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Interaction of purine bases and nucleosides with serum albumin
Journal
of
MOLECULAR STRUCTURE ELSEVIER
Journal of Molecular Structure 410-41 I (1997) 27-29
Interaction of puke
bases and nucleosides with serum albumin A. Sulkowska*,
A. Michnik
Biophysical Chemistry Department, Medical University School, Jagiellon’ska 4, 41-200 Sosnowiec, Poland
Received 26 August 1996; revised I7 October 1996; accepted I8 October 1996
Abstract The proton NMR spectra of alkyl derivatives of adenine and adenosine have been studied. High-resolution (400 MHz) proton spectra were recorded at 300 K at increasing concentrations of serum albumin. The dependence of the chemical shifts and the line width of the individual spectral lines on the protein concentration provides some detailed information about the nature of the complexes between the puxine derivatives and albumin. Comparison of data for the methylated and non-methylated purine bases and nucleosides indicates the formation of non-specific complexes with serum albumin. However, the presence of the ethyl group in 8-ethyl-9N-methyladenine means that in the adenine derivative-serum albumin complex the ethyl chain preserves its dominant role in binding. An advantage of our model is that the ?T--?T interaction between the adenine ring and the amino acids of the protein can be replaced by hydrophobic interaction in the case of complexation of the ethyl adenine derivative. 0 1997 Elsevier Science B.V. Keywords:
Aliphatic adenine derivatives;
Proton NMR; Hydrophobic
1. Introduction In natural materials numerous unusual bases occur in addition to the five major bases-adenine, guanine, cytosine, thymine and uracil. Some of these unusual substituted bases are found only in the nucleic acids of bacteria and viruses, but many are also found in the DNA and transfer RNAs. Both bacterial and human DNA contain, for example, significant quantities of 5methylcytosine; bacteriophages contain 5-hydroxymethylcytosine. Several unusual bases have been discovered in the messenger RNA molecules of mammalian cells: N6-methyladenine, N6-dimethyladenine and N’-methylguanine. The function of these substituted bases is not fully understood. Proteins that interact with DNA bring about or stop information transfer (transcription, replication and
* Corresponding
author.
0022-2860/97/$17.00
Bovine serum albumin
recombination). The DNA-interacting proteins can interact with specific nucleotide sequences or sites, or with the DNA non-sequence-specifically. The specific interaction of a protein with a sequence of DNA nucleotides must involve several attachment sites. Any single interaction cannot be expected to provide a sufficient free energy change to account for the tightness and specificity of the interactions observed. The simultaneous interaction of a number of functional groups on the two partners must be involved. In our work we would like to attempt an approach to the physico-chemical basis of the interaction specificities, working from small molecule and model system considerations and dealing with certain biologically relevant systems in detail to define classes of interaction mechanisms. Knowledge of the tertiary structure of bovine serum albumin (BSA) as a model globular protein [l] let us
0 1997 Elsevier Science B.V. All rights reserved. I-O
PI/ SOO22-2860(96)0968
interactions:
28
A. Suikowska,
A. MichnWJournal
of Molecular
approach the problem of purine ligand-globular protein interaction. We have considered specific and non-specific binding of purine bases and purine nucleosides to a protein model: native and denatured BSA.
Adenine (6-aminopurine) from Aldrich Chemical Co., N6-methyladenine (6_methylaminopurine), lmethyladenine (6-amino- 1-methylpurine), 1-methyladenosine (6-amino- 1-methylpurine 9-ribofuranoside) from Serva, adenosine (6-aminopurine 9-ribofuranoside) from Fluka Chem. AG and N6-methyladenosine (6-methylaminopurine 9-ribofuranoside) from Sigma Chemical Co. were used as received without further purification. Crystallized and lyophilized BSA fraction V was purchased from PPH POCh WSiS, Krakow. D20 (99.75% isotopic purity) was obtained from the Institute of Nuclear Research (Swierk). All solutions were prepared by dissolution of BSA and/or purine
of half line width Av liz + ud (Hz) and relative broadening
Ligand
Adenine N6-methyladenine
I-methyladenine
ðyl-9N-methyladenine
H(8) H(2) H(8) H(2) CH3(Nh) H(8) H(2) CHj(1) H(2) CHj(9) CH2(8)
Adenosine N6-methyladenosine
I-methyladenosine
CH#3) H(8) H(2) H(8) H(2) CH q(N’) H(8) H(2) CH3(1)
a Maximum deviation 2 0.15 Hz. b Line width not measurable.
