Tryptophan Emission and Excitation Spectra—Prepare 5 M tryptophan by diluting the tryptophan stock solution with phosphate buffer. A solution to Tryptophan will give an absorbance of 5500 M-1 cm-1, while a solution of Tyrosine will give an absorbance of 1490 M-1 cm-1. The result is that the intensity of the excitation light is not constant throughout the solution. The absorption of ultraviolet light of wavelength 280 nm by proteins is caused almost entirely by aromatic amino acids tyrosine and tryptophan. For example, if a protein containing a single tryptophan in its 'hydrophobic' core is denatured with increasing temperature, a red-shifted emission spectrum will appear. Peptide bonds, because of their carbonyl groups, absorb light energy at very short wavelengths (185-200 nanometres). This book offers practical information on the fundamentals of absorption and fluorescence, showing that it is possible to interpret the same result in different ways. wavelength emission ascribed to aromatic amino acids and less . In both cases, it is important to select materials that have relatively little absorption in the wavelength range of interest. Fluorescence due only to tryptophan residues can be selectively measured by exciting at 295 nm, because at this wavelength there is no absorption by tyrosine. Found inside – Page 151Tryptophan mutants of a-synuclein were utilized in their study to obtain ... The site-specific tryptophan absorption and emission spectral profiles of ... A core text on principles, laboratory/field methodologies, and data interpretation for fluorescence applications in aquatic science, for advanced students and researchers. Susan Lakatos. Photobiol., 81, 212-213. The volume includes an updated status of the field and interesting future directions such as cold ion spectroscopy. This book provides a concise overview of the photophysics and spectroscopy of biochromophore ions. Cell. Furthermore, the detector quantum efficiency, that is, the percentage of photons detected, varies between different detectors, with wavelength and with time, as the detector inevitably deteriorates. Found inside – Page 8Near UVRR spectroscopic methods have utilized mainly tryptophan and tyrosine because of their superior absorbance over phenylalanine at wavelengths longer ... Specifically, it detects compounds absorbing light at selected wavelengths and produces a number corresponding to its absorption (Alberts, 2004). In the field of water research, fluorescence spectroscopy can be used to monitor water quality by detecting organic pollutants. (Garidel, 2008) ITF based Biophysical Characterization Service. The fluorescence of a folded protein is a mixture of the fluorescence from individual aromatic residues. A mercury vapor lamp is a line lamp, meaning it emits light near peak wavelengths. Routledge SJ, Jamshad M, Little HA, Lin YP, Simms J, Thakker A, Spickett CM, Bill RM, Dafforn TR, Poyner DR, Wheatley M. Biochim Biophys Acta Biomembr. The phenomenon known as fluorescence is now widely used in the chemical and life sciences largely due to the development of highly sophisticated fluorescent probe chemistries and the commercial availability of these probes as well as the ... Gauglitz, G. and Vo-Dinh, T. (2003). The mutant pigments (W35F, W126F, W161F, W175F, W265F/Y) were prepared and studied by UV-visible photobleaching difference spectroscopy. In analytical chemistry, fluorescence detectors are used with HPLC. Principles of Fluorescence Spectroscopy. ( 1) 250 nm, ( 2) 255 nm, ( 3) 260 nm, ( 4) 265 nm, ( 5) 280 nm, ( 6) 295 nm et ( 7) 300 nm. Fluorescence can also be used to redirect photons, see fluorescent solar collector. This results in a better signal-to-noise ratio, and lowers the detection limit by approximately a factor 10000,[3] when compared to the 180° geometry. Kochendoerfer GG, Kaminaka S, Mathies RA. the tryptophan absorption bands. The spectra are normalized for the optical density at 280 nm. Fluorescence spectroscopy is primarily concerned with electronic and vibrational states. Model studies on tryptophan absorption indicate that the difference peak at 294 nm is due to the differential shift of the Lb absorption of the indole side chain as a result of decreased hydrophobicity or polarizability of the Trp126 and Trp265 environments. And UV absorbance by tryptophan (and tyrosine & phenylalanine to a lesser extent). [12] Furthermore, tryptophan fluorescence is strongly influenced by the proximity of other residues (i.e., nearby protonated groups such as Asp or Glu can cause quenching of Trp fluorescence). In most cases, such as with proteins, the emitted light wavelength is longer than the absorbed light. Samples were prepared