Jean-Luc Dimicoli

INTERACTION OF OLIGOPEPTIDES CONTAINING AROMATIC AMINO ACIDS WITH NUCLEIC ACIDS. FLUORESCENCE AND PROTON MAGNETIC RESONANCE STUDIES

1. introduction A study of interactions between amino acids and nucleic acid bases is required to explain the nature and specificity of recognition of nucleic acids by enzymes and proteins and to shed some light on the origin of the genetic code. We previously reported that aromatic amino acids could form complexes with nucleic acid bases either in frozen aqueous solutions [ 1,2] or in concentrated fluid solutions [3]. More recently, evi- dence has been provided for the interaction of nucleo- tides with immobilized amino acids [4]. A study of complex formation between tyramine [2] or trypt- amine [5] and nucleic acids in single-stranded or double-stranded conformations demonstrated a direct interaction of the phenol or indole rings with the purine and pyrimidine bases. Proton magnetic reso- nanee studies provided evidence for intercalation of the indole ring between thebases of single-stranded poly A [2, 5,6] and of double-stranded DNA [7]. This led us to propose that aromatic amino acids could be involved in the binding of enzymes or proteins to nucleic acids by direct interaction with the bases [8]. Such a proposal was also made in the case of trypt- ophan upon indirect evidence based upon changes in the denaturation temperature of DNA [9]. A study of the binding of oligopeptides containing aromatic amino acids to nucleic acids was undertaken to provide evi- dence for this direct interaction with the bases. In order to increase the affinity of the oligopeptides for nucleic acids, the aromatic amino acid was linked to lysine res- idues. The techniques of fluorescence and proton magne- tic resonance were used to investigate the binding process. 6 2. Experimental Most of the experimental conditions can be found in earlier publications [2,3,5]. The oligopeptides were purchased either from Schwarz-Mann (Lys Trp Lys, Trp Lys, Lys Tyr Lys) or from Cycle Chemical Corporation (Lys Tyramide, Lys Lys Lys). Calf thy- mus DNA samples used in the PMR experiments were sonicated until the sedimentation coefficient was de- creased to about 7. All measurements were carried out in a buffer containing 1 mM Na cacodylate and 1 mM NaCl at pH 7. The temperature was maintained at 20”. Tryptophan-containing peptides were excited at 295 nm and fluorescence intensity measured at 370 run. For tyrosine-containing peptides, the corres- ponding wavelengths were 275 and 3 10 nm. Proton magnetic resonance experiments were carried out in Dz 0 with a Briiker HFX 90 MHz spectrometer. Chem- ical shifts were measured with respect to an external reference (HMS). 3. Results 3.1.

SELF-ASSOCIATION OF DAUNORUBICIN

with cytoxic and antimitotic activities. Presently it is used in the treatment of acute leukemia [ 1 ] . A considerable body of evidence has been accum- ulated which is in agreement with the existence of an intercalative complex with the DNA [2], which results in inhibition of both enzymatic RNA and DNA synthesis [3,4]. The study of the physico-chemical properties of the DNA-Daunorubicin complex [5] led us to a preliminary study of the behaviour of this molecule in solution. It could be expected that, due to its planar aromatic ring, strong interactions between molecules could exist as already shown for many compounds such as actinomycin [6], proflavine [7], purine derivatives [8-IO] or acridine orange [ 1 l] . This paper reports results of a study on the effects of the antibiotic concentration investigated by various physicochemical methods: ultraviolet and visible ab- sorption, circular dichroism (CD) and nuclear magnetic resonance (NMR). From the comparison between the results yielded by these different methods a model for the self-association of Daunorubicin is proposed.

Interactions of tyrosine and tyramine with nucleic acids and their components. Fluorescence, nuclear magnetic resonance and circular dichroism studies.

Abstract Interactions between tyrosine or tyramine and DNA, poly(A), nucleosides or nucleotides have been investigated by fluorescence, proton magnetic resonance and circular dichroism. 1. 1. In frozen aqueous solutions at 77°K, the fluorescence of tyrosine and tyramine is quenched upon interaction with purine or pyrimidine derivatives. The fluorescence of pyrimidines is much more affected than that of purines. 2. 2. In aqueous solutions at room temperature, the PMR signals of the purine and pyrimidine rings are shifted upfield in the presence of tyramine. The PMR signals of tyramine are also shifted to higher fields in the presence of purine derivatives. These PMR results demonstrate that complex formation in aqueous solutions involves a stacking of the aromatic rings of both molecules. 3. 3. Tyramine interacts with native double-stranded DNA. The fluorescence of tyramine is quenched in the complex and the circular dichroism spectrum of DNA is affected by the binding of tyramine. A binding constant of approx. 10 3 M −1 has been determined from a study of the competition between serotonin and tyramine for binding to DNA. Electrostatic contribution to the binding process is demonstrated by its dependence upon ionic strength. 4. 4. Tyramine binds to single-stranded poly(A) at pH 7. Circular dichroism and PMR studies indicate that the interactions between the adenine rings in poly(A) are decreased upon binding of tyramine. The proton resonance signals of tyramine are shifted upfield in the presence of poly(A), indicating that the phenol ring of tyramine is stacked with the adenine rings of poly(A). These results are discussed with respect to the role that could be played by tyrosine residues in enzymes or proteins in their interaction with nucleic acids. A direct interaction with the bases is suggested and conformational changes of the nucleic acid are expected as a result of such an interaction.

