Modification of arginine and lysine in proteins with 2,4-pentanedione.

Primary amines react with 2,4-pentanedione at pH 6-9 to form enamines, N-alkyl-4-amino-3-penten-2-ones. The latter compounds readily regenerate the primary amine at low pH or on treatment with hydroxylamine. Guanidine and substituted guanidines react with 2,4-pentanedione to form N-substituted 2-amino-4,6-dimethylpyrimidines at a rate which is lower by at least a factor of 20 than the rate of reaction of 2,4-pentanedione with primary amines. Selective modification of lysine and arginine side chains in proteins can readily be achieved with 2,4-pentanedione. Modification of lysine is favored by reaction at pH 7 or for short reaction times at pH 9. Selective modification of arginine is achieved by reaction with 2,4-pentanedione for long times at pH 9, followed by treatment of the protein with hydroxylamine. The extent of modification of lysine and arginine side chains can readily be measured spectrophotometrically. Modification of lysozyme with 2,4-pentanedione at pH 7 results in modification of 3.8 lysine residues and less than 0.4 arginine residue in 24 hr. Modification of lysozyme with 2,4-pentanedione at pH 9 results in modification of 4 lysine residues and 4.5 arginine residues in 100 hr. Treatment of this modified protein with hydroxylamine regenerated the modified lysine residues but caused no change in the modified arginine residues. One arginine residue seems to be essential for the catalytic activity of the enzyme.     

Synthesis and application of chemically reactive proteins by the reversible modification of protein amino groups with exo-cis-3,6-endo-epoxy-4,5-cis-epoxyhexahydrophthalic anhydride.

The reversible reaction of exo-cis-3,6-endo-epoxy-4,5-cis-epoxyhexahydrophthalic anhydride (EEHPA) with free protein amino groups is described. The free protein amino groups of lysozyme can be completely blocked through the reaction of the anhydride EEHPA. The chemically less reactive epoxy groups in EEHPA-modified lysozyme remain intact during modification of the protein and can be used for many subsequent chemical reactions. Hydrolysis of the modified inactive lysozyme at pH 2.5 results in deblocking and almost complete recovery of the enzymic activity of the protein. The epoxy groups in EEHPA-modified proteins have a great many potential uses: disaggregation of supramolecular structures, conversion of hydrophobic membrane proteins or tryptic peptides into water-soluble coloured proteins or peptides, inhibition of tryptic cleavage at lysine residues, synthesis of chemically reactive proteins or enzymes for affinity chromatography or immobilized-enzyme technology, two-dimensional separation techniques for complex protein mixtures, detection of specific protein-binding sites for organic substrates or tumour diagnostics, synthesis of defined artificial glycoproteins for biophysical and cytochemical studies and chemical synthesis of radioactively labelled proteins.     

Effect of alkylation with different sized substituents on thermal stability of lysozyme.

The amino groups of hen egg white lysozyme were reductively alkylated by the reaction with aliphatic aldehydes of various chain lengths and with two aldehydes of different steric hindrance at pH 7.5 and 4 degrees for 3 h. About four of the original six lysine residues were modified by the reaction with acetaldehyde, n-butylaldehyde or n-hexylaldehyde. About three lysine residues were 2,2-dimethylpropylated with trimethylacetaldehyde while a single residue was modified with benzaldehyde. The thermal stabilities of these alkylated lysozymes were investigated by differential scanning calorimetry (DSC) at different acidic pH values. Alkylation thermally destabilized the proteins, depending not only on the extent of modification but also on the size of the substituent. The alkylated derivatives were 8-19 kJ/mol less stable than native lysozyme at 25 degrees and pH 3.0. The temperature dependences of the activities of the alkylated lysozymes against ethylene glycol chitin indicated that the orders of the optimum temperatures and the maximum activities were exactly the same as the order of the thermal stabilities.     

On the reaction of papain and succinylpapin with diazo-1-H-tetrazole.

