Determination of the rates of formation and hydrolysis of the Schiff bases formed by pyridoxal 5′-phosphate and copolypeptides containing l-lysine

We determined the apparent rate constants of formation (k₁) and hydrolysis (k₂) of the Schiff bases formed between pyridoxal 5′-phosphate (PLP) and l-lysine and l-alanine copolymers of different compositions, as well as those formed between PLP and l-lysine and l-glutamic acid copolymers, at various pH values, a temperature of 25 °C and an ionic strength of 0.1 M. The k₁ values obtained in neutral and acidic media were independent of the copolymer composition. The efficiency of the intramolecular acid catalysis for the formation of the Schiff bases was found to be somewhat higher than that of PLP—primary amine systems (the slope of the Brøwted plot was α=0.77). The most stable of the Schiff bases studied was that with a protonated imine nitrogen and phosphate group and a unprotonated pyridine nitrogen.     

A 13C n.m.r. study of the B6 vitamins and of their aldimine derivatives

The vitamins, pyridoxine, pyridoxal, pyridoxamine, pyridoxal-5′-phosphate and pyridoxamine-5′-phosphate, have been studied in aqueous solution over a pH range of 2–12 by ¹³C nuclear magnetic resonance spectroscopy. Resonance assignments are made primarily by the spin–spin coupling constants of carbons with protons and with phosphorus. The proton–carbon coupling constants show a marked conformational dependence in the hemiacetal form of pyridoxal. Furthermore, the H-6? C-5 coupling constant in the vitamins is much smaller than the corresponding constant in pyridine. This may be due either to an effect of the C-5 substituent in vitamins or to a different electronic configuration of the zwitterionic hydroxypyridine ring. The addition of manganese to a solution of pyridoxal phosphate causes line broadenings consistent with the interaction of the metal ion with this vitamin at the formyl and phenolic oxygens. The chemical shifts of the aromatic carbons of pyridoxine have been calculated, as a function of pH, by summing shielding parameters which were estimated empirically from pyridine derivatives. The calculated shifts agree well with the experimental data for C-3, C-5 and C-6, less well for C-2, and poorly for C-4. The deviation from additivity for C-4 indicates a preferred orientation for the 4-hydroxymethyl substituent caused by internal hydrogen bonding between the substituents at C-3 and C-4. Evidence is presented for the existence of the free aldehyde form of pyridoxal at alkaline pH. Aldimine complexes of pyridoxal and pyridoxal phosphate with amines and amino acids have also been studied. Characteristic chemical shift changes caused by both pyridinium and aldimine nitrogen deprotonations are seen. Additionally, the chemical shifts of carbons of the pyridine ring are dependent upon the structure of the imine, especially when the aldimine nitrogen is protonated. We conclude that this dependency is due to steric effects in an aldimine complex which is constrained by internal hydrogen bonding. We also discuss the merits of carbons 3 and 4 as possible sites of cofactor labeling for enzymatic studies.     

Rapid photodynamics of vitamin B6 coenzyme pyridoxal 5'-phosphate and its Schiff bases in solution

The active form of vitamin B6, pyridoxal 5'-phosphate (PLP), is an important cofactor for numerous enzymes in amine and amino acid metabolism. Presented here is the first femtosecond transient absorption study of free PLP and two Schiff bases, PLP-valine and PLP-alpha-aminoisobutyric acid (AIB), in solution. Photoexcitation of free PLP leads to efficient triplet formation with an internal conversion rate that increases with increasing pH. The measured excited-state kinetics of the PLP-valine Schiff base exhibits a dramatic deuterium dependence as a result of excited-state proton transfer (ESPT) of the Calpha hydrogen in the amino acid substrate. This is consistent with formation of the key reaction carbanionic intermediate (quinonoid), which is resonance stabilized by the electron-deficient, conjugated pi system of the Schiff base/pyridine ring. The transient absorption signals of the PLP-Schiff base with alpha-methylalanine (2-aminoisobutyric acid), which does not have a Calpha proton, lack an observable deuterium effect, verifying ESPT formation of the quinonoid intermediate. In contrast to previous studies, no dependence on the excitation wavelength of the femtosecond kinetics is observed with PLP or PLP-valine, which suggests that a rapid (<250 fs) tautomerization occurs between the enolimine (absorbing at 330 nm) and ketoenamine (absorbing at 410 nm) tautomers in solution.     