Interactions entre acides aminés et acides nucléiques. III — Etude par absorption et résonance magnétique nucléaire de la formation de complexes entre le tryptophane et les constituants des acides nucléiques(*)

Summary Absorption and proton magnetic resonance studies show that tryptophan (and other indole derivatives) can form molecular complexes with bases, nucleosides or nucleotides in aqueous solutions. In most cases these complexes have a 1:1 stoichiometry and absorb at longer wavelengths than the two components. This new absorption is ascribed to electron transfer from the indole ring of tryptophan to the purine or the pyrimidine ring of the nucleic acid component. Pyrimidines are found to be better electron acceptors than purines and protonated derivatives better acceptors than the corresponding unprotonated species. The upfield shifts of the purine and pyrimidine protons due to complex formation with tryptophan indicate that the aromatic rings are stacked parallel to each other in the complex. Apparent association constants as well as enthalpy and entropy variations have been determined from both absorbance and proton magnetic resonance measurements. Values obtained under different experimental conditions are dicussed with respect to self-association of tryptophan molecules and contribution from complexes of different stoichiometries. The results obtained in fluid solutions are compared to those previously obtained in aggregates formed in frozen aqueous mixtures.

Hydrogen bonding of amino acid side chains to nucleic acid bases

of tbe s~ci~~~ty of ~~~r~ct~ons b&tweeri nucieic acids and proteins is a Fur&mental problem of motecuia~ bioiogy. Many sttadihs have been made on the ~~~ctro~~t~~ ~t~ra~t~o~ ~~~w~~~ basic ~ly~~~t~d~s and iiucleic acids [I, 2) _ More recently, results on the forniation of sta&d cdmpkxes between aromatic amino acids and ~o~y~uc~e~t~de bases have beefi reported [3-41. Anqther type of force might be of imgortarke in .nucIeic acid-protege ~tera~t~o~s, narqe!y _ hydrogen bonding_ Study of this type of force, on model compounds, is very difficult in aqueous sclutions duct to competition of hydrogen bond&g to the sokent. However, nucleic acid derivatives have been recently shown to form hydrogen bonds in concentrated aqueous scllutbns .[?I . ~re~im~~ary i~v~sti~t~o~ on the exisierke of such interiactions can be ma& in oe~anic solvents. ~~is,m~t~od has been s~ce~ssf~~y a~~~jed-t~ the study cf specifiic pairing of nucleic acid bases either by infraired (IQ spectroscogy [X-l i ] or by proton magnetic resonmce ~~~~~ [ 12-t s]l We wish to repart preliminary results on the interactions between derivatives of adenine or ttracg, and compounds bearing chemical functions idenltical to several amino acid side chains. The experiments are performed i?x cbkxofonm sok6tiQns aEd by~ro~~~-bo~~ ZLXCia~on is investigated by llip and BMR SpectroscopieS. 9~~t~y~ad~~i~e (A) and L -cydohexyfuracif { U) were obtained from Cyclochemical Co_, Los Angeles, axed used without further purification_ Chloroform ws ~st~~ed after being dried over GIC~~, and then passed thzoragh an aturnina gel column 20 cm long. p-Cresol, indole, and imidaxole were pwified by subknation. Bntyric acid was distifled over CaCiz _ ~~etbyl~~a~id~ne was prepared from its hydrochloride by addition of ~NaOHH ina ethanol and &led under vacuum wirh P,O, . -Infra-red spectra were recorded with a Beckman I& I2 double beam spectrophotometer, using A@ cells. Proton magnetic resonance spectra were obtained with a Briiker HFX 90 MHz spectrometer. TMS was used as an intend reference.

Nucleic acids-protein interactions. Conformational changes induced by the binding of aromatic amines to polyadenylic acid

The binding of Tryptamine, Serotonine, Phenylethylamine and Histamine to poly(A) in its single stranded form at pH 7 leads to a decrease of its circular dichroism (C.D) amplitude without any appreciable alteration of the shape of the C.D. spectrum. The magnitude of the effect depends on the size of the aromatic ring and decreases in the order : tryptamine greater than tyramine greater than phenylethylamine greather than histamine. A method is described which allows the calculation of association constants from C.D. data. The C.D. amplitude decreases linearly with concentration of bound molecules. Binding of aromatic amines to poly(A) leads to a change in the proton chemical shifts of both the amine and the poly(A) protons. Quantitative analysis of P.M.R. data demonstrates that the shifts of poly(A) protons are linearly related to the concentration of bound molecules.

NMR and enzymatic investigation of the interaction between elastase and sodium trifluoroacetate

At pH 5.5, sodium trifluoroacetate is a potent competitive inhibitor of porcine elastase (Ki = 2.6 mM) and human leukocyte elastase (Ki = 9.3 mM). For both enzymes the Ki increases strongly with pH. Sodium fluoride is inactive on pancreatic elastase and sodium acetate is a weak inhibitor of this enzyme. Trifluoroethanol inhibits both enzymes but is less active than trifluoroacetate in acidic pH conditions. Bovine trypsin and alpha-chymotrypsin are resistant to the action of sodium trifluoroacetate and trifluoroethanol. The interaction between sodium trifluoroacetate and pancreatic elastase is also demonstrated by 19F NMR spectroscopy. Trifluoroacetyltrialanine is able to displace trifluoroacetate from its complex with pancreatic elastase. In addition, a method using turkey ovomucoid for the active site titration of leukocyte and pancreatic elastase is described.