Carnosine binding: characterization of steric and charge requirements for ligand recognition.

The steric and charge requirements for binding of l-carnosine (β-alanyl-l-histidine) by bovine serum albumin were investigated with proton magnetic resonance (¹HMR) spectrometry. The histidinyl side chain of the dipeptide is responsible for primary recognition by the binding site. Furthermore, recognition is specific to a particular orientation of the histidinyl side chain that is determined by the other amino acid residue of the dipeptide. It was found that, although salts do not have a great effect on the binding of carnosine to bovine serum albumin, this binding cannot be measured by equilibrium dialysis in the presence of salt because of formation of a complex Donnan equilibrium. Carnosine, which has been postulated to have a role in olfaction, binds to the crude particulate fraction of nasal olfactory epithelium in the same steric orientation as it does to bovine serum albumin. Therefore, we have used the binding of carnosine to bovine serum albumin as a model system to test potential competitive inhibitors of carnosine binding that ultimately could be tested for activity in the olfactory pathway. It was found that the binding of carnosine to bovine serum albumin is a good model of nonspecific binding of carnosine to tissue preparations but not of the specific binding of carnosine to the nasal olfactory epithelium. In addition to requiring the proper conformation of the histidinyl residue, the binding to olfactory epithelium also appears to require recognition of the β-alanyl residue and of substituents on the imidazole ring. Evidence is provided that the carnosine binding by the nasal olfactory epithelium demonstrated by ¹HMR spectroscopy does not occur with the mature olfactory receptor neurons.     

Unfolded transferrin polypeptide chain is immunologically crossreactive with similar derivatives of serum albumin and alpha-fetoprotein

Radioimmunoassays utilizing reduced and carboxymethylated (RC) proteins as antigens reveal a cross-reactivity between alpha-fetoprotein (AFP) and albumin. Similar assays were used to study the relationships of AFP and albumin to other serum proteins. Of the several serum proteins tested, transferrin showed the most similarity with AFP and albumin. There was no cross-reactivity of the native proteins, but antisera prepared against RC-albumin and RC-AFP bound 125I-labeled RC-transferrin at high titers, and antiserum to RC-transferrin bound labeled RC-AFP but not RC-albumin. Inhibition assays utilizing binding of 125I-RC-AFP or 125I-RC-transferrin to anti-RC-albumin showed that the RC derivatives of AFP, albumin, and transferrin were equally efficient inhibitors, whereas other serum proteins inhibited much less. The serum vitamin D carrier protein (Gc protein) showed intermediate reactivity. The reactivities of the antisera to RC-albumins with RC-transferrin and RC-Gc protein were corroborated by immunostaining of proteins separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and transferred to nitrocellulose filters. These antisera stained the bands formed by RC derivatives of albumin, AFP, transferrin, and Gc protein, but not other proteins tested. AFP and albumin are known to have amino acid sequence homology. Our results suggest that transferrin and possibly also Gc protein may be structurally related to AFP and albumin.     

2-Iodoestradiol binds with high affinity to human sex hormone binding globulin (SHBG)

Human sex hormone binding globulin (SHBG) binds a set of steroids that differ slightly from each other in structure. Dihydrotestosterone and testosterone are bound with high affinity by SHBG whereas estradiol is bound with a lower affinity. In this work we have studied the binding to human SHBG of the derivatives obtained by substituting iodine in the aromatic A-ring of estradiol. Three A-ring iodinated estradiol derivatives, 2-iodoestradiol, 4-iodoestradiol and 2,4-di-iodoestradiol, were obtained by treating 17 beta-estradiol with NaI and Chloramine T and separating the reaction products by HPLC. Their structures were confirmed by mass spectrometry and 1H-NMR. The corresponding radioactive compounds were obtained with use of Na[125I] in the same synthesizing procedure. Incubation of whole serum, serum albumin and purified SHBG with each of the three [125I]iodoestradiols followed by agarose gel electrophoresis showed only 2-iodoestradiol to have a strong binding to SHBG. This steroid was also bound to albumin, but with a lower affinity. Besides SHBG and albumin, there were no other binders of 2-iodoestradiol in human serum. The affinity constant for the binding of 2-iodoestradiol to purified human SHBG at 37 degrees C and physiological pH was determined by a dextran-coated charcoal method to be 2.4 x 10(9) M-1 (i.e. exceeding that of dihydrotestosterone). It was found that 0.9 mol of 2-iodoestradiol was bound per mol of SHBG dimer (93 kDa) at saturation, and that 2-iodoestradiol competed with dihydrotestosterone for the same binding site of SHBG. It was concluded that 2-iodoestradiol has a remarkably high affinity for human SHBG, and that its gamma-emitting 125I-analog is useful for binding studies of human SHBG.     