Role of the pyridine nitrogen in pyridoxal 5'-phosphate catalysis: activity of three classes of PLP enzymes reconstituted with deazapyridoxal 5'-phosphate

Pyridoxal 5'-phosphate (PLP; vitamin B(6))-catalyzed reactions have been well studied, both on enzymes and in solution, due to the variety of important reactions this cofactor catalyzes in nitrogen metabolism. Three functional groups are central to PLP catalysis: the C4' aldehyde, the O3' phenol, and the N1 pyridine nitrogen. In the literature, the pyridine nitrogen has traditionally been assumed to be protonated in enzyme active sites, with the protonated pyridine ring providing resonance stabilization of carbanionic intermediates. This assumption is certainly correct for some PLP enzymes, but the structures of other active sites are incompatible with protonation of N1, and, consequently, these enzymes are expected to use PLP in the N1-unprotonated form. For example, aspartate aminotransferase protonates the pyridine nitrogen for catalysis of transamination, while both alanine racemase and O-acetylserine sulfhydrylase are expected to maintain N1 in the unprotonated, formally neutral state for catalysis of racemization and β-elimination. Herein, kinetic results for these three enzymes reconstituted with 1-deazapyridoxal 5'-phosphate, an isosteric analogue of PLP lacking the pyridine nitrogen, are compared to those for the PLP enzyme forms. They demonstrate that the pyridine nitrogen is vital to the 1,3-prototropic shift central to transamination, but not to reactions catalyzed by alanine racemase or O-acetylserine sulfhydrylase. Not all PLP enzymes require the electrophilicity of a protonated pyridine ring to enable formation of carbanionic intermediates. It is proposed that modulation of cofactor electrophilicity plays a central role in controlling reaction specificity in PLP enzymes.     

A POSSIBLE MECHANISM FOR THE ROLE OF N-RETINYLIDENE PHOSPHATIDYL ETHANOLAMINE IN THE REGENERATION OF RHODOPSIN

Abstract— A series of molecular orbital calculations on a model Schiff base comparable to protonated N -retinylidene phosphatidyl ethanolamine isomers has been made. The effect of the charged oxygen atoms of the phosphate moiety on the distribution of positive charge along the polyene chain of these isomers has been calculated. The stabilizing coulombic energy of interaction of these opposite charges and the possibility of free rotation around carbonxarbon double bonds in the electronically excited state has led to the conclusion that an 11- cis Schiff base isomer is the most probable product of the photoisomerization of an all-trans Schiff base.
The formation of a stable unprotonated all-trans Schiff base in aqueous detergent dispersion and its subsequent conversion to the protonated form, both with absorption spectra in conformity with the literature, is demonstrated.     

Absorption studies of neutral retinal Schiff base chromophores

The neutral retinal Schiff base is connected to opsin in UV sensing pigments and in the blue-shifted meta-II signaling state of the rhodopsin photocycle. We have designed and synthesized two model systems for this neutral chromophore and have measured their gas-phase absorption spectra in the electrostatic storage ring ELISA with a photofragmentation technique. By comparison to the absorption spectrum of the protonated retinal Schiff base in vacuo, we found that the blue shift caused by deprotonation of the Schiff base is more than 200 nm. The absorption properties of the UV absorbing proteins are thus largely determined by the intrinsic properties of the chromophore. The effect of approaching a positive charge to the Schiff base was also studied, as well as the susceptibility of the protonated and unprotonated chromophores to experience spectral shifts in different solvents.     

Photophysical and Photochemical Studies of Pyridoxamine

The absorption and fluorescence emission of pyridoxamine were studied as function of pH and solvent properties. In the ground state, pyridoxamine exhibits different protonated forms in the range of pH 1.5–12. Fluorescence studies showed that the same species exist at the lowest singlet excited state but at different pH ranges. The phenol group is by ca. 8 units more acidic in the excited state than in the ground state. On the other hand, the pyridine N‐atom is slightly more basic in the lowest excited state than in the ground state. Excitation spectra and emission decays in the pH range of 8–10 indicate the protonation of the pyridine N‐atom by proton transfer from the amine group, in the ground and singlet excited states. Spectroscopic studies in different solvents showed that pyridoxamine in the ground or excited states exhibits intramolecular proton transfer from the pyridine N‐atom to the phenol group, which is more favorable in solvents of low hydrogen‐bonding capacity. The cationic form with the protonated phenolic group, which emits at shorter wavelength, is the dominant species in nonprotic solvents, but, in strong proton‐donor solvents, both forms exist. The fluorescence spectra of these species exhibit blue shift in protic solvents. These shifts are well‐correlated with the polarity and the H‐donor ability of the solvent.     