1. The reaction of papain and succinylpapain with diazo-1-H-tetrazole was investigated under different conditions. The extent of modification of the amino acids histidine, tyrosine, tryptophan and lysine was determined spectrophotometrically and/or by amino acid analysis. 2. Only one of the two histidine residues present in the enzyme reacts with diazo-1-H-tetrazole forming a monoazo derivative. The pH dependence of the coupling reaction reveals a normal pK of this reactive histidine. There are several arguments suggesting that this may be histidine 159 near the essential SH-group of papain. 3. All five tryptophan residues of the protein react with the diazonium ion below pH 7 forming a monoazo derivative with an absorption maximum at 370 nm, above pH 7 only four residues couple with diazo-1-H-tetrazole. The reaction of one tryptophan and one histidine are correlated as can be concluded from the pH dependence of the coupling rate of both amino acids and the parallel impairment of the catalytic acitivity. 4. 10-11 tyrosine residues out of 19 react with diazo-1-H-tetrazole to give bisazo compounds. 5 residues involved in hydrogen bridges form monoazo compounds. Only 12 tyrosines can be acylated by acetylimidazole. A relationship between the extent of modification of tyrosine and the activity of the enzyme could not be found.     

Chemical modification of buried lysine residues of bovine serum albumin and its influence on protein conformation and bilirubin binding.

Using a double modification technique about 20% of the lysine residues of bovine serum albumin (BSA) which are not easily accessible in the native protein have been modified. The technique involved approximately 80% modification of lysine residues of BSA with citraconic anhydride followed by chemical modification of the remaining lysine residues with acetic anhydride, succinic anhydride, potassium cyanate, or O-methylisourea. Finally, these preparations were decitraconylated under mild acidic conditions to yield acetylated, succinylated, carbomylated or guanidinated BSA. All of these preparations were found to be homogeneous with respect to charge and size. The spectral, hydrodynamic and bilirubin binding properties of these preparations are described. In contrast to most of the highly modified proteins these preparations with the exception of succinylated BSA are very similar to native BSA in their spectral and hydrodynamic properties. However, the equilibrium association constant (Ka) with bilirubin measured by fluorescence quenching was decreased by about 100-fold in acetylated, carbamylated and succinylated BSA, but only 3-fold in guanidinated BSA. Since conformationally acetylated and carbamylated BSAs are identical to guanidinated BSA we conclude that the decrease in Ka in these preparations is solely due to loss of positive charge on 'critical' lysine residues. The results support a binding model for BSA in which bilirubin binding site is buried and the protein undergoes a series of relaxational changes in conformation upon interaction with bilirubin.     

The reaction of protein amino groups with methyl 5-iodopyridine-2-carboximidate. A possible general method of preparing isomorphous heavy-atom derivatives of proteins

1. The synthesis of methyl 5-iodopyridine-2-carboximidate and its reaction with amino groups of model compounds and performic acid-oxidized insulin are described. The reagent was designed to introduce heavy atoms into specific sites in proteins. 2. Specific reaction with the amino groups of oxidized insulin can be achieved under reasonably mild conditions giving rise to the corresponding N-monosubstituted amidines. 3. The extent of reaction of this reagent with protein amino groups can be readily determined by difference spectroscopy. Modification of lysine residues inhibits tryptic cleavage at such residues, and this can be of assistance in establishing the site of modification in the primary structure. 4. Evidence is presented to show that methyl 5-iodopyridine-2-carboximidate can react specifically, at pH5.0, with the aromatic amino group of 3-amino-l-tyrosine; the final product of this reaction is a 2-arylbenzoxazole. 5. The use of this reagent as a general method for preparing heavy-atom isomorphous derivatives of proteins is discussed.     

Unravelling the mechanism of the interactions of oxazolidine A and E with collagens in ovine skin

Cross-linking agents play an important part in the physical properties of collagen based biomaterials. Oxazolidines are novel aldehydic tanning agents that are widely used to stabilise collagens in the leather industry. The exact mechanism through which they cross-link collagens is, however, not well understood. When they are combined with vegetable tannins, it is thought that oxazolidines form carbocationic intermediates through ring opening, which are then able to interact with the amino acid side chains of collagens and flavonoid ring systems of vegetable tannins. In this study, the interactions of oxazolidines, with collagens, have been investigated using a number of analytical techniques. High pressure liquid chromatography (HPLC) analysis of oxazolidine tanned collagen samples showed that there is an irreversible reaction with tyrosine side chains. Mass spectrometry (MS) revealed the formation of a Schiff's base adduct with lysine residues, which was reversible in nature. MS analysis of reaction of oxazolidines with a model peptide Suc-Ala-Phe-Lys-AMC in presence of NaCNBH(3), indicated the formation of a product with an increase in molecular weight of 28 kD characteristic of the addition of two methyl groups to lysine. Differential scanning calorimetry showed a synergistic effect for combination tannage, with best results being obtained when vegetable tan was added prior to the aldehydic tanning agents. Circular dichroism (CD) studies of collagen in presence of the more reactive oxazolidine A showed that there was a loss in ellipticity simply because of aggregation of collagen molecules rather than a change in the secondary structure. Based on the results obtained, a scheme has been proposed to explain the possible mechanism of action of oxazolidines with the collagen amino acid side chains.     