Transformation of anthracycline antibiotics and their semisynthetic derivatives as affected by the liver aldo-keto reductase of rats

Formation of 13-dihydro derivatives of rubomycin (daunorubicin), carminomycin, doxorubicin and some of their semisynthetic derivatives under the effect of pure aldo-keto reductase from the rat liver was studied. Attachment of an oxy group to C-14 markedly retarded formation of the 13-dihydro derivatives while attachment of the bulky radicals to the same position prevented their formation. Binding of the anthracycline antibiotics to human serum albumin had no impact on the fermentative reaction rate. Rubomycin, carminomycin and doxorubicin significantly differed in their lipophilic properties and capacity for binding to serum albumin.     

Species-dependent binding of serum albumins to the streptococcal receptor protein G

The interaction of the serum albumin binding domain from streptococcal protein G to serum albumins isolated from different species was investigated. The highest affinity to protein G was found for serum albumins from rat, man and mouse. A medium binding was found for serum albumin from rabbit, cow, hen and horse, while little or no binding was found for ovalbumin and serum albumin from sheep. The interaction between human serum albumin and protein G showed rapid binding kinetics at the temperatures 7, 22 and 37 degrees C. Furthermore, the ability of different serum albumins to function as affinity ligands when covalently coupled to a solid support was tested. The results show that protein G derivatives could be eluted at different pH depending on the origin of the serum albumin. It was also possible to elute the streptococcal receptor efficiently from the mouse serum albumin matrix with human serum albumin. Based on these results, a gene fusion system for recovery of sensitive proteins by affinity purification is described, where high yields are obtained under mild elution conditions.     

Molecular recognition of indole derivatives by polymers imprinted with indole-3-acetic acid: A QSPR study

Three molecularly imprinted polymers (MIPs) were prepared using the phytohormone indole-3-acetic acid (IAA) as a template molecule, 4-vinylpyridine (MIP-1 and MIP-2) or N,N-dimethylaminoethyl methacrylate (MIP-3) as functional monomers, ethylenglycol dimethacrylate as a cross linker and acetonitrile (MIP-1), a methanol–water mixture (MIP-2) or chloroform (MIP-3) as porogens. Retention factors for IAA and 29 indole derivatives were determined by high-performance liquid chromatography, using the molecularly imprinted polymers as stationary phases and acetonitrile as an eluent. High correlations between selectivity factors of above mentioned polymers indicate that their retention mechanisms are basically the same. A quantitative structure–property relationships analysis revealed that the presence of the terminal carboxyl group on the 3-side chain plays an essential role in the binding of the indole derivatives to the polymers. The derivatives without the carboxyl group exhibit a drastically lower affinity toward the polymers. Another factor which favors the binding is electronic density of indole nucleus. Substituents with electro-withdrawing properties enhance the binding, while electro-donating substituents have the opposite effect. The length of the 3-side chain also affects the binding. Indole-3-carboxylic acid having the carboxyl group directly attached to the ring as well as the derivatives whose side chain is longer than that of IAA bind to the polymers with a lower affinity.     

The biosynthesis of rat serum albumin. Metabolism of the NH2-terminal hexapeptide of proalbumin

Proalbumin differs from serum albumin in containing a leading hexapeptide segment, Arg-Gly-Val-Phe-Arg-Arg. This propeptide is removed in the Golgi complex immediately prior to secretion of the albumin, but its fate and possible functions are unknown. We have tested for the presence of the propeptide and its immediate catabolic products in rat liver and plasma and have studied both the disappearance of 3H-propeptide after intravenous injection and the breakdown of synthetic propeptide by rat liver cell components and plasma in vitro. We found no detectable propeptide or its two pentapeptide derivatives in rat liver or plasma at a sensitivity of less than 1 microM. Injected 3H-propeptide was completely cleared from blood within 2 min. No binding of free propeptide to serum albumin was observed. Liver cell fractions as well as blood plasma degraded added propeptide, with the highest activity being observed in smooth microsomes, the Golgi-enriched fraction, and plasma membrane. These preparations chiefly removed the terminal arginine residues, whereas enzymes in the cytosol degraded the peptide completely to amino acids. The activity in plasma resided largely in an alpha-globulin with molecular mass of about 280,000 Da which appears to be carboxypeptidase N. We conclude that the liberated propeptide is quickly broken down within the liver cell and does not accumulate in an amount sufficient to exert feedback or other effects on albumin synthesis.     