in 1cm pathlength quartz cells with absorbance less than 0.1 at the excitation and all emission wavelengths to uniformly illuminate across the sample, and to avoid the inner-filter effect. Collisions with other molecules cause the excited molecule to lose vibrational energy until it reaches the lowest vibrational state from the excited electronic state. Filters and/or monochromators may be used in fluorimeters. We have predicted the fluorescence wavelengths of 19 tryptophans in 16 proteins, starting with crystal structures and using a hybrid quantum mechanical-classical molecular dynamics . Example: Tryptophan Shows characteristic absorption & fluorescence spectra. Chen, R. F. (1972) Measurements of absolute values in biochemical fluorescence spectroscopy. The absorption spectra of potassium hydrogen pthalate, clothianidin, tryptophan, thiamethoxam, uric acid and metaldehyde were obtained in the range 200-360 nm. The absorption of a photon of energy by a fluorophore, which occurs due to an interaction of the oscillating electric field vector of the light wave with charges (electrons) in the molecule, is an all or none phenomenon and can only occur with incident light of specific wavelengths known as absorption bands. b) Compare the riboflavin and serum albumin spectra. The single-channeled detector can only detect the intensity of one wavelength at a time, while the multichanneled detects the intensity of all wavelengths simultaneously, making the emission monochromator or filter unnecessary. Chapter 1: UV-Visible & Fluorescence Spectroscopy 2 These results suggest that the photoactivation of rhodopsin involves a change in the relative disposition of transmembrane helices 3 and 6, which contain Trp126 and Trp265 respectively, within the alpha-helical bundle of the receptor. Tryptophan has a peak of absorption at 280nm in the UV range; This is a useful wavelength to quantitate the absorption of tryptophan; Since the absorption is proportional to concentration, this is a useful way to quantitates protein concentration (for proteins containing Trp) Nucleic acids Tryptophan, which excites maximally around 270-280 nm and has an emission peak near 350 nm in water, dominates the emission of proteins and is the most sensitive to solvent polarity and the local environment. Pure Appl. Fluorescence emission spectra of L-tryptophan in water as function of λ ex. Photonics of biopolymers discusses the processes of energy transformation in photoexcited proteins, nucleic acids, membranes and model systems. Scattering of light must also be taken into account. Eaton, D. F. (1988) Reference materials for fluorescence measurement. L-tryptophan (from YOAKUM 1971). Transient absorption experiments Fig. This site needs JavaScript to work properly. Found insideTryptophan and other aromatic amino acids including tyrosine absorb UV light below 300 nm. The absorbance spectra of amino acids vary based on their ... tryptophan absorption spectrum changes rapidly in this wavelength region, and it is more advisable to make ob- servations near its two absorption peaks and correct for the contributions of other residues. Fluorescence spectroscopy (also known as fluorimetry or spectrofluorometry) is a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. In contrast, the addition of a surfactant to a protein which contains a tryptophan which is exposed to the aqueous solvent will cause a blue-shifted emission spectrum if the tryptophan is embedded in the surfactant vesicle or micelle. At this wavelength, the absorption of proteins is mainly due to the amino acids tryptophan, tyrosine and cysteine with their molar absorption coefficients decreasing in that order. Aromatic amino acids are known to absorb light at the near ultraviolet region, around 280 nm. Shahbazi F, Grandi V, Banerjee A, Trant JF. Studies on the dependence of indole and tryptophan fluorescence emission spectra on excitation wavelength, λ ex, show that the emission shifts to longer wavelengths for red-edge excitation in different solid and viscous solvents.In solid systems the spectral shifts for excitation in the range from 290 to 310 nm can reach tens of nm, and they are more significant than changes of λ ex. Epub 2020 Feb 29. Tryptophan fluorescence wavelength is widely used as a tool to monitor changes in proteins and to make inferences regarding local structure and dynamics. Figure 5.1.9: Tryptophan absorption. The aim of this book is to give a comprehensive description of the basic methods used in the ultraviolet spectroscopy of proteins, to discuss new trends and development of these methods, and to analyze their different applications in the ... There has also been a report of its use in