410-411
(1997) 27-29
derivative in D20. For the molar concentration calculation the molecular weight of 66 000 for BSA was used. Proton NMR spectra were taken with a Bruker DPX 400 MHz spectrometer at 300 K. Chemical shifts in ppm were measured with respect to a water signal of 4.80 ppm. For the quantitative comparison of the effect of protein on the NMR line width relative broadening X values were calculated as described in [2].
2. Experimental
Table I Dependence (% w/v)
Structure
3. Results Chemical shifts, line widths and relative broadening for the proton NMR lines of the purine derivatives are presented in Table 1 Table 2. With increasing BSA concentration gradual broadening of the lines can be observed (Table 1) and any changes in the position of the line sets of the ligands (Table 2). For aromatic protons of adenine and adenosine the effect of line broadening was especially strong
factor X of the adenine derivative purine protons on BSA concentration
2% BSA
0% BSA
1% BSA
3% BSA
Av I/Z
A,J I/Z
X
Au I/Z
X
Au i/z
X
2.30 4.08 3.51 3.95 3.99 2.50 2.89 3.05 I .38 1.38 1.75 1.25 I .85 1.85 2.33 3.1 I 7.50 0.90 1.15 1.20
4.16 5.64 4.04 4.03 5.5 1 2.74 2.89 4.16 2.63 3.13 4.63 3.75 3.14 2.73 3.04 3.20 9.96 1.26 1.35 I .67
I .80 I .38 I.15 1.02 1.38 1.10 1.oo I .36 I .90 2.27 2.65 3.00 1.70 1.48 I .30 I .03 1.33 1.37 1.17 1.39
4.47 6.01 4.37 4.45 b 3.15 3.47 b 4.25 4.88 7.75 6.25 4.44 2.81 2.60 3.27 b I .34 1.61 b
1.94 1.47 1.25 1.13 b I .26 1.20 b 3.08 3.54 4.43 5.00 2.41 1.I7 1.12 1.05 b 1.49 1.39 b
6.78 7.60 5.51 5.73 b 4.20 4.35 b 4.50 5.82 7.75 7.20 5.47 4.97 3.78 4.34 b 1.50 1.71 2.80
2.94 I .86 I .57 I .47 b 1.68 I.51 b 3.26 4.20 4.43 5.76 2.96 2.69 1.69 I .40 b 1.67 I .48 2.33
A. Suikowska, A. MichniWJournal
Table 2 Chemical shifts 6 (ppm)” of purine derivatives (0:3.5% w/v)-D20 systems at 300 K Ligand
H(8)
H(2)
Adenine N6-methyladenine 1-methyladenine 8-ethyl-9N-methyladenine
8.11 8.10 8.05
8.07 8.08 7.99 7.93
Adenosine N6-methyladenosine I-methyladenosine
8.22 8.21 8.11
8.10 8.10 7.99
of Molecular Structure 410-411 (1997) 27-29
in the ligand-BSA
CHx
Ligand/BSA molar ratio
1IO:330 2.92 3.5 1 I .35h 2.87’ 3.50d 2.85 3.51
1 lo:330 110:330 1 IO:330
1 IO:660 220~660 190: 1320
a DzO position 4.800 ppm. ’ C(8)-CH1 triplet. ’C(8)-CH2 quartet. d N(9)-CH?.
compared with their methyl derivatives (except for 8ethyl-9Nmethyladenine). For all ligands the relative broadening factor X for the H(8) proton was higher than for the H(2) proton (Table 1). When compare relative broadening factors (Table 1) one can see that an order of broadening tendency of adenine and adenosine derivative protons should be as follows: CHs(8) = CHs(9) > H(8) > H(2). The methyl peaks of N6-methyladenine, l-methyladenine and N6-methyladenosine were overlapped by the protein signals in the aliphatic region. For g-ethyl9Nmethyladenine the relative broadening of the aliphatic protons attached to the imidazole ring was greater than in other ligands having methyl in the pyrimidine ring. It is apparent that the long-chain derivative (8-ethyl-9Nmethyladenine) undergoes greater broadening than the other ligands.