Positions in human serum albumin which involve the indole binding site. Sequence of 107-residue fragment.

The first 107 residues of Fragment C of human serum albumin have been sequenced and two positions at which affinity labels block the indole site determined. Histidine 23 is the position of blockage by bromoacetyl-L-tryptophan and lysine 67 is the position of blockage by 5-dimethylaminonaphthalene-1-sulfonyl chloride and probably pyridoxal-5'-phosphate. The presence of an indole ligand at the binding site markedly reduces incorporation of the label into the above lysyl residue, and in the case of 5-dimethylaminonaphthalene-1-sulfonyl chloride, increases incorporation into three other positions, lysine residues 13, 39, and 84. It is concluded that binding of the indole ligand on the site brings about conformational changes in the albumin structure exposing new reactive positions for 5-dimethylaminonaphthalene-1-sulfonyl chloride. There is a large accumulation of basic and hydrophobic residues and no glycine, serine, threonine, valine, aspartate, or cysteine residues in the sequence 10 to 43. Lysine 71 has been identified by amino acid analyses and sequence studies as the position acetylated by acetylsalicylic acid (Hawkins, D. R., Pinckard, N., Crawford, C. P., and Farr, R. S. J. Clin. Invest. (1969) 48, 536), establishing the structural relationships of two major ligand binding sites on albumin. The lone tryptophan is at position 86. Evidence indicates that within residues 1 to 86 of Fragment C and within residues of the A-Phe fragment (Mr equals approximately 10,000), the latter known to be adjacent to Fragment C in the whole albumin structure, exists the major binding sites of all ligands for human serum albumin.     

Detergents as probes of hydrophobic binding cavities in serum albumin and other water-soluble proteins

As an extension of our studies on the interaction of detergents with membranes and membrane proteins, we have investigated their binding to water-soluble proteins. Anionic aliphatic compounds (dodecanoate and dodecylsulfate) were bound to serum albumin with high affinity at nine sites; related nonionic detergents (C12E8 and dodecylmaltoside) were bound at seven to eight sites, many in common with those of dodecanoate. The compounds were also bound in the hydrophobic cavity of beta-lactoglobulin, but not to ovalbumin. In addition to the generally recognized role of the Sudlow binding region II of serum albumin (localized at the IIIA subdomain) in fatty acid binding, quenching of the fluorescence intensity of tryptophan-214 by 7,8-dibromododecylmaltoside and 12-bromododecanoate also implicate the Sudlow binding region I (subdomain IIA) as a locus for binding of aliphatic compounds. Our data document the usefulness of dodecyl amphipathic compounds as probes of hydrophobic cavities in water-soluble proteins. In conjunction with recent x-ray diffraction analyses of fatty acid binding as the starting point we propose a new symmetrical binding model for the location of nine high-affinity sites on serum albumin for aliphatic compounds.     

Determination of binding affinities of retinoids to retinoic acid-binding protein and serum albumin

Binding affinities of retinoic acid and its synthetic analogues to intracellular retinoic acid-binding protein, which is a possible candidate for mediating their biological function, and to serum albumin, the plasma transport protein, were evaluated. A quantitative method involving elimination of interfering serum albumin by immunoprecipitation was developed to measure the binding efficiency of these retinoids, some of which are active in modifying epithelial differentiation and preventing tumorigenesis. Two cyclopentenyl analogues of retinoic acid and 13-cis-retinoic acid showed, like retinoic acid, a binding efficiency of 100% for the cellular binding protein. With the phenyl, dichlorophenyl and trimethylmethoxyphenyl analogues of retinoic acid, the binding efficiency increased as the substituents on the aromatic ring increased; thus the trimethylmethoxyphenyl analogue binds almost as efficiently as retinoic acid itself. However, the trimethylmethoxyphenyl analogue with a sulphur atom on the side chain has a much decreased binding affinity. The correlation noticed between the binding efficiency of these retinoids and their biological activity in differentiation and/or in the control of tumorigenesis particularly enhances the confidence in the present method of determining the relative binding efficiencies. None of the vitamins, hormones and cofactors tested, showed appreciable affinity for the retinoic acid-binding site. Studies on binding of retinoic acid and its analogues to serum albumin indicate that no correlation exists between binding affinity for albumin and their biological potency.     