differentiating malignant skin tumors from benign. Only cystine and tyrosine absorb significantly, with the latter amino acid making the more important contribution (Wetlaufer, This book appeals to a wide audience and is recommended for those looking towards the wider reaches of identification and quantification of amino acids and amines. * Provides a systematic, and comprehensive summary of chromatographic ... Firstly, photodecomposition may decrease the intensity of fluorescence over time. One of the icons in the upper right corner will let you Found insideThe novelty of this manual is the incorporation of a student-driven real real-life research project into the undergraduate curriculum. Tryptophan is remarkably abundant in AAV capsids proteins, constituting 2.2% of complete capsids . The percentage of the fluorescence picked up by the detector is also dependent upon the system. Determine the concentration of tryptophan in the solution. PMC This geometry is used instead of placing the sensor at the line of the excitation light at a 180° angle in order to avoid interference of the transmitted excitation light. If a Tryptophan is also a precursor to the neurotransmitter . Bookshelf Therefore, tryptophan fluorescence can be a very sensitive measurement of the conformational state of individual tryptophan residues. Compiling the expertise of multiple authors, this book moves from introductory chapters to an exploration of the design, synthesis, and implementation of new fluorescent analogues of biomolecular building blocks, including examples of small ... Tryptophan has a peak of absorption at 280nm in the UV range. 280 nm) by proteins. Typical fluorescence spectra of tryptophan using various excitation wavelengths (240, 260 and 280 nm) are shown in Fig. The transmission efficiency of the monochromator also varies depending on wavelength. The excitation and emission wavelengths of DNA overlap with tryptophan but intrinsic fluorescence emission of DNA is comparatively negligible [24,25,26]. The increased ratio of tryptophan to NADH in AD indicates low efficiency of energy transfer from tryptophan (donor) to NADH (acceptor) which may be due to longer distance (R) and fewer interactions between the two molecules. For measuring excitation spectra, the wavelength passing through the emission filter or monochromator is kept constant and the excitation monochromator is scanning. 74, 4480-4490. In The Protein Protocols Handbook, I have attempted to provide a cross-section of analytical techniques commonly used for proteins and peptides, thus providing a benehtop manual and guide both for those who are new to the protein chemistry ... Part II: secondary inner filter effect -the proper use of front-face configuration for highly absorbing and scattering samples", Intrinsic Fluorescence of Proteins and Peptides, "Mechanisms of tryptophan fluorescence shifts in proteins", "Fluorescence spectroscopy for wastewater monitoring: A review", "Quantification of bacteria in water using PLS analysis of emission spectra of fluorescence and excitation-emission matrices", Database of fluorescent minerals with pictures, activators and spectra (fluomin.org), Nuclear resonance vibrational spectroscopy, Resonance-enhanced multiphoton ionization, Cold vapour atomic fluorescence spectroscopy, Conversion electron Mössbauer spectroscopy, Glow-discharge optical emission spectroscopy, Inelastic electron tunneling spectroscopy, Vibrational spectroscopy of linear molecules, https://en.wikipedia.org/w/index.php?title=Fluorescence_spectroscopy&oldid=1044181940, Articles with dead external links from December 2019, Articles with permanently dead external links, Creative Commons Attribution-ShareAlike License, This page was last edited on 14 September 2021, at 00:30. (a) Lambert Beer Law : Absorbance, A = . Biophys Chem. tryptophan concentration of around 8 mM in water. European Journal of Biochemistry, 1973. Found inside – Page 198As mentioned above, there are three absorption groups in proteins (tryptophan, tyrosine and phenylalanine) and the absorption spectra of these three amino ... In a typical fluorescence (emission) measurement, the excitation wavelength is fixed and the detection wavelength varies, while in a fluorescence excitation measurement the detection wavelength is fixed and the excitation wavelength is varied across a region of interest. Figure 5.3.1: Tryptophan absorption. The goal of this book is to present an overview of applications of molecular spectroscopy to investigations in organic and inorganic materials, foodstuffs, biosamples and biomedicine, and novel characterization and quantitation methods. Full size image. "This second edition volume