Peroxidation of lysozyme treated with Cu(II) and hydrogen peroxide

When lysozyme was treated with Cu(II) and H₂O₂ at pH 7.4, the protein underwent polymerization as well as changes in its fluorescent characteristics. Upon prolonged incubation, most of the protein aggregates were degraded into smaller peptides. Amino acid analysis indicated that the basic amino acid residues were most susceptible to the oxidation. Tryptophan residues were converted to N-formylkynurenine and kynurenine, and lysine residues were deaminated to form α-aminoadipic acid δ-semi- aldehyde. During Cu(II)H₂O₂ treatment, the formation of carbonyl groups was accompanied by the loss of free amino groups in the protein. Succinylation of free amino groups protected lysine residues from oxidation by Cu(II)H₂O₂, but failed to prevent polymerization. The studies with the modified lysozyme suggest that Cu(II)H₂O₂ can oxidize various amino acid residues in addition to lysine to generate different types of carbonyl compounds and these carbonyl compounds may be responsible for the formation of crosslinks in the polymerization process.     

A quantitative determination by capillary gas-liquid chromatography of neutral and amino sugars (as O-methyloxime acetates), and a study on hydrolytic conditions for glycoproteins and polysaccharides in order to increase sugar recoveries

Pyridine borane has been reported as a superior reagent over a wide pH range, 5-9, for the reductive methylation of amino groups of proteins with formaldehyde [J. C. Cabacungan , A. I. Ahmed , and R. E. Feeney (1982) Anal. Biochem. 124, 272-278]. It has also been reported to reduce tryptophan to dihydrotryptophan and to inactivate lysozyme in trifluoroacetic acid [M. Kurata , Y. Kikugawa , T. Kuwae , I. Koyama , and T. Takagi (1980) Chem. Pharm . Bull 28, 2274-2275]. In the present study the specificity of pyridine borane for the two different modifications under different reaction conditions has been demonstrated, and extended to the application to the synthesis of protein containing reductively attached carbohydrates. In the acid reduction, pyridine borane selectively reduced all six tryptophans in lysozyme to dihydrotryptophan while all other amino acids remained intact. On similar treatment no cleavage of the carbohydrate moiety from chicken ovomucoid, and no losses of activity of ovomucoid or ribonuclease, two proteins devoid of tryptophan, were observed. Nearly complete methylation of the lysines of lysozyme, chicken ovomucoid, and ribonuclease was achieved with formaldehyde at pH 7.0 after 2 h at room temperature, with the retention of full activity of the protein without any destruction of tryptophan. The same chemistry was applied to covalently attach glucose and lactose to bovine serum albumin. Parameters, including pH, temperature, and methanol, that affect the reactions were investigated. Incremental additions of pyridine borane during the course of the reactions increased the rate of modification. The covalent attachment of sugar to the epsilon-amino group of lysine was demonstrated by the synthesis of N-alpha- acetylglucitollysine and comparison with acid hydrolysates of the bovine serum albumin-sugar derivatives.     

Reactivity of histidine and lysine side-chains with diethylpyrocarbonate -- a method to identify surface exposed residues in proteins

The chemical modification of amino acid side-chains followed by mass spectrometric detection can reveal at least partial information about the 3-D structure of proteins. In this work we tested diethylpyrocarbonate, as a common histidyl modification agent, for this purpose. Appropriate conditions for the reaction and detection of modified amino acids were developed using angiotensin II as a model peptide. We studied the modification of several model proteins with a known spatial arrangement (insulin, cytochrome c, lysozyme and human serum albumin). Our results revealed that the surface accessibility of residues is a necessary, although in itself insufficient, condition for their reactivity; the microenvironment of side-chains and the dynamics of protein structure also affect the ability of residues to react. However the detection of modified residues can be taken as proof of their surface accessibility, and of direct contact with solvent molecules.     

Conversion of lysine to N(epsilon)-(carboxymethyl)lysine increases susceptibility of proteins to metal-catalyzed oxidation.