Effect of molecular parameters on the binding of phenoxyacetic acid derivatives to albumins

The interaction of 12 phenoxyacetic acid derivatives with human and serum albumin as well as with egg albumin was studied by charge-transfer reversed-phase (RP) thin-layer chromatography (TLC) and the relative strength of interaction was calculated. Each phenoxyacetic acid derivative interacted with human and bovine serum albumins whereas no interaction was observed with egg albumin. Stepwise regression analysis proved that the lipophilicity of the derivatives exert a significant impact on their capacity to bind to serum albumins. This result supports the hypothesis that the binding of phenoxyacetic acid derivatives to albumins may involve hydrophobic forces occurring between the corresponding apolar substructures of these derivatives and the amino acid side chains.     

Bovine serum albumin. Study of the fatty acid and steroid binding sites using spin-labeled lipids.

Three spin-labeled derivatives of stearic acid and two derivatives of palmitic acid have been used to study the structure of the strong fatty acid binding site of bovine serum albumin. The steroid and indole binding sites have been studied using spin-labeled derivatives of androstol and indole, respectively. Paramagnetic resonance and fluorescence quenching data suggest that the fatty acid, steroid, and indole binding sites may be identical. The mobility of the nitroxyl group at C-8 of palmitic acid bound to albumin at a 1:1 molar ratio is unaffected when the carboxyl group is esterified. When the nitroxyl group is located at C-5 on this acid its motion is detectably increased by esterification of the carboxyl group but the magnitude of this change is small. This result suggests that the carboxyl group may play a minor role in the binding of fatty acids to the strongest fatty acid binding site of albumin. When stearic acid derivatives bearing the nitroxide at C-5, C-12, and C-16 are bound to albumin at a ligand to albumin ratio of 1, the order of mobility at 0-30 degrees is C-16 greater than C-12 congruent to C-5. Although motion at the methyl terminus is always greater than at the COOH terminus in the range 0-60 degrees, a simple monotonic increase in chain motion between the two termini is not observed. Arrhenius plots of the motion parameters for these bound fatty acids show two abrupt changes in slope. The temperature ranges for these changes are 15-23 degrees and 38-45 degrees. These results suggest that when one mole of spin-labeled fatty acid is bound to albumin, the protein undergoes a conformational change in each of these temperature ranges.     

Correlations between functional porphyrin positions and accumulation in cancer cells

Porphyrin is accumulated in tumours due to its interaction with protein. Cancer therapy with porphyrin as a carrier molecule is attracting attention. Porphyrin displays two functional sites termed β- and meso-positions. A correlation between the functional position on the porphyrin molecule and the ability to accumulate in cancer cells is observed in the present study. The accumulation of porphyrin derivatives was determined by measuring fluorescence intensity after incubation for 2 and 24 h. The accumulation of cancer cells depended on the position and length of functional groups. Estimated binding constants between porphyrin and bovine serum albumin suggest that the position of functional groups leads to changes in binding affinity and influences the accumulation of porphyrin derivatives in cancer cells.     

A soluble auxin-binding protein, ABP57. Purification with anti-bovine serum albumin antibody and characterization of its mechanistic role in the auxin effect on plant plasma membrane H+-ATPase

