| dc.description.abstract | The present work has its origin in a recent report describing a new reaction of o-benzoquinone (oBQ) with the thioether group of methionine. The reactions of o-quinones, in general, with proteins have great biological relevance, but the chemistry of these reactions is rather complex and an integrated picture is difficult to obtain. Some of the functional groups that are known to be involved in these reactions are the amino, imino, and sulfhydryl groups. All these reactions take place at a fast rate. The need to evaluate the importance of this new reaction was felt and that has resulted in the work which has been presented in this thesis.
The thesis concerns itself with:
Section I:
The reaction of o-benzoquinone with methionine, which has already been described, has been investigated further.
Section II:
Exploiting this reaction for the chemical modification of biomolecules of which methionine is a constituent. A well-characterized enzyme, RNase A, has been chosen for this purpose.
Section III:
Investigating whether this reaction is a general reaction of o-quinones.
Thus, the first two sections involved oBQ. The third section involved some biologically important o-quinones, namely the o-quinones of DOPA, adrenaline, noradrenaline, and homoprotocatechuic acid.
Crystalline oBQ was prepared by the oxidation of catechol. Freshly prepared oBQ, obtained as bright red crystals, was found to be stable in a suspension of anhydrous ether at 0 °C for more than four hours. The quinone was found to have characteristic absorption spectra in solvents such as ether, chloroform, hexane, and glacial acetic acid, with absorption maxima in the region of 380 nm. From the constancy of the absorption spectra with time, the quinone was found to be relatively stable at room temperature in those solvents.
The quinone was less stable in aqueous solvents as well as in solvents like alcohol and formic acid. In aqueous solutions at pH below 2, the quinone was stable for periods up to 10 minutes. Above this pH, the rate of decomposition increased progressively with pH. In view of this, all modification studies have been confined to acidic pH.
Some spectral studies have been carried out with o-benzoquinone and its N-acetyl methionine (NAM) derivative to define their structure in further detail. In acidic solutions, the oBQ derivative of NAM was found to have an absorption spectrum with max at 250 nm and 237 nm, the molar extinction coefficients being 7.57 × 10³ and 1.14 × 10 , respectively. At pH above 6.0, spectral changes characteristic of phenolic ionization were observed. These changes were found to be of two kinds:
A reversible ionization of a phenolic group with pK 6.9
An irreversible ionization above pH 9.0
An NMR spectrum of this derivative was recorded in deuterated glacial acetic acid and was found to be in agreement with the proposed structure, especially with reference to the position of the substituent.
Infrared spectra of o-benzoquinone were recorded in chloroform solution. It shows a doublet in the 1700 cm ¹ region. One of these is presumably due to olefinic group frequency, since o-quinones do not show full resonance. NMR spectra of o-benzoquinone were recorded in both deuterated glacial acetic acid and chloroform. Both spectra showed two multiplets characteristic of an AA BB case, a compound containing two pairs of magnetically nonequivalent protons. The electron density distribution is therefore asymmetrical in the ring.
o-Benzoquinone specifically reacts with methionine residues of the enzyme RNase A at acidic pH. Modified enzymes show characteristic spectral changes, especially around 250 nm. Difference spectral studies have been designed to calculate the rate constant of the reaction of o-benzoquinone with the enzyme. It was found that the reaction is a second-order reaction with a rate constant of the order of 10² M ¹ min ¹.
Thus, compared to the rate of free methionine with o-benzoquinone in aqueous acetic acid, which is 187 M ¹ min ¹, the reaction with methionine residues in RNase A is slower. This difference reflects the limited accessibility of methionine residues in the enzyme conformation.
It was observed that at pH 3, RNase A does not react with o-benzoquinone. Since free methionine reacts with o-benzoquinone even at pH 3, the non-reactivity of the methionine residues of the enzyme at this pH suggests that the side chains of these residues are not exposed to the solvent, and hence are not available to the reagent. Thus, there is a distinct change in the conformation of the enzyme around pH 3.