Metal-catalyzed oxidation (MCO) of proteins leads to the conversion of some amino acid residues to carbonyl derivatives, and may result in loss of protein function. It is well documented that reactions with oxidation products of sugars, lipids, and amino acids can lead to the conversion of some lysine residues of proteins to N(epsilon)-(carboxymethyl)lysine (CML) derivatives, and that this increases their metal binding capacity. Because post-translational modifications that enhance their metal binding capacity should also increase their susceptibility to MCO, we have investigated the effect of lysine carboxymethylation on the oxidation of bovine serum albumin (BSA) by the Fe(3+)/ascorbate system. Introduction of approximately 10 or more mol CML/mol BSA led to increased formation of carbonyls and of the specific oxidation products glutamic and adipic semialdehydes. These results support the view that the generation of CML derivatives on proteins may contribute to the oxidative damage that is associated with aging and a number of age-related diseases.     

Glycation of amino groups in protein. Studies on the specificity of modification of RNase by glucose

Ribonuclease A has been used as a model protein for studying the specificity of glycation of amino groups in protein under physiological conditions (phosphate buffer, pH 7.4, 37 degrees C). Incubation of RNase with glucose led to an enhanced rate of inactivation of the enzyme relative to the rate of modification of lysine residues, suggesting preferential modification of active site lysine residues. Sites of glycation of RNase were identified by amino acid analysis of tryptic peptides isolated by reverse-phase high pressure liquid chromatography and phenylboronate affinity chromatography. Schiff base adducts were trapped with Na-BH3CN and the alpha-amino group of Lys-1 was identified as the primary site (80-90%) of initial Schiff base formation on RNase. In contrast, Lys-41 and Lys-7 in the active site accounted for about 38 and 29%, respectively, of ketoamine adducts formed via the Amadori rearrangement. Other sites reactive in ketoamine formation included N alpha-Lys-1 (15%), N epsilon-Lys-1 (9%), and Lys-37 (9%) which are adjacent to acidic amino acids. The remaining six lysine residues in RNase, which are located on the surface of the protein, were relatively inactive in forming either the Schiff base or Amadori adduct. Both the equilibrium Schiff base concentration and the rate of the Amadori rearrangement at each site were found to be important in determining the specificity of glycation of RNase.     

In-gel derivatization of proteins for cysteine-specific cleavages and their analysis by mass spectrometry

As a potential tool for proteomics and protein characterization, in-gel cysteine- and arginine-specific cleavage is demonstrated by means of trypsin or endoproteinase Lys-C for six model proteins (lysozyme, alpha-lactalbumin, beta-lactoglobulin, ribonuclease A, albumin, and transferrin), ranging in size from 14 kDa to 79 kDa. Chemical modifications of cysteine (aminoethylation with bromoethylamine or N-(iodoethyl)-trifluoroacetamide, and subsequent guanidination) and lysine (acetylation) prior to tryptic digestion releases peptides delineated by cysteine or arginine residues. Peptide products are analyzed by MALDI-TOF-MS, ESI-MS, and ESI- and MALDI-MS/MS (with a quadrupole time-of-flight instrument). Complications induced by acrylamide alkylations of cysteines were avoided by substituting lower pH bis-tris polyacrylamide gels for tris-glycine. Sequence coverages from 35 to 86% were obtained and amino acid compositions of generated peptides could be confirmed by comprehensive y- and b-ion series. Detailed information about, in particular, cysteine rich proteins after gel electrophoresis were obtained. The chemistries for modification and cleavage specificities at cysteine residues provide an alternative means to characterize and identify proteins separated by gel electrophoresis.     

Phosphorylation of yeast protein: Reduction of ribonucleic acid and isolation of yeast protein concentrate

Yeast nucleoproteins were chemically phosphorylated with phosphorus oxychloride (POCL(3)). Studies using (31)P nuclear magnetic resonance (NMR) spectroscopy, stability to pH and lysine estimation all indicated that the epsilon-amino group of lysine was the principal functional group phosphorylated. Phosphorylation of ca. 30% of the lysine residues resulted in removal of more than 85% of contaminant ribonucleic acid from protein precipitated at pH 4.2. Phosphorylation did not alter the amino acid composition of yeast proteins and was reversible under acidic conditions. Based on the data, a method for the preparation of phosphorylated yeast protein with low levels of nucleic acid is proposed.     