Photophysical Study of Pyridoxal 5'-Phosphate and Its Schiff Base with n-Hexylamine

Abstract— Phosphopyridoxal enzymes exhibit a wide variety of reactions. The activation mechanism proceeds with the formation of Schiff bases that undergo loss or transfer of one or more protons. Changes in the fluorescent properties of the pyridoxal 5'-phosphate and its Schiff base may signal changes in the protonation state and/or in the microenvironment of the cofactor. In this paper the average fluorescence lifetime of pyridoxal 5'-phosphate (PLP) as a function of pH was studied in a medium of 0.1 M ionic strength at 25°C. The ionic species of PLP with an unprotonated hydrogen atom were found to exhibit fluorescence lifetimes in the region of 100 ps on excitation at λ= 390 nm. The average fluorescence lifetimes for PLP in its Schiff bases with n-hexylamine in media of low polarity are reported.     

Kinetic and mechanism of reactions of L-α-Glutamic acid and L-Glutamine with pyridoxal

The kinetics and mechanisms of condensation of pyridoxal with L-α-glutamic acid and L-glutamine were studied by UV spectroscopy and polarimetry. L-α-Glutamic acid reacts with pyridoxal to form a Schiff base whose subsequent hydrolysis gives rise to pyridoxamine and α-ketoglutaric acid. The reaction of Lglutamine with pyridoxal involves the Γ-NH₂ group and affords a Schiff base whose subsequent hydrolysis gives rise to pyridoxamine and L-α-glutamic acid.     

A fluorescent molecular sensor for pH windows in traditional and polymeric biocompatible micelles: comicellization of anionic species to shift and reshape the ON window

A new approach is presented to obtain fluorescent sensors for pH windows that work in water and under biomimetic conditions. A single molecule that features all-covalently linked components is used, thus making it capable of working as a fluorescent sensor with an OFF/ON/OFF response to pH value. The components are a tertiary amine, a pyridine, and a fluorophore (pyrene). The forms with both protonated bases or both neutral bases quench the pyrene fluorescence, whereas the form with the neutral pyridine and protonated amine groups is fluorescent. The molecular sensor is also equipped with a long alkyl chain to make it highly hydrophobic in all its protonated and unprotonated forms, that is, either when neutral or charged. Accordingly, it can be confined at any pH value either in traditional (i.e., low-molecular-weight) nonionic surfactant micelles or inside polymeric, biocompatible micellar containers. Relevant for future applications in vivo, thanks to its strong hydrophobicity, no leakage of the molecular sensor is observed from the polymeric biocompatible micelles. Due to the proximity of the pyridine and amine functions in the molecular structure and the poor hydration inside the micelles, the observed pK(a) values are low so that the ON window is positioned at very low pH values. However, the window can be shifted to biologically relevant values by comicellization of anionic species. In particular, in the micelles of the nonionic surfactant TritonX-100, a shift of the ON window to pH 4-6 is obtained by addition of the anionic sodium dodecyl sulphate surfactant, whose negative charge promotes the stability of the protonated forms of the pyridine and amine fragments. In the case of the polymeric micelles, we introduce the use of the amphiphilic polystyrene sulfonate anionic polyelectrolyte, the comicellization of which induces a shift and sharpening of the ON window that is centered at pH 4.     

Release of enzyme strain during catalysis reduces the activation energy barrier

Several mechanisms have been considered as principal factors in enhancing the catalytic reaction velocity of enzymes: approximation, covalent catalysis, general acid-based catalysis, and strain. Among them, the strain on the substrate and/or the enzyme is often found to be brought about on association of the substrate and the enzyme. If this strain is released in the transition state, it contributes to enhancing the k(cat) value, although it does not change the k(cat)/K(m) value. In aspartate aminotransferase, however, we found by analysis of the Schiff base pK(a) values that the unliganded enzyme carries a strain in the protonated Schiff base formed between the coenzyme pyridoxal phosphate and a lysine residue. This bond is cleaved in most of the reaction intermediates, including the transition state. As a result, the activation energy between the free enzyme plus substrate and the transition state is decreased by 16 kJ/mol, equal to the value of the strain energy. The net effect of this strain is enhancement (10(3)-fold) of the catalytic efficiency in terms of k(cat)/K(m), the more important indicator of the catalytic efficiency at low concentration of the substrate.     