4. Discussion The relaxation time and the width of the NMR spectral line depends on the motion of the molecule which gives rise to the line. If the motion of the molecule is restricted, the relaxation time of the nuclei is shortened and the width of its NMR spectral lines is increased. For adenine the increase in line width of the pyrimidine ring proton H(2) with increased BSA concentration is more limited than for the imidazole ring proton H(8) (Table 1).
29
The results presented above demonstrate that the aromatic ring of the purine bases and their nucleosides are able to interact with serum albumin in ligandserum albumin complexes. The relative broadening coefficient order: XCH3 > Xn(s) > Xn(z), can be explained by the higher tendency of an aliphatic group (CH,) and imidazole ring (H(8)) than other protons of of adenine and adenosine to associate with macromolecule. From the unchanged values of the chemical shifts (Table 2) one can conclude that after formation of the adenine derivative-BSA complexes no changes in intermolecular distance occurred. The BSA polypeptide chain contains approximately 600 amino acid residues, and the loop region of each subdomain contains three helical segments, about 30% of the surface being hydrophobic. In our experiment about 0.2-2 adenine derivative molecules fall to each amino acid side-chain of the protein. The phenol ring of tyrosine is able to form stacked complexes with adenine bases in singlestranded poly A, but in Lys-Tyr-Lys-DNA complexes tyrosine does not intercalate between the base pairs [3]. This suggests the possibility of competition between different types of binding during association. Considering the marked and selective broadening of the NMR lines of the purine aliphatic groups of 8ethyl-9N-methyladenine in the presence of BSA we can assume that they reflect the existence of weak hydrophobic association between ethyl or/and methyl groups of the ligand and the hydrophobic grooves of albumin. The results indicate the involvement of the aliphatic chain of the adenine derivatives as the primary binding site, and the imidazole ring of the ligands as the secondary binding site. The quantitative pattern of relative line broadening suggests that the length of the aliphatic chain of the purine derivatives plays a particular role in the interaction with albumin.
References [I] A. Sulkowska, B. Lubas and T. Wilczok, Radiat. Res., 85 (1981) 1. [2] A. Sulkowska and A. Miehnik, J. Mol. Struct., 348 (1995) 73. [3] Y. Ohta, T. Tanaka, Y. Baba and A. Kagemoto, J. Phys. Chem., 90 (1986) 4438.
of
MOLECULAR STRUCTURE ELSEVIER
Journal of Molecular Structure 410-41 I (1997) 27-29
Interaction of puke
bases and nucleosides with serum albumin A. Sulkowska*,
A. Michnik
Biophysical Chemistry Department, Medical University School, Jagiellon’ska 4, 41-200 Sosnowiec, Poland
Received 26 August 1996; revised I7 October 1996; accepted I8 October 1996
Abstract The proton NMR spectra of alkyl derivatives of adenine and adenosine have been studied. High-resolution (400 MHz) proton spectra were recorded at 300 K at increasing concentrations of serum albumin. The dependence of the chemical shifts and the line width of the individual spectral lines on the protein concentration provides some detailed information about the nature of the complexes between the puxine derivatives and albumin. Comparison of data for the methylated and non-methylated purine bases and nucleosides indicates the formation of non-specific complexes with serum albumin. However, the presence of the ethyl group in 8-ethyl-9N-methyladenine means that in the adenine derivative-serum albumin complex the ethyl chain preserves its dominant role in binding. An advantage of our model is that the ?T--?T interaction between the adenine ring and the amino acids of the protein can be replaced by hydrophobic interaction in the case of complexation of the ethyl adenine derivative. 0 1997 Elsevier Science B.V. Keywords:
Aliphatic adenine derivatives;
Proton NMR; Hydrophobic
1. Introduction In natural materials numerous unusual bases occur in addition to the five major bases-adenine, guanine, cytosine, thymine and uracil. Some of these unusual substituted bases are found only in the nucleic acids of bacteria and viruses, but many are also found in the DNA and transfer RNAs. Both bacterial and human DNA contain, for example, significant quantities of 5methylcytosine; bacteriophages contain 5-hydroxymethylcytosine. Several unusual bases have been discovered in the messenger RNA molecules of mammalian cells: N6-methyladenine, N6-dimethyladenine and N’-methylguanine. The function of these substituted bases is not fully understood. Proteins that interact with DNA bring about or stop information transfer (transcription, replication and
* Corresponding
author.