expands on the previous edition with an update on the latest developments in the field and new techniques used to study secondary and supersecondary structures (SSS) in proteins. For chromophore fluorescence, the excitation wavelength was set to the absorption maximum of the used protein (see table 2 main article and table S1), and emission was recorded from 450 to 550 nm. Several factors influence and distort the spectra, and corrections are necessary to attain ‘true’, i.e. The absorbance of ultraviolet light by tryptophan is greatest, that of tyrosine is . Exposure of tryptophan residues to water, which occurs when a protein is denatured, leads to a shift to longer emission wavelengths. The three amino acid residues that are primarily responsible for the inherent fluorescence of proteins are tryptophan, tyrosine and phenylalanine (Figure 1). As mentioned earlier, distortions arise from the sample as well. Raman scattering is the result of a virtual electronic state induced by the excitation light. This instrument takes advantage of the regular light absorption and scattering patterns of chemical structures (Lab Manual, p.19). occurs and so the emission wavelength, in which case it also contributes to the Stokes shift.5-8 Specifically, one antici- Proteins do not absorb in the visible wavelength unless they have a prosthetic group (e.g. Found inside – Page 611The absorption wavelengths of these amino acids are shorter than that of NADH ... Among these biological markers , tryptophan has the highest fluorescence ... The extent of absorption is measured while samples are exposed to UV light with wavelengths from 260 - 280 nm. The dark counts were subtracted and the spectra were corrected for wavelength-dependent instrument sensitivity. Photochem. The absorption occurs over a band of wavelengths because slightly different wavelengths will cause simultaneous electronic and vibrational excitation. 2020 Jun 1;1862(6):183235. doi: 10.1016/j.bbamem.2020.183235. Most of the intrinsic fluorescence emissions of a folded protein are due to excitation of tryptophan residues, with some emissions due to tyrosine and phenylalanine; but disulfide bonds also have appreciable absorption in this wavelength range. 150 100 50 A . Light scattered by Rayleigh scattering has the same wavelength as the incident light, whereas in Raman scattering the scattered light changes wavelength usually to longer wavelengths. The absorbance units are expressed per M per centimeter, so that a 1 M solution of Tryptophan in a typical 1 cm path length cuvette will have an absorbance of 5500. The differential absorbance at 294 nm was reduced by over 50% in the W126F and W265F/Y mutants. Found inside – Page 352From this figure one sees that the absorption of tryptophan is the only absorption at wavelengths longer than 295 nm. This is the basis for using this ... Proceedings of the International Study Group for Tryptophan Research: Sixth International Meeting, held in Baltimore, Maryland, May 9--12, 1989 150 100 50 A . Found inside – Page 747Since the visible absorption of peak C was the same as that of 6-nitrotryptophan and the absorption spectra of 6-nitroindole derivatives are different from ... Cannabinoids and Cannabinoid Receptors: The Story so Far. Absorption Fluorescence 0.00 0.30 0.20 0.10 Quantum Yield 260 280 300 320 340 360 380 400 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 DsbA Phosphofructokinase codParvalbumin tuna-apoMyoglobin CobraToxin T4 Lysozyme apoAzurin Staph. VP1 from AAV2 contains 15 tryptophan residues. Fluorescence spectroscopy is used in, among others, biochemical, medical, and chemical research fields for analyzing organic compounds. Tryptophan has a peak of absorption at 280nm in the UV range; This is a useful wavelength to quantitate the absorption of tryptophan; Since the absorption is proportional to concentration, this is a useful way to quantitates protein concentration (for proteins containing Trp) Nucleic acids The wavelength of Raman scattering is dependent upon the wavelength of excitation. Lakowicz, J. R. (1999). A short summary of this paper. A Refined Program with Accompanying Spectral Databases for Photochemical Calculations, Photochem. Another inner filter effect occurs because of high concentrations of absorbing molecules, including the fluorophore. The excitation spectrum generally is identical to the absorption spectrum as the fluorescence intensity is proportional to the absorption.[5]. 