RETINYLIDENE-OPSIN SCHIFF BASE CHROMOPHORES AND THEIR ACCESSIBILITY TO WATER

We propose a conceptual model to form the basis of understanding of the retinylidene-opsin Schiff base (SB) chromophores. The first suggestion is that the protonated imine is H-bonded to water, and this complex is similar in H-bonding to a hydronium ion-water dimer (H₅O₅₊). The second proposal is a limited accessibility of water to the SB region of the chromophore. We scrutinize data from anion-sensitive and UV-absorbing visual pigments and find consistency with expectations based on the model: (1) Hydration plays a fundamental role in the photochemistry. (2) Protonation of the imine nitrogen is not obligatory. The latter point is supported by spectroscopic evidence from UV-absorbing visual pigments, in which unprotonated SB chromophores are inferred. Further support is derived from thermodynamic analysis of the temperature dependence of protonation in SB models and in a UV-absorbing visual pigment. We discuss H-bonding opportunities for retinylidene-opsin SB: the strong H-bonds to ions, the weak H-bonds of C-H groups, serving as H-bond donors, and of the π-bonds, in which π-electrons act as H-bond acceptors.     

吡啶团簇的飞秒光电离和从头计算研究

用飞秒激光电离飞行时间质谱研究了吡啶分子团簇在400 nm波长下的多光子光电离,实验观测到一系列的质子化和非质子化团簇离子.结果表明,质子转移也能发生在弱氢键结合的分子间.通过分析离子峰宽和离子信号强度随气源压力的变化,得到质子化团簇离子来源于大团簇离子的碎裂,而非质子化团簇离子是中性团簇直接电离的结果.从头计算结果表明,吡啶团簇是通过弱氢键C-H…N 结合在一起的,并且团簇离子离解倾向于生成质子化产物.     

Reaction mechanisms between methylamine and a few Schiff bases: Ab initio potential energy surfaces of a catalytic step in semicarbazide sensitive amino oxidases (SSAO)

The potential energy surfaces for the transamination reaction catalyzed by SSAO were explored for some of the possible reactants considered in a preliminary investigation (Comput Chem 2000, 24 , 311). The proton transfer to methylamine (as a model of the catalytic base belonging to the enzyme active site)—either from the keto or enol form of the reactant Schiff bases with one of the possible cofactors, pyridoxal phosphate, PLP (using as a model the pyridoxal ring protonated at N)—was investigated. The enol form seems to be preferred in the region of the neutral intermediate, because even the keto form undergoes a spontaneous rearrangement to the enol form once the C_α proton is delivered to methylamine, producing methylammonium. When the proton is returned back to the Schiff base (on C₁), the adduct is about 1.4 kcal/mol more stable than the reactants, while a canonical electron distribution is obtainable only for the enol form. The proton transfer to methylamine was also studied in the presence of the other possible cofactor (para or ortho) topaquinone, TQ. A steep uphill pathway, similar to the keto‐pyridoxal Schiff base one, is obtained using the Schiff base with pTQ, which requires a rearrangement to the final intermediate. On the contrary, using the oTQ structures with the quinonoid O on the same side of methylamine, the proton abstracted from the Schiff base goes spontaneously onto the other quinonoid oxygen. The effect on the barrier heights produced by the presence of a variety of functional groups in the vicinity of the pyridoxal ring nitrogen was also examined. © 2001 John Wiley & Sons, Inc. Int J Quant Chem, 2001     

Light-enhanced catalysis by pyridoxal phosphate-dependent aspartate aminotransferase

