Über die quantitative Berücksichtigung der Tyndall‐Absorption im UV‐Absorptionsspektrum von proteinen

Schauenstein, E.; Bayzer, H.

doi:10.1002/pol.1955.120168104

Cited by 38 publications

(8 citation statements)

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“…According to these measurements the decadic extinction coefficient of the native protein at 276 nm (pH 7.0) was 1440 f 75 (SE) M-' cm-', which is insignificantly different from that of free tyrosine (t = 1405 M-' cm-'; Fasman, 1976). Optical density due to light scattering was routinely subtracted by exponential extrapolation from measurements at 330-370 nm (Schauenstein & Bayzer, 1955). Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) was performed in 18% acrylamide and 0.48% N,N'methylenebis(acrylamide), and otherwise as described by Laemmli (1 970).…”

Section: Methodsmentioning

confidence: 99%

“…According to these measurements the decadic extinction coefficient of the native protein at 276 nm (pH 7.0) was 1440 ±75 (SE) M"1 cm"1, which is insignificantly different from that of free tyrosine (e = 1405 M"1 cm"1; Fasman, 1976). Optical density due to light scattering was routinely subtracted by exponential extrapolation from measurements at 330-370 nm (Schauenstein & Bayzer, 1955).…”

Section: Methodsmentioning

confidence: 99%

“…When the tyrosine residues were removed enzymatically, the exicted-state lifetime of the phenylalanine residues nearly doubled. Because of energy transfer, the tyrosine emission had two apparent fluorescence decay lifetimes; one lifetime (3.9 ns) was that of tyrosine while the second (12.1 ns) corresponded to the excited state of phenylalanine.x e histone-like protein HTa' is abundantly associated with the DNA of the Archaebacterium Thermoplasma acidophilum (Searcy, 1975). It functions to protect DNA from thermal denaturation (Stein & Searcy, 1978).…”

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confidence: 99%

“…x e histone-like protein HTa' is abundantly associated with the DNA of the Archaebacterium Thermoplasma acidophilum (Searcy, 1975). It functions to protect DNA from thermal denaturation (Stein & Searcy, 1978).…”

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confidence: 99%

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Phenylalanine-to-tyrosine singlet energy transfer in the archaebacterial histone-like protein HTa

Searcy¹,

Montenay-Garestier²,

Hélène³

1989

Biochemistry

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The Archaebacterium Thermoplasma acidophilum has a histone-like protein (HTa) abundantly associated with its deoxyribonucleic acid. Each native tetrameric complex of HTa contains 20 phenylalanine residues, 4 tyrosine residues, and no tryptophan. When the protein was excited by radiation at 252 nm, which is a wavelength absorbed predominantly by phenylalanine, the fluorescent emission was mostly from tyrosine. According to the excitation spectrum for this tyrosine fluorescence, the cause was energy transfer from phenylalanine, which occurred with about 50% efficiency. When the tyrosine residues were removed enzymatically, the excited-state lifetime of the phenylalanine residues nearly doubled. Because of energy transfer, the tyrosine emission had two apparent fluorescence decay lifetimes; one lifetime (3.9 ns) was that of tyrosine while the second (12.1 ns) corresponded to the excited state of phenylalanine.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Phenylalanine-to-tyrosine singlet energy transfer in the archaebacterial histone-like protein HTa

Searcy¹,

Montenay-Garestier²,

Hélène³

1989

Biochemistry

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“…The other method used the absorbance spectra obtained at neutral pH and E(277 nm) = 0.22 (mg/ml)-', which was calculated from E277(Tyr) = 1350 M-1 cm-' (13) and 11.1 Tyr/ mol. In order to obtain agreement with the biuret and high pH determination, the light scattering was measured, extrapolated, and subtracted from the 277 nm value (14).…”

Section: Methodsmentioning

confidence: 99%

Intramolecular crosslinking of tropomyosin via disulfide bond formation: Evidence for chain register

1976

Proc. Natl. Acad. Sci. U.S.A.