0022-2860/97/$17.00
Bovine serum albumin
recombination). The DNA-interacting proteins can interact with specific nucleotide sequences or sites, or with the DNA non-sequence-specifically. The specific interaction of a protein with a sequence of DNA nucleotides must involve several attachment sites. Any single interaction cannot be expected to provide a sufficient free energy change to account for the tightness and specificity of the interactions observed. The simultaneous interaction of a number of functional groups on the two partners must be involved. In our work we would like to attempt an approach to the physico-chemical basis of the interaction specificities, working from small molecule and model system considerations and dealing with certain biologically relevant systems in detail to define classes of interaction mechanisms. Knowledge of the tertiary structure of bovine serum albumin (BSA) as a model globular protein [l] let us
0 1997 Elsevier Science B.V. All rights reserved. I-O
PI/ SOO22-2860(96)0968
interactions:
28
A. Suikowska,
A. MichnWJournal
of Molecular
approach the problem of purine ligand-globular protein interaction. We have considered specific and non-specific binding of purine bases and purine nucleosides to a protein model: native and denatured BSA.
Adenine (6-aminopurine) from Aldrich Chemical Co., N6-methyladenine (6_methylaminopurine), lmethyladenine (6-amino- 1-methylpurine), 1-methyladenosine (6-amino- 1-methylpurine 9-ribofuranoside) from Serva, adenosine (6-aminopurine 9-ribofuranoside) from Fluka Chem. AG and N6-methyladenosine (6-methylaminopurine 9-ribofuranoside) from Sigma Chemical Co. were used as received without further purification. Crystallized and lyophilized BSA fraction V was purchased from PPH POCh WSiS, Krakow. D20 (99.75% isotopic purity) was obtained from the Institute of Nuclear Research (Swierk). All solutions were prepared by dissolution of BSA and/or purine
of half line width Av liz + ud (Hz) and relative broadening
Ligand
Adenine N6-methyladenine
I-methyladenine
ðyl-9N-methyladenine
H(8) H(2) H(8) H(2) CH3(Nh) H(8) H(2) CHj(1) H(2) CHj(9) CH2(8)
Adenosine N6-methyladenosine
I-methyladenosine
CH#3) H(8) H(2) H(8) H(2) CH q(N’) H(8) H(2) CH3(1)
a Maximum deviation 2 0.15 Hz. b Line width not measurable.
410-411
(1997) 27-29
derivative in D20. For the molar concentration calculation the molecular weight of 66 000 for BSA was used. Proton NMR spectra were taken with a Bruker DPX 400 MHz spectrometer at 300 K. Chemical shifts in ppm were measured with respect to a water signal of 4.80 ppm. For the quantitative comparison of the effect of protein on the NMR line width relative broadening X values were calculated as described in [2].
2. Experimental
Table I Dependence (% w/v)
Structure
3. Results Chemical shifts, line widths and relative broadening for the proton NMR lines of the purine derivatives are presented in Table 1 Table 2. With increasing BSA concentration gradual broadening of the lines can be observed (Table 1) and any changes in the position of the line sets of the ligands (Table 2). For aromatic protons of adenine and adenosine the effect of line broadening was especially strong
factor X of the adenine derivative purine protons on BSA concentration
2% BSA
0% BSA
1% BSA
3% BSA
Av I/Z
A,J I/Z
X
Au I/Z
X
Au i/z
X
2.30 4.08 3.51 3.95 3.99 2.50 2.89 3.05 I .38 1.38 1.75 1.25 I .85 1.85 2.33 3.1 I 7.50 0.90 1.15 1.20
4.16 5.64 4.04 4.03 5.5 1 2.74 2.89 4.16 2.63 3.13 4.63 3.75 3.14 2.73 3.04 3.20 9.96 1.26 1.35 I .67
I .80 I .38 I.15 1.02 1.38 1.10 1.oo I .36 I .90 2.27 2.65 3.00 1.70 1.48 I .30 I .03 1.33 1.37 1.17 1.39
4.47 6.01 4.37 4.45 b 3.15 3.47 b 4.25 4.88 7.75 6.25 4.44 2.81 2.60 3.27 b I .34 1.61 b
1.94 1.47 1.25 1.13 b I .26 1.20 b 3.08 3.54 4.43 5.00 2.41 1.I7 1.12 1.05 b 1.49 1.39 b
6.78 7.60 5.51 5.73 b 4.20 4.35 b 4.50 5.82 7.75 7.20 5.47 4.97 3.78 4.34 b 1.50 1.71 2.80
2.94 I .86 I .57 I .47 b 1.68 I.51 b 3.26 4.20 4.43 5.76 2.96 2.69 1.69 I .40 b 1.67 I .48 2.33
A. Suikowska, A. MichniWJournal
Table 2 Chemical shifts 6 (ppm)” of purine derivatives (0:3.5% w/v)-D20 systems at 300 K Ligand
H(8)
H(2)
Adenine N6-methyladenine 1-methyladenine 8-ethyl-9N-methyladenine
8.11 8.10 8.05
8.07 8.08 7.99 7.93
Adenosine N6-methyladenosine I-methyladenosine
8.22 8.21 8.11
8.10 8.10 7.99
of Molecular Structure 410-411 (1997) 27-29
in the ligand-BSA
CHx
Ligand/BSA molar ratio
1IO:330 2.92 3.5 1 I .35h 2.87’ 3.50d 2.85 3.51
1 lo:330 110:330 1 IO:330
1 IO:660 220~660 190: 1320
a DzO position 4.800 ppm. ’ C(8)-CH1 triplet. ’C(8)-CH2 quartet. d N(9)-CH?.
