilized by the interaction with the protein. On interaction of sodium dodecyl sulphate with phycocyanin the resonance peaks of the surfactant were not only broadened but also significantly shifted upfield 27 indicating very strong interaction of the entire hydrocarbon chains of the surfactant with the protein. On this background, the unchanged positions of the n.m.r. lines of alkylureas in the presence of BSA reflect rather weak association of the denaturant molecules with the protein. The results show a higher decrease in motional freedom of the long-chained and non-branched alkyls of ureas, increasing generally in the order given by equation (2). As shown by the results for t-butylurea (Figure 2e) branching of the hydrocarbon portion leads to its weaker immobilization. The correlation of our n.m.r, results with the effectiveness of the alkylureas as protein denaturants allows us to suggest that their greater denaturation ability can be explained by stronger association of particular alkyl chains with the polypeptide chains of proteins. On the other hand, marked selectivity in the linewidth increase observed for the individual segments of the aikyl

chains of alkylureas on their interaction with protein can be attributed to non-equivalent immobilization of various segments during formation of the alkylurea-BSA complexes. The spectral differences observed for alkylurea molecules on binding to the protein reflect the nature of their primary binding sites. The hypothetical complexes between non-branched molecules and a polypeptide chain can a priori involve (Figure 51): (1) hydrophobic association; (2) multiple hydrogen bonds leading to binding of the polypeptide chains; and (3) hydrogen bonds with polar fragments of the peptide linkages or with the side chains of hydrophilic amino acids. Our results show that the lines of 0t-methylenes (next to NH-) are more broadened at lower protein concentrations than those attributable to other groups. This suggests that the amide nitrogen is also involved in the interaction of alkylureas with protein in such a way which promotes hydrophobic association for the ~methylenes adjacent to NH-. Thus, our data support the structure of intermolecular complexes with proposed bridging (Figure 5Ib) since that can cause stronger immobilization of the proton groups located in close proximity to the amide nitrogen and hence, greater broadening of their n.m.r, lines. For t-butylurea, the weaker relative broadening of the n.m.r, lines can be explained by sterically weakened possibilities of association through hindrance of both the packing of the molecules with branched alkyl chains between the hydrophobic amino acid residues and the formation of additional hydrogen bonds with bridging. This restriction was taken into account in the presented scheme of the tbutylurea-BSA complex (Figure 511).

Int. J. Biol. Macromol., 1986, Vol 8, October

291

IH n.m.r, study of monoalkylureas association with BSA: A. Michnik and B. Lubas H O I R- N -C -NH

i

ALKYL UREAR:CH3 IVl

Alkylureas are stronger denaturing agents for proteins than the respective alcohols 2'2s. Generally, in our experiments their association seems stronger compared with the association of alcohols having the same aliphatic chains (Figures 3 and 4). Thus, the pattern of association of alkylureas and alcohols with BSA as studied by 1H n.m.r. is consistent with the denaturation effectiveness of the two kinds of denaturants as well as with their association constants evaluated from model calculations. Basing on a correlation of 1H n.m.r, data for monovalent and divalent alcohols we have demonstrated

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,

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Figure 2 Linewidth of n.m.r, signals of the monoalkylureas as a function of BSA concentration in D20, pH 5.5+0.1; (a) methylurea, 0.5 M ; (b) ethylurea, 0.5 M ; (c) 1-propylurea, 0.4 M ; (d) 1-butylurea, 0.3M; (e) t-butylurea, 0.6M. Each point represents the average of 25 measurements (five independently prepared samples, five n.m.r, registrations each). The error indicated is the calculated standard deviation. The analysed lines are as shown in Table 1 Signals of protons

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:

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i

i

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Figure 3 Dependence of the relativebroadening of alkyl n.m.r. signals for monoalkylureas (alkyl indicated) on the alkylurea/BSA molar ratio (solid curves). Broken curves represent the analogous dependence for monovalent alcohols taken for comparison from our previous paper la

2.0

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t-Butyl Methylenes

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1.5 2.0 Relative broadening

2.5

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Figure 4 Quantitative ranking in the association effectivity with BSA of various segments of monoalkylureas (solid bars) and monovalent alcohols (broken bars) as judged from relative line broadening for denaturant/BSA molar ratio of 750:1

292

Int. J. Biol. Macromol., 1986, Voi 8, October

1H n.m.r, study of monoalkylureas association with BSA: A. Michnik and B. Lubas H I