The mechanisms of pyridoxal 5'-phosphate (PLP)-dependent enzymes require substrates to form covalent "external aldimine" intermediates, which absorb light strongly between 410 and 430 nm. Aspartate aminotransferase (AAT) is a prototypical PLP-dependent enzyme that catalyzes the reversible interconversion of aspartate and α-ketoglutarate with oxalacetate and glutamate. From kinetic isotope effects studies, it is known that deprotonation of the aspartate external aldimine C(α)-H bond to give a carbanionic quinonoid intermediate is partially rate limiting in the thermal AAT reaction. We show that excitation of the 430-nm external aldimine absorption band increases the steady-state catalytic activity of AAT, which is attributed to the photoenhancement of C(α)-H deprotonation on the basis of studies with Schiff bases in solution. Blue light (250 mW) illumination gives an observed 2.3-fold rate enhancement for WT AAT activity, a 530-fold enhancement for the inactive K258A mutant, and a 58600-fold enhancement for the PLP-Asp Schiff base in water. These different levels of enhancement correlate with the intrinsic reactivities of the C(α)-H bond in the different environments, with the less reactive Schiff bases exhibiting greater enhancement. Time-resolved spectroscopy, ranging from femtoseconds to minutes, was used to investigate the nature of the photoactivation of C(α)-H bond cleavage in PLP-amino acid Schiff bases both in water and bound to AAT. Unlike the thermal pathway, the photoactivation pathway involves a triplet state with a C(α)-H pK(a) that is estimated to be between 11 and 19 units lower than the ground state for the PLP-Val Schiff base in water.     

Chemical transformations of pyridoxal and pyridoxal 5′-phosphate condensation products with amino acids

The mechanism of chemical transformations of pyridoxal and pyridoxal 5′-phosphate condensation products with amino acids is studied by kinetic measurements. The Schiff bases are shown to be fairly stable in neutral media. In acid media, the Schiff bases are hydrolyzed into the initial components. In alkaline media, cleavage of α-hydrogen from the amino acid fragment and structural rearrangement into the quinoid form followed by hydrolysis of the latter with elimination of pyridoxamine and keto acid take place. The rate constants of the chemical transformations of the Schiff bases are found to depend on the pH of the medium. It is shown for the first time that the phosphate group in the pyridoxal 5′-phosphate fragment catalyzes the α-hydrogen cleavage and strongly accelerates alkaline decomposition of the Schiff bases.     

15N nuclear magnetic resonance studies of acid-base properties of pyridoxal-5'-phosphate aldimines in aqueous solution

By use of 15N NMR spectroscopy, we have measured the pKa values of the aldimines 15N-(pyridoxyl-5'-phosphate-idine)-methylamine (2a), N-(pyridoxyl-5'-phosphate-15N-idine)-methylamine (2b), and 15N-(pyridoxyl-idine)-methylamine (3). These aldimines model the cofactor pyridoxal-5'-phosphate (PLP, 1) in a variety of PLP-dependent enzymes. The acid-base properties of the aldimines differ substantially from those of the free cofactor in the aldehyde form 1a or in the hydrated form 1b, which were also investigated using 15N NMR for comparison. All compounds contain three protonation sites, the pyridine ring, the phenol group, and the side chain phosphate (1, 2) or hydroxyl group (3). In agreement with the literature, 1a exhibits one of several pKas at 2.9 and 1b at 4.2. The 15N chemical shifts indicate that the corresponding deprotonation occurs partially in the pyridine and partially in the phenolic site, which compete for the remaining proton. The equilibrium constant of this ring-phenolate tautomerism was measured to be 0.40 for 1a and 0.06 for 1b. The tautomerism is essentially unaltered above pH 6.1, where the phosphate group is deprotonated to the dianion. This means that the pyridine ring is more basic than the phenolate group. Pyridine nitrogen deprotonation occurs at 8.2 for 1a and at 8.7 for 1b. By contrast, above pH 4 the phosphate site of 2 is deprotonated, while the pyridine ring pKa is 5.8. The Schiff base nitrogen does not deprotonate below pH 11.4. When the phosphate group is removed, the pKa of the Schiff base nitrogen decreases to 10.5. The phenol site cannot compete for the proton of the Schiff base nitrogen and is present in the entire pH range as a phenolate, preferentially hydrogen bonded to the solvent. The intrinsic 15N chemical shifts provide information about the hydrogen bond structures of the protonated and unprotonated species involved. Evidence is presented that the intramolecular OHN hydrogen bond of PLP aldimines is broken in aqueous solution. The coupling between the inter- and intramolecular OHN hydrogen bonds is also lost in this environment. The pyridine ring of the PLP aldimines is not protonated in aqueous solution near neutral pH. The basicity of the aldimine nitrogens would be even lower without the doubly negatively charged phosphate group. Protonation of both the Schiff base and pyridine nitrogens has been discussed as a prerequisite for catalytic activity, and the implications of the present findings for PLP catalysis are discussed.     