Structure-sweetness relationship in egg white lysozyme: role of lysine and arginine residues on the elicitation of lysozyme sweetness

Lysozyme is one of the sweet-tasting proteins. To clarify the structure-sweetness relationship and the basicity-sweetness relationship in lysozyme, we have generated lysozyme mutants with Pichia systems. Alanine substitution of lysine residues demonstrated that two out of six lysine residues, Lys13 and Lys96, are required for lysozyme sweetness, while the remaining four lysine residues do not play a significant role in the perception of sweetness. Arginine substitution of lysine residues revealed that the basicity, but not the shape, of the side chain plays a significant role in sweetness. Single alanine substitutions of arginine residues showed that three arginine residues, Arg14, Arg21, and Arg73, play significant roles in lysozyme sweetness, whereas Arg45, Arg68, Arg125 and chemical modification by 1,2-cyclohexanedione did not affect sweetness. From investigation of the charge-specific mutations, we found that the basicity of a broad surface region formed by five positively charged residues, Lys13, Lys96, Arg14, Arg21, and Arg73, is required for lysozyme sweetness. Differences in the threshold values among sweet-tasting proteins might be caused by the broadness and/or the density of charged residues on the protein surface.     

Mass spectrometric determination of early and advanced glycation in biology

Protein glycation in biological systems occurs predominantly on lysine, arginine and N-terminal residues of proteins. Major quantitative glycation adducts are found at mean extents of modification of 1–5 mol percent of proteins. These are glucose-derived fructosamine on lysine and N-terminal residues of proteins, methylglyoxal-derived hydroimidazolone on arginine residues and N^ε-carboxymethyl-lysine residues mainly formed by the oxidative degradation of fructosamine. Total glycation adducts of different types are quantified by stable isotopic dilution analysis liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring mode. Metabolism of glycated proteins is followed by LC-MS/MS of glycation free adducts as minor components of the amino acid metabolome. Glycated proteins and sites of modification within them – amino acid residues modified by the glycating agent moiety - are identified and quantified by label-free and stable isotope labelling with amino acids in cell culture (SILAC) high resolution mass spectrometry. Sites of glycation by glucose and methylglyoxal in selected proteins are listed. Key issues in applying proteomics techniques to analysis of glycated proteins are: (i) avoiding compromise of analysis by formation, loss and relocation of glycation adducts in pre-analytic processing; (ii) specificity of immunoaffinity enrichment procedures, (iii) maximizing protein sequence coverage in mass spectrometric analysis for detection of glycation sites, and (iv) development of bioinformatics tools for prediction of protein glycation sites. Protein glycation studies have important applications in biology, ageing and translational medicine – particularly on studies of obesity, diabetes, cardiovascular disease, renal failure, neurological disorders and cancer. Mass spectrometric analysis of glycated proteins has yet to find widespread use clinically. Future use in health screening, disease diagnosis and therapeutic monitoring, and drug and functional food development is expected. A protocol for high resolution mass spectrometry proteomics of glycated proteins is given.     

Chemical modifications of ribonuclease U1.

In order to obtain information on the nature of the amino acid residues involved in the activity of ribonuclease U1 [EC 3.1.4.8], various chemical modifications of the enzyme were carried out. RNase U1 was inactivated by reaction with iodoacetate at pH 5.5 with concomitant incorporation of 1 carboxymethyl group per molecule of the enzyme. The residue specifically modified by iodoacetate was identified as one of the glutamic acid residues, as in the case of RNase T1. The enzyme was also inactivated extensively by reaction with iodoacetamide at pH 8.0 with the loss of about one residue each of histidine and lysine. When RNase U1 was treated with a large excess of phenylglyoxal, the enzymatic activity and binding ability toward 3'-GMP were lost, with simultaneous modification of about 1 residue of arginine. The reaction of citraconic anhydride with RNase U1 led to the loss of enzymatic activity and modification of about 1 residue of lysine. The inactivated enzyme, however, retained binding ability toward 3'-GMP. These results indicate that there are marked similarities in the active sites of RNases T1 and U1.     

The buoyant titration of native and carbamylated bovine serum mercaptalbumin.