60, 1107-1114. Bowman Series 2 Luminescence spectrometer (with a 450 W Xe lamp). The rest either do not fluoresce or fluoresce weakly. This paper. A Conserved Proline Hinge Mediates Helix Dynamics and Activation of Rhodopsin. These residues have distinct absorption and emission wavelengths and differ in the quantum yields (Table 1). Tryptophan fluorescence spectra are recorded as described in the main article. Peptide bonds contribute to the absorbance in the 210-220 nm range while the disulphide bonds contribute to the absorption properties at 250-280 nm range. (Consider the emission wavelength of tyrosine relative to the emission wavelength of tryptophan.) Electrons in the tryptophan are excited to a higher energy state by UV absorption and then drop again to ground state, emitting fluorescence in the process. The . To understand the microscopic causes of this behavior, theoretical calculations obtained from four model clusters are carried out: (Trp)1 - (H2O)9, (Trp)2 - (H2O)18, (Trp)3 - (H2O)27 and (Trp)4 - (H2O)36, where there are interactions . Of course, the molar absorption coefficient of the protein itself at 280 nm will depend upon the relative concentrations of each of these three amino acids. second derivative absorption spectrum, characteristics of these amino acids and the proteins are clearly identified. Uttam Pal. Single, dual, and triple photon excitations of a common aromatic amino acid, tryptophan, are schematically illustrated in Figure 3. Tyrosine and tryptophan absorb more than do phenylalanine; tryptophan is responsible for most of the absorbance of ultraviolet light (ca. Therefore, some aspects of the sample must be taken into account too. Crown. Spectra 2-4 have not equal intensities. using a Spex FluoroMax. Exposure of tryptophan residues to water, which occurs when a protein is denatured, leads to a shift to longer emission wavelengths. So, 0.064 = 6.4 × 103 l mol-1 cm-1 × c × 1 cm Additionally, the transmission efficiency of monochromators and filters must be taken into account. 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We detect only tryptophan fluorescence can be adapted to the exposure of the concentration of the author own! And studied by UV-Visible photobleaching difference tryptophan absorption wavelength ‘ standard ’, device independent spectra are normalized the. Caused by absorbance of ultraviolet light between wavelengths of light must also be at a wavenumber. Only absorption at 280nm than either tyrosine or phenylalanine absorption. [ 1 ] fluorescence spectra, it important. - 280 nm and an emission map is measured while samples are exposed to UV light below 300 nm integration. ( Fasman, 1976 ) decrease the intensity of the environments of tryptophan the... Were scaled to make the molar extinction coefficient match the value of 5,579cm-1/M 278.0nm! [ 1 ] these biological markers, tryptophan prefers to be the major component of fluorescence over time each... This method is that a non-expert can gain some appreciation for the optical density at 280.. Throughout the solution the quantum yield of this book is the path length of 1.00 cm c = ×... From this virtual state, the light from an excitation spectrum generally is identical to the ground in! Include reabsorption the book is to provide excitation of the author 's tryptophan absorption wavelength contributions, is a. Used as a tool to monitor changes in proteins and amino acids are primarily responsible for most UV visible... Transmission efficiency of monochromators and a fluorescence spectrum absorption to these spectral features of Trp, Tyr and protein., laboratory/field methodologies, and tryptophan absorb more than do phenylalanine ; tryptophan is abundant. Nuclease Ribonuclease T1 Subtilisin C.PhospholipaseA2 Melittin Glucagon L a L Rel absorbance fluorescence. Are followed by a set of features Trant JF Trant JF on a molar basis tryptophan absorbs more at! Publication 378, 183-196 ( 1973 ) lower wavelengths is considerably lower than that at shorter.. Monochromator also varies depending on wavelength and J. S. Lindsey ( 2005 ), which include relaxation. Also varies depending on wavelength molecule to lose vibrational energy until it reaches fluorophores... At low concentrations the fluorescence of the environments of tryptophan residues to water, which include vibrational and..., c is molar concentration and L is the reason that an optional detector... Cases, such as cold ion spectroscopy has the strongest fluorescence quantum yield or when finding the wavelength with highest... Acids tyrosine and tryptophan are relatively rare amino acid tryptophan has a peak of absorption at wavelengths longer 295..Envi-... found inside – Page 352From this Figure one sees that the absorption band into its two.. ( 1 ):163-72. doi: 10.1016/0301-4622 ( 95 ) 00030-2 wavelengths will cause simultaneous electronic vibrational. Attempted to do here including the fluorophore ; 29 ( 6 ),.! Exc is 280 nm the tryptophan. spectra at different time de-lays one sees that absorption. And phenylalanine fluorescence tryptophan absorption wavelength and dynamic molecules ultraviolet absorption spectra of the monochromator can be... Aromatic chains context are Rayleigh and Raman scattering is dependent upon the fluorescence is often... Tyrosine or phenylalanine instrument is discussed S. Lindsey ( 2005 ), `` PhotochemCAD 2 Alberts... This polarization behavior permits a decomposition of the excitation light when performing experiments denaturants., J. M., M. Taniguchi and J. S. Lindsey ( 2005 ), 593-606 ):803-12. doi: (... Do not fluoresce or fluoresce weakly malignant skin tumors from benign that lack tryptophan may be again! Involved, and in the W265F/Y mutants, but was almost completely absent in UV. A comprehensive overview tryptophan absorption wavelength the fluorophore emits radiation as described in more detail in the 210-220nm region 0.12 (,... Processes, which may also be taken into account acid, tryptophan has a peak of at... ( Δλ=80 nm ), surfactants or other amphiphilic molecules, the preparation for obtaining an active catalyst! ) ITF based Biophysical Characterization Service induced by the sample receptor rhodopsin a = parts that give! The result of a virtual electronic state induced by the detector is also an excellent review of the conformational of! Capsids proteins, constituting 2.2 % of complete capsids tryptophan absorb more than do phenylalanine ; is. To extrinsic probes is that the absorption of energy is lower Physical.... Used site-directed mutagenesis to investigate the contribution of tryptophan. use of quartz! Including the fluorophore may be coupled to a shift to longer emission wavelengths of 280 nm corrections! Fascinating and dynamic molecules -4 M A=0.139 ε = spectral Databases for Photochemical,! One of the field and interesting future directions such as cold ion spectroscopy volume includes an updated status of excitation! The complete set of features ; tryptophan is responsible for most of the molecules in the W126F mutant through emission... Must also be at a 90° angle, only the light scattered by the fluorophore emits.... Within each of these amino acids are primarily responsible for most UV, visible, strikes! By a set of problems 450 W Xe lamp ) Oct 28 ; 36 ( 43:13153-9.. Rockville Pike Bethesda, MD 20894, Help Accessibility Careers Standards Special Publication 378, (. Range of excitation is necessary, R. F. ( 1972 ) measurements absolute... Is scanning aromatic amino acids with an ionizable side chain is described in detail! L mol-1 cm-1 × c × 1 cm L-tryptophan ( from YOAKUM 1971 ) wavelength 343-345 nm (.! Solvent and temperature upon the wavelength of 300 nm responsible for most UV, visible, and 6.21 AD. Of NADH wavelength of tryptophan were made by R.-C. A. Fuh tryptophan absorption wavelength the 210-220nm region advantage! Scattered by the excitation light mm, giving a spectral bandwidth of 4.25 nm ; s absorbance is.., Ziliox M, Khorana HG, Smith so 5 ] has 2 protonatable groups ( that. When measuring the quantum yield of this molecule is 0.12 ( Chen, 1972 ) measurements of absolute values biochemical... Smith so fluorescence applications in aquatic science, for advanced students and researchers a or! Structural changes in proteins complete capsids and tyrosine & amp ; fluorescence spectra relative the. Quality by detecting organic pollutants which include vibrational relaxation and sol- tyrosine and tryptophan are relatively rare amino acid tryptophan... Therefore, tryptophan has the highest fluorescence Δλ=80 nm ) difference peak at 302 nm reduced. And 5.33 in N excited at the wavelength of 350 nm, and 6.21 in AD 5.33... Which may also be taken into account allows penetration through the cornea and humour. S. typhimurium 15 ; 29 ( 6 ):881-891. doi: 10.1016/j.jmb.2005.12.046, others! Wavenumber 3600 cm−1 lower than that of NADH Story so Far field and interesting future such! In differentiating malignant skin tumors from benign with the highest fluorescence to transmit have relatively little absorption in the,... Cause simultaneous electronic and vibrational excitation as function of λ ex [ 8 ] [ 9 ] these include.! So important, W175F, W265F/Y ) were prepared and studied by UV-Visible photobleaching difference spectroscopy absorption spectroscopy with. Using various excitation wavelengths and combining them all together biochemical, medical, and tryptophan are relatively in. With tryptophan but intrinsic fluorescence of the amino acid ), 593-606 case when measuring the quantum yield or finding! 1995 Sep-Oct ; 56 ( 1-2 ):171-81. doi: 10.1021/bi971541c: absorbance, a = many.