compared with their methyl derivatives (except for 8ethyl-9Nmethyladenine). For all ligands the relative broadening factor X for the H(8) proton was higher than for the H(2) proton (Table 1). When compare relative broadening factors (Table 1) one can see that an order of broadening tendency of adenine and adenosine derivative protons should be as follows: CHs(8) = CHs(9) > H(8) > H(2). The methyl peaks of N6-methyladenine, l-methyladenine and N6-methyladenosine were overlapped by the protein signals in the aliphatic region. For g-ethyl9Nmethyladenine the relative broadening of the aliphatic protons attached to the imidazole ring was greater than in other ligands having methyl in the pyrimidine ring. It is apparent that the long-chain derivative (8-ethyl-9Nmethyladenine) undergoes greater broadening than the other ligands.
4. Discussion The relaxation time and the width of the NMR spectral line depends on the motion of the molecule which gives rise to the line. If the motion of the molecule is restricted, the relaxation time of the nuclei is shortened and the width of its NMR spectral lines is increased. For adenine the increase in line width of the pyrimidine ring proton H(2) with increased BSA concentration is more limited than for the imidazole ring proton H(8) (Table 1).
29
The results presented above demonstrate that the aromatic ring of the purine bases and their nucleosides are able to interact with serum albumin in ligandserum albumin complexes. The relative broadening coefficient order: XCH3 > Xn(s) > Xn(z), can be explained by the higher tendency of an aliphatic group (CH,) and imidazole ring (H(8)) than other protons of of adenine and adenosine to associate with macromolecule. From the unchanged values of the chemical shifts (Table 2) one can conclude that after formation of the adenine derivative-BSA complexes no changes in intermolecular distance occurred. The BSA polypeptide chain contains approximately 600 amino acid residues, and the loop region of each subdomain contains three helical segments, about 30% of the surface being hydrophobic. In our experiment about 0.2-2 adenine derivative molecules fall to each amino acid side-chain of the protein. The phenol ring of tyrosine is able to form stacked complexes with adenine bases in singlestranded poly A, but in Lys-Tyr-Lys-DNA complexes tyrosine does not intercalate between the base pairs [3]. This suggests the possibility of competition between different types of binding during association. Considering the marked and selective broadening of the NMR lines of the purine aliphatic groups of 8ethyl-9N-methyladenine in the presence of BSA we can assume that they reflect the existence of weak hydrophobic association between ethyl or/and methyl groups of the ligand and the hydrophobic grooves of albumin. The results indicate the involvement of the aliphatic chain of the adenine derivatives as the primary binding site, and the imidazole ring of the ligands as the secondary binding site. The quantitative pattern of relative line broadening suggests that the length of the aliphatic chain of the purine derivatives plays a particular role in the interaction with albumin.
References [I] A. Sulkowska, B. Lubas and T. Wilczok, Radiat. Res., 85 (1981) 1. [2] A. Sulkowska and A. Miehnik, J. Mol. Struct., 348 (1995) 73. [3] Y. Ohta, T. Tanaka, Y. Baba and A. Kagemoto, J. Phys. Chem., 90 (1986) 4438.