However, it was found that RNase S, S-protein, and S-peptide all react with o-benzoquinone at pH 3. The reactivity of the methionine residues of RNase S at pH 3 could be attributed either to the dissociation of RNase S into S-protein and S-peptide, both of which are reactive toward the quinone, or to the presence of one or more methionine residues in undissociated RNase S in an environment favorable for reaction.
It was also observed that partially modified RNase derivatives obtained by reaction with o-benzoquinone at pH 1 further react with oBQ at pH 3. Thus, modification of one or more methionine residues introduces conformational changes in the enzyme molecule, which become evident in the availability of methionine residues at pH 3 for reaction.
This observation provides an excellent method for determining how the conformation of RNase A is influenced upon reaction with o-benzoquinone at different methionine residues. However, a prerequisite for such studies is the availability of well-defined individual derivatives.
To obtain various modified derivatives, the enzyme was reacted with o-benzoquinone at pH 1.0. At high concentrations of oBQ, all the methionine residues of the enzyme react to form their sulfonium derivatives. Amino acid analysis of this derivative shows the absence of methionine residues. Moving boundary electrophoresis shows that this derivative has additional positive charge compared to RNase A. The derivative was found to be completely inactive.
A partially modified derivative preparation was obtained by limiting the concentration of o-benzoquinone. Attempts were made to separate the derivative(s) from unreacted RNase A on Amberlite (X2-64) columns using phosphate buffer at pH 6.3. However, the derivative was unstable at this pH and lyophilized preparations were pink in color.
Therefore, separation procedures involving lower pH were used. It was found that the derivatives separate from unreacted RNase A on Sephadex G-50 columns. A partially modified derivative was obtained using this procedure. Spectral titration showed that the phenolic group of the introduced chromophore ionizes at a lower pH compared with the phenolic group of the oBQ-NAM derivative, probably due to the different environments of the chromophore.
The freshly isolated derivative was found to be about 20 % active, but it lost activity on storage. It showed further increase in activity with S-protein, indicating that the added S-protein can combine with the S-peptide of the derivative, and the S-protein portion of the derivative is modified.
Amino acid analysis of these derivatives has been carried out, and some preliminary attempts have been made to locate the site of modification.
The other o-quinones chosen for reaction with methionine were the o-quinones of DOPA, adrenaline, noradrenaline, and homoprotocatechuic acid. These o-quinones have never been isolated previously. Conventional methods were attempted to obtain them but were unsuccessful.
However, it was found that these o-quinones are produced as transient intermediates when the respective catechols are oxidized with ceric sulfate in dilute sulfuric acid. The oxidations were carried out in the presence of NAM or methionine to trap these quinones in the form of derivatives.
A procedure was designed to isolate these derivatives from the reaction mixture. The UV spectra of all these derivatives were found to be similar to the oBQ-NAM derivative spectrum.
The derivative of dopaquinone was studied in detail. Both alkaline and acid hydrolysis gave methionine, and analysis showed that one molecule of o-quinone reacts with one molecule of methionine
. Electrophoresis of the NAM derivative at pH 2 did not show the expected increased mobility due to positive charge on the sulfonium group. However, the derivative obtained with N-acetyl methionine amide showed increased mobility. Thus, the free carboxyl group of methionine may interact with other parts of the molecule.
Spectral titration of the derivative showed that the phenolic group ionizes at a lower pH than the pKa value of the oBQ-NAM derivative.
On the basis of these studies, the following structure can be tentatively assigned to this derivative.
From the similarity in their spectra, it is evident that the o-quinones of adrenaline, noradrenaline, and homoprotocatechuic acid also react with methionine to give similar products.
In conclusion, the work indicates that o-quinones can influence both the structure and function of biomolecules by reacting with their methionine residues. It is shown that this reaction is a general reaction of o-quinones, since all the o-quinones studied reacted in a similar fashion.
It is therefore likely that this reaction plays an important role in various biological processes involving reactions between o-quinones and proteins. | |