ABP(57) is an auxin-binding protein that possesses receptor function. In this study, a protocol for ABP(57) purification was developed on the basis of cross-reactivity shown between ABP(57) and antisera raised against bovine serum albumin, which enabled us to purify ABP(57) with a high yield and to further characterize it. ABP(57) activates plant plasma membrane H(+)-ATPase (PM H(+)-ATPase) via direct interaction. The binding of indole-3-acetic acid (IAA) to the primary binding site on ABP(57) caused a marked increase in the affinity of ABP(57) for PM H(+)-ATPase, which was accompanied by a change in ABP(57) conformation. Meanwhile, additional IAA binding to the secondary site on ABP(57) nullified the initial effect without inducing further conformational change. When ABP(57) with IAA occupying only the primary site interacted with PM H(+)-ATPase, no IAA could access the secondary site. These results suggest that IAA-induced biphasic alteration in the affinity of ABP(57) for PM H(+)-ATPase correlates with a bell-shaped dose response of the enzyme to IAA. There is also a possibility that, whereas the stimulation phase of the response is associated with a conformational change of ABP(57), the destimulation phase probably results from hindrance arising directly from the presence of IAA at the secondary site.     

QSAR of adenosine A3 receptor antagonist 1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives using chemometric tools

Considering the potential of selective adenosine A3 receptor subtype ligands in the development of prospective therapeutic agents, an attempt has been made to explore physicochemical requirements of 1,2,4-triazolo[4,3-a]quinoxalin-1-one derivatives for A3 receptor binding. In this study, lipophilicity (logP), physicochemical substituent constants (pi, MR, sigma p) of phenyl ring substituents, and Wang-Ford charges of common atoms of the quinoxaline nucleus (calculated from molecular electrostatic potential surface of energy-minimized geometry using AM1 technique) were used as independent variables along with suitable dummy parameters. The best multiple linear regression (MLR) equation obtained from factor analysis (FA-MLR) as the preprocessing step could explain and predict 72.6% and 65.3%, respectively, of the variance of the binding affinity. The same equation also emerged as the best equation in the population of 100 equations obtained from genetic function approximation (GFA-MLR). The results suggested that presence of an electron-withdrawing group at the para position of the phenyl ring would be favorable for the binding affinity. Again, the presence of a nitro group at position R1 increases the binding affinity. When factor scores were used as predictor variables in the principal component regression analysis, the resultant model showed 78.6% explained variance and 63.1% predicted variance. The best equation derived from G/PLS could explain and predict 74.4% and 64.8%, respectively. The results have suggested the importance of Wang-Ford charges of atoms C15 and C19, apart from positive contributions of electron-withdrawing para substituents of the variance of the phenyl ring and nitro group at the R1 position.     

Binding of Progesterone to Nerve Cell Membranes of Rat Brain Using Progesterone Conjugated to 125I-Bovine Serum Albumin as a Ligand

Radioiodinated bovine serum albumin conjugated to progesterone was used as a probe to examine binding parameters of steroids to membrane preparations from rat brain tissue. The binding of 11 alpha-hydroxyprogesterone-11-hemisuccinate-125I-bovine serum albumin conjugate reached saturation after 30 min of incubation at 5 degrees C. Several bovine serum albumin-conjugated steroids were then tested for competition displacement studies. Among these steroid conjugates, the bovine serum albumin conjugate at position 3 of progesterone had the highest affinity, with an estimated inhibition constant of 28.5 +/- 2.1 nM (n = 3), whereas bovine serum albumin itself and the 17 beta-estradiol 6-(O-carboxy-methyl)oxime-bovine serum albumin conjugate showed no specific displacement. In addition, the binding sites were localized in an axolemma-enriched fraction of rat brainstem. Specific binding was obtained in tissues from cerebral cortex, brainstem, cerebellum, corpus striatum, and hypothalamus, but little or no binding occurred in uterus, ovary, liver, and spleen. The present data indicate that progesterone-125I-bovine serum albumin conjugate can be used as a ligand to study progesterone-membrane receptor interactions.     

Effect of pH on the transient reduction of pig-plasma benzylamine oxidase by benzylamine derivatives.

1. The transient kinetics of reduction of the 470-nm absorption band in benzylamine oxidase by substrate at different pH values between 6 and 10 have been studied by stopped-flow techniques, and substituent effects on kinetic parameters for the reduction process have been examined using a series of ring-substituted benzylamine derivatives as the substrates. 2. Reduction of the enzyme by substrate takes place in two kinetically distinguishable steps, with the intermediate formation of an enzyme-substrate complex in which the substrate appears to be covalently bound through its amino group to the prosthetic group of the enzyme, possibly in the form of an amine-pyridoxal Schiff-base. 3. The apparent stability of the enzyme-substrate complex shows no obvious dependence on the electronic properties of the amine substrates, but is strongly pH-dependent in a way suggesting that substrate-binding involves the non-protonated amines, exclusively, and requires the presence of the acid form of an ionizing group in the enzyme with apparent pKa of 8.8. 4. Reduction of the enzymatic 470-nm chromophore and release of the aldehyde product of the catalytic process are rate-limited by the same monomolecular reaction step involving the enzyme-substrate complex. Rate constants for the rate-limiting reaction exhibit no significant dependence on pH between 6 and 10, but correlate with Hammett sigma-values for the ring-substituted benzylamine derivatives tested, yielding a phi-value of + 0.3.     