FLUORESCENCE AND PHOTOCHEMISTRY OF OLIGOCYTIDYLIC AND POLYCYTIDYLIC ACIDS IN AQUEOUS SOLUTION

Abstract —In addition to the monomer-like fluorescence, a long-wavelength emission (Λ^max_em= 410 nm) has been detected in the dinucleoside 5'-5' pyrophosphate (CppC) at room temperature. This emission looks very similar to that previously reported for the acidic forms of Poly C (Poly C. Poly C⁺ and Poly I. Poly C. Poly C⁺). Only the monomer-like emission (Λ^max_em= 330 nm) can be detected in neutral Poly C, acidic CppC, and the neutral or protonated forms of the dinucleoside phosphate CpC.A correlation between the room temperature fluorescence of oligo and polycytidylic acids and their photochemical behaviour is found. Irradiation of all the polymeric samples at both neutral and acid pH results in the formation of minor photoproducts. They have been characterized by their absorbance (in the range 300–400 nm) and their fluorescence spectra. The same product is obtained in all cases where the monomer-like fluorescence only is detected. Distinct products are formed in neutral CppC and in the acidic Poly C forms.
The results are discussed with respect to the conformation of the oligo and polycytidylic acids and possible relationships between the 410–420 nm emission and adduct formation. An excimer is proposed as a common, intermediate excited state in both radiative deactivation and adduct formation in neutral CppC and the acidic Poly C forms.     

A model for enzyme-substrate interaction in alanine racemase.

We report on a theoretical model for the complex of the enzyme alanine racemase with its natural substrate (L-alanine) and cofactor (pyridoxal 5'-phosphate). Electrostatic potentials were calculated and ionization states were predicted for all of the ionizable groups in alanine racemase. Some rather unusual charge states were predicted for certain residues. Tyr265' has an unusually low predicted pK(a) of 7.9 and at pH 7.0 has a predicted average charge of -0.37, meaning that 37% of the Tyr265' residues in an ensemble of enzyme molecules are in the phenolate form. At pH 8-9, the majority of Tyr265' side groups will be in the phenolate form. This lends support to the experimental evidence that Tyr265' is the catalytic base involved in the conversion of L-alanine to D-alanine. Residues Lys39 and Lys129 have predicted average charges of +0.91 and +0.14, respectively, at pH 7.0. Lys39 is believed to be the catalytic base for the conversion of D-alanine to L-alanine, and the present results show that, at least some of the time, it is in the unprotonated amine form and thus able to act as a base. Cys311', which is located very close to the active site, has an unusually low predicted pK(a) of 5.8 and at pH 7.0 has a predicted average charge of -0.72. The very low predicted charge for Lys129 is consistent with experimental evidence that it is carbamylated, since an unprotonated amine group is available to act as a Lewis base and form the carbamate with CO(2). Repeating the pK(a) calculations on the enzyme with Lys129 in carbamylated form predicts trends similar to those of the uncarbamylated enzyme. It appears that the enzyme has the ability to stabilize negative charge in the region of the active site. Implications for selective inhibitor design are discussed.     

INVESTIGATION INTO THE SPECTROSCOPY AND PHOTOISOMERIZATION OF A SERIES OF POLY (ETHYLENE GLYCOL) PEPTIDE SCHIFF BASES OF 11-cis RETINAL

Abstract— The 11-cis and all-trans isomers of a series of poly(ethylene glycol)-oligopeptide - Schiff bases as models for rhodopsin were synthesized and studied. Absorption data for certain of the PEG-peptide Schiff bases demonstrated that no intramolecular hydrogen-bonding (or protonation) occurs between the Schiff base and an acidic amino acid residue, as was previously thought. Photoisomerization of the 11-cis protonated and unprotonated Schiff bases were examined using both steady state and laser flash techniques. Also with 355 nm excitation (and additionally 532 nm in one case), an approximate 40% increase in quantum yield of isomerization (φ) occurred for all protonated PEG-peptide Schiff bases compared to the H⁺-n-butylamine counterparts (in methanol). In one case, a > 100% increase in φ was found in dichloromethane. These data show that PEG-oligopeptide Schiff bases are still further improved models for rhodopsin compared to their n-butylamine analogs.