H I

1T

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C--C--N--C o

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H

I C

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Figure 5 Schematic representationof the postulatedstructureof hydrophobic complexes which may be formed between long-chained (1)or branched (IS)monoalkylureas with hydrophobic amino acid residues of BSA polypcptide chain

that hydrogen bonds are probably not of primary importance in the association of alcohols with BSA. For glycols, which act as weaker denaturing agents than monovalent alcohols and alkylureas 2s the association ability reflected in our n.m.r, data was also decreased ~s. Thus, it appeared that hydrogen bonds can give a contribution to the association only by extra stabilization of the hydrophobic interactions. The present data for alkylureas and their comparison with those previously obtained for alcohols demonstrate the especially strong influence of the neighbouring polar groups on the adjacent ~-methylenes. Therefore, one can conclude that out data provide evidence for a mixed mechanism of alkylurea binding to protein involving the hydrophobic interaction of alkyl fragments with the amino acid sites of a protein additionally stabilized by hydrogen bonds in which the amide nitrogen participates. Examination of the quantitative pattern of n.m.r, line broadening for various non-polar fragments of the alkylurea chains gives information mainly about differences in strength and extent of their binding. However, some difficulties in the interpretation of data should be pointed out. They mostly concern changes in internal rotation on moving away from the head group of the particular ureas as well as free rotation in their --CH a units. Alas, the n.m.r, data alone do not reflect these molecular events and other methods, possibly further studies on model systems, are required to provide an interpretation of the phenomena observed. Acknowledgements

The authors are indebted to Professor G. Barone for the discussion of the results and to Dr A. Wtodawer for the gift of some alkylureas. This investigation was supported by the Polish Academy of Sciences, project PW 09.7.7.1.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Herskovits, T. T., Jaillett, H. and Gadegbeku, B. J. Biol. Chem. 1970, 245, 4544 EIbaum, D. and Herskovits, T. T. Biochemistry 1974, 13, 1268 EIbaum, D., Pandolfelli, E. R. and Herskovits, T. T. Biochemistry 1974, 13, 1278 Elbaum, D., Harrington, J. P., Bookchin, R. M. and Nagel, R. L. Biochim. Biophys. Acts 1978, 534, 228 Bhat, R. K. and Herskovits, T. T. Biochemistry 1975, 14, 1572 Herskovits, T. T. and Harrington, J. P. Biochemistry 1975, 14, 4964 Warren, J. R. and Gordon, J. A. Biochim, Biophys. Acta 1976, 420, 387 Herskovits, T. T., Behrens, C. F., Sinta, P. D. and Pandolfelli, E. R. Biochim. Biophys. Acta 1977, 490, 192 Bonner, O. D., Bednarek, J. M. and Arisman, R. K. J. Am. Chem. Soc. 1977, 99, 2898 Bonner, O. D. Physiol. Chem. Phys. 1978, 10, 399 Barone, G., Rizzo, E. and Vitagliano, V. J. Phys. Chem. 1970, 74, 2230 Barone, G., Rizz0, E. and Volpe, V. J. Chem. Eng. Data 1976, 21, 59 Barone, G., Castronuovo, G., Della Volpe, C. and Ella, V. J. Sol. Chem. 1977, 6, 117 Barone, G., Castronuovo, G., Crexenzi, V., Elia, V. and Rizzo, E. J. Sol. Chem. 1978, 7, 179 Barone, G., Castronuovo, G., Elia, V. and Menna, A. J. Sol. Chem. 1979, 8, 157 Barone, G., Cacace, P., Castronuovo, G. and Elia, V. J. Chem. Soc. Farad. 7~'ans. 1 1981, 77, 1569 Philip, P. R., Perron, G. and Desnoyers, J. E. Can. J. Chem. 1974, 52, 1709 Lubas, B., Soltysik-Rasek, M. and Lesniewska, I. Biochemiso3' 1979, 18, 4943 Jardetzky, O. Adr. Chem. Phys. 1964, 7, 499 Jardetzky, O. and Wade-Jardetzky, N. G. Mol. Phannacol. 1965, 1, 214 Fischer, J. J. and Jardetzky, O. J. Am. Chem. Soc. 1965, 8% 3237 Jardetzky, O. and Roberts, G. C. K. "NMR in Molecular Biology', Academic Press, New York, 1981 Soltysik-Rasek, M., Le~niewska, I. and Lubas, B. Int. J. Biol. Macromol. 1985, 7, 235

Int. J. Biol. Macromol., 1986, Vol 8, October

293

I H n.m.r, study o f monoalkylureas association with BSA." A. Michnik and B. Lubas 24 25 26

294

Lubas, B. and Soitysik-Rasek, M. Int. J. Biol. Macromol. 1985, 7, 242 Subramanian, S., Sarma, T. S., Bala Subramanian, D. and Ahluwalia, J. C. J. Phys. Chem. 1971, 75, 815 Arsenault, M. A., Freeman, C. D. and Hooper, D. L. 'Applied

Int. J. Biol. M a c r o m o l . ,

1986, V o l 8, O c t o b e r

27 28

Spectroscopy', 1974, 28, 197 Rosenberg, R. M., Crespi, H. L. and Katz, J. J. Biochim. Biophys Acta 1969, 175, 31 Herskovits, T. T., Gadegbeku, B. and Jaillet, H. J. Biol. Chem 1970, 245, 2588