Regioselective esterification and etherification of cellulose: a review

Deep understanding of the structure-property relationships of polysaccharide derivatives depends on the ability to control the position of the substituents around the monosaccharide ring and along the chain. Equally important is the ability to analyze position of substitution. Historically, both synthetic control and analysis of regiochemistry have been very difficult for cellulose derivatives, as for most other polysaccharide derivatives. With the advent of cellulose solvents that are suitable for chemical transformations, it has become possible to carry out cellulose derivatization under conditions sufficiently mild to permit increasingly complete regiochemical control, particularly with regard to the position of the substituents around the anhydroglucose ring. In addition, new techniques for forming cellulose and its derivatives from monomers, either by enzyme-catalyzed processes or chemical polymerization, permit us to address new frontiers in regiochemical control. We review these exciting developments in regiocontrolled synthesis of cellulose derivatives and their implications for in-depth structure-property studies.     

Influence of aspirin and iron(III) tetrasulfonated phthalocyanine on bilirubin binding by human serum albumin

The interaction of bilirubin with aspirin-modified human serum albumin (HSA) and the influence of iron tetrasulfonated phthalocyanine on bilirubin binding by the native protein has been studied by difference spectroscopy and circular dichroism measurements. Spectroscopic studies of the systems containing bilirubin and aspirin-modified HSA compared to the analogous systems with the native protein have shown that selective acetylation of albumin at lysine 199 inhibits bilirubin binding by this protein. In both cases, interaction between bilirubin and albumin leads to complex formation at a molar ratio of ligand to protein of 2:1. The studies of the reaction of bilirubin with fragments of albumin produced by reaction with CNBr have demonstrated that one of the strong bilirubin binding sites is located in the M fragment and is close to the high-affinity binding site of aspirin. The other one was found in fragment C. Acetylation of albumin brings about marked conformational change in the protein, which probably accounts for the decrease in its ability to react with anti-HSA antibody. Bilirubin does not change the secondary structure of albumin but, like aspirin, lowers its antigenicity. It has been suggested that the decrease in antigenic properties in this case results from cooperation of the closely neighboring antigenic and bilirubin-binding sites. The studies of the influence of iron(III) tetrasulfonated phthalocyanine on bilirubin binding by HSA suggest that there is no competition between strong sites for iron(III) tetrasulfonated phthalocyanine and bilirubin, but these compounds compete for some of the weaker sites.     

Absorption and fluorescence spectra of ring-substituted indole-3-acetic acids

The absorption and fluorescence spectra of indole-3-acetic acid (1), a plant growth regulator (auxin) and experimental cancer therapeutic, 29 ring-substituted derivatives and the 7-aza analogue (1H-pyrrolo[2,3b]pyridine-3-acetic acid) are compared. Two to four absorbance maxima in the 260-310-nm range are interpreted as overlapping vibronic lines of the 1La<--1A and 1Lb<--1A transitions. Two further maxima in the 200-230-nm region are assigned to the 1Ba<--1A and 1Bb<--1A transitions. 4- and 7-Fluoroindole-3-acetic acid exhibit blue shifts with respect to 1, most other derivatives show red shifts. All indole-3-acetic acids studied, with the exception of chloro-, bromo- and 4- or 7-fluoro-derivatives, fluoresce at 345-370 nm when excited at 275-280 nm. 7-Azaindole-3-acetic acid emits at 411 nm. The fluorescence quantum yield of 6-fluoroindole-3-acetic acid significantly exceeds that of 1 (0.3); the other derivatives have lower quantum yields. The plant-growth promoting activity of the ring-substituted indole-3-acetic acids studied correlates with the position of the 1Bb<--1A transition band.