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The work reported below is an extension of these early observations using a different approach. We have found that it is possible to completely crosslink reduced tropomyosin by reaction with 5,5'-dithiobis(2-nitrobenzoate) (NbS)2. This aromatic disulfide causes the formation of S-S bonds between the corresponding cysteines of the two parallel chains within the same molecule by a two-step disulfide exchange process. Since this process requires the close proximity of two SH groups, these studies provide strong evidence for the model in which the chains are in register. In addition, from our consistent observations that only two crosslinked dimer chain species of approximately equal concentration are formed, it appears that the tropomyosin molecule is composed of only aa and a/3 chains.Tropomyosin is a rod-shaped molecule of 68,000 daltons whose subunits are made up of two parallel a-helical polypeptide chains of about equal molecular weight (1). It is one of the components of the thin filaments of skeletal muscle, and is involved in the regulation of muscle contraction (1). Recently, considerable progress has been made in characterizing the two subunits (2) and in analyzing the amino-acid sequence (3-5), and it is clear that rabbit skeletal tropomyosin consists of two kinds of chains, a and fl, which differ slightly in amino-acid composition and sequence (2-5). The a chain, which has one cysteine, is present in about a 3-fold excess over the ,3 chain, which contains two cysteines (2). Although the precise arrangement of chains is not known, there is some evidence to suggest that tropomyosin is not composed of a random mixture of a and /3 chains (6). Sodek et al. proposed a model for the interacting chains based on the observation that hydrophobic sidechains are predominantly located in two series which repeat every seventh residue in the sequence (7). This appears to allow maximum hydrophobic interaction along a line between the two a-helical chains when they are in register or when one chain is translated 7, 14, 21, ... residues with respect to the other. Although this model showed favorable contacts when the two chains were in register, the 14-residue staggered model was preferred because it appeared to optimize hydrophobic interactions and account for end-to-end aggregation.Evidence has accumulated over the years that unless care is taken to prevent heavy metal ion contamination, the SH groups of tropomyosin are readily oxidized to S-S (8-10). MATERIALS AND METHODSPreparation of Reduced Tropomyosin. Tropomyosin was prepared by the method of Bailey (11) as modified by Greaser and Gergely (12). Tropomyosin at about 5 mg/ml was dissolved in buffer (0.05 M Na phosphate, 0.01 M Na acetate, 1.0 M NaCl, 1 mM EDTA) at pH 7.5, containing 20 mM dithiothreitol, filtered through a Millipore HAWP 0.45 ,um filter, and warmed to 450 for 1 hr. After cooling, the pH was immediately reduced to 4.6 by addition of a few drops of HC1, and the isoelectric precipitate was collected by centrifugation for 15 min at 15,000 rpm. After di...

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Correction of light‐scattering errors in spectrophotometric protein determinations

1971

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SynopsisErrors diie to light scat t.eriiig are R feat lire of spectrophotometric met,hods of protein determiriation. The experimental miidit ions may he chosen to allow calculation of the scatter coiitribrition, which is obtained eiserltially from the linear relationship between log turbidity and log incident wavelength, together with knowledge of the slope of such fruictions Using this iriformation, the scatter cwrrections may be obtained by varioiis simple and rapid methods, applicable to single or mult icomponent protein solut,ions.The apparent absorbance of protein so1ut.ions embraces a component due to light, scattering, and wit,h lipoproteins, for example, this scattered component, may greatsly exceed that, of t,he true absorbance. Protein concent,rat,ions may be estimated spectrophotometrically from t'he absorbance at. 280 rim or occasionally 254 nm in flow-cell syst,ems, from the 215/225 nm absorbance difference'z2 or by the Lowry m e t h~d ,~ giving color development estimated at 650 nm. The turbidity contribution with the Lowry method may be reduced by heating or pre-incubat'ing at high pH4J arid with the related but, less specific biuret method by incorporating det,ergent,G but. the errors are not generally severe, as the scatter decreases with increase in wavelength. In the ultraviolet range, the use of a close differ~n c e measurement reduces the scattering contributions, which are most severe when absorbance measurements are restricted to one wave1engt.h.The scat,teririg component in the ultraviolet can be determined from the \vell-knon.n Rayleigh relationship that states that, turbidity or optical density is inversely proport,ional to the wavelength of the incident light raised to a power Ti., this exponent, having a value of between 4 and commonly 2 , dcperidirig on the relshonship between t,he incident, light wavelength and the size, shape and riat,ure of interface of the dissolved macrorn~lecules.~ Thus, det.ermination of t.urbidit,y values at. a series of wavelengths in the visible range allows extrapolation to the ult,raviolet and subtraction of the scnt.t,ercd component, to give t,rue absorbance. This ext,rapolation may be derived from a calculation of the Rayleigh scattering constarit and the wivelength or more empirically from graphical plots of log (t,urbidit,y) versus log (incident. wavelength). lo, l 1 These latter extrapolat.ions are very close to line:wl2 and t,Iw slopes :ire t,he \v:ivelengt,h cxponmts 12448 0

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Über die quantitative Berücksichtigung der Tyndall‐Absorption im UV‐Absorptionsspektrum von proteinen

Cited by 38 publications

References 10 publications

Phenylalanine-to-tyrosine singlet energy transfer in the archaebacterial histone-like protein HTa

Phenylalanine-to-tyrosine singlet energy transfer in the archaebacterial histone-like protein HTa

Intramolecular crosslinking of tropomyosin via disulfide bond formation: Evidence for chain register

Correction of light‐scattering errors in spectrophotometric protein determinations

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