The buoyant density titration curves of native and carbamylated bovine serum mercaptalbumin were measured throughout the pH range 5.3-12.7. Large increments in the buoyant density were observed above pH 10, with inflection pH values of 11.2 and 11.4 for native and carbamylated bovine serum mercaptalbumin, respectively. For the modified protein in which 25 out of 58 lysine residues were carbamylated, the buoyant densities were 0.048 g/ml higher at neutral pH and 0.024 g/ml higher at the extrapolated pH 13. The carbamyl groups apparently produce a larger residual density at pH 13 than they did in the case of ovalbumin. Homopolymer buoyant density titration data were demonstrated to be of value in calculating the contributions of titratable residues to the buoyant density of both proteins. The buoyant density increment at high pH was due largely to the deprotonation of the lysines as indicated by the diminished change in buoyant density between pH 10 and 12.7 for the modified protein. These density changes were attributable primarily to a gain of cesium ions. The limited modification of the lysine residues under mild reaction conditions and the rather high intrinsic dissociation constant of tyrosine residues in mercaptalbumin may indicate a preferential modification of easily accessible lysine residues. Phenolic deprotonation is facilitated by the neutralization of normally charged lysine residues and demonstrates ionic interactions between internal lysines and certain carboxyl and tyrosine residues thereby stabilizing the native state of the protein.     

Alkaline pH-dependent differential unfolding characteristics of mesophilic and thermophilic homologs of dimeric serine hydroxymethyltransferase

Environmental variables such as pH can significantly influence the folding and stability of a protein molecule. In the present investigation, we compared the alkaline pH-induced unfolding of two homologous serine hydroxymethyltransferase from mesophilic Bacillus subtilis (bsSHMT) and thermophilic Bacillus stearothermophilus (bstSHMT) using various biophysical techniques. The thermophilic enzyme bstSHMT was found to be more resistant to alkaline denaturation compared to its mesophilic counterpart, bsSHMT. Unfolding studies using domain-swapped chimera, constructed by swapping the C-terminal domain of these two wild-type proteins, revealed that C-terminal domain plays a pivotal role in the folding, stability and subunit interaction of these proteins. Primary amino acid sequence analysis of the proteins showed that bsSHMT has six unconserved lysine residues in C-terminal domain, which are absent in bstSHMT. Chemical modification of lysine side chains resulted in stabilization of monomers, only in case of bsSHMT. Moreover, comparison between homology model of bsSHMT with the crystal structure of bstSHMT revealed that a small stretch of 11 amino acids at the end of C-terminal domain was found protruding outside the molecule as a flexible coiled structure in bsSHMT. Taken together these findings suggest that possibly the presence of these non-identical lysine moieties and a small extension of C-terminal domain may be responsible for low stability of bsSHMT under alkaline pH condition.     

π-Cation interactions as the origin of the weak absorption at 532 nm observed in tryptophan-containing polypeptides

We have previously reported that bovine serum albumin (BSA) and other proteins that do not contain prosthetic groups exhibited a weak light absorption in the visible, only detectable by pulsed laser-induced optoacoustic spectroscopy (LIOAS). Human serum albumin (HSA) exhibited signals 25% higher than those observed with BSA. Signals comparable to those obtained with BSA were observed with poly(L-Trp, L-Lys), poly(L-Trp, L-Arg) or poly(L-Trp, L-Orn) at pH 7.0. No signals were obtained when tryptophan was replaced by other amino acids or when free tryptophan or the tripeptide Lys-Trp-Lys was assayed (pH 7.0). Tryptophan in HCl 5 N produced LIOAS signals similar to those produced by tryptophan-containing copolymers. Moreover, the absorption peak could be observed in a UV-VIS spectrophotometer. Therefore, the LIOAS signals obtained with BSA, HSA, and tryptophan-containing random copolymers may be attributed to a new transition of the indole moiety of their tryptophan residues when "protonated". Tryptophan residues of proteins are known to participate in π-cation interactions, which are important in protein stability and function. As a matter of fact, HSA and BSA contain an internal tryptophan in close proximity to lysine and arginine residues and therefore suitable for π-cation interactions. The strength of this type of interaction strongly depends on distances and relative orientations of both amino acid residues. Accordingly, these interactions should be highly sensitive to conformational changes. Based on preliminary results that have shown that LIOAS signal at 532 nm depended on the aggregation state of BSA and/or on the oxidation state of its Cys-34, we postulate that the LIOAS signal observed with proteins and tryptophan-containing polypeptides are related to Trp-Lys or Trp-Arg interactions and that the intensity of the signal depends on the strength of such interactions.