A comparative Raman spectroscopic study of cholinesterases

doi:10.1016/0300-9084(91)90167-Y

Biochimie

Volume 73, Issue 11, November 1991, Pages 1375-1386

https://doi.org/10.1016/0300-9084(91)90167-Y Get rights and content

Abstract

We report Raman spectra of various cholinesterases: lytic tetrameric forms (G₄) obtained by tryptic digestion of asymmetric acetylcholinesterase (AChE) from Torpedo californica and Electrophorus electricus, a PI-PLC-treated dimeric form (G₂) of AChE from T marmorata, and the soluble tetrameric form (G₄) of butyrylcholinesterase (BuChE) from human plasma. The contribution of different types of secondary structure was estimated by analyzing the amide I band, using the method of Williams [1]. The spectra of cholinesterases in 10 mM Tris-HCl (pH 7.0) indicate the presence of both α-helices (about 50%) and β-sheets (about 25%), together with 15% turns and 10% undefined structures. In 20 mM phosphate buffer (pH 7.0), the spectra indicated a smaller contribution of α-helical structure (about 35%) and an increased β=sheet content (from 25 to 35%). This shows that the ionic milieu profoundly affects either the conformation of the protein (AChE activity is known to be sensitive to ionic strength), or the evaluation of secondary structure, or both. In addition, we analyzed vibrations corresponding to the side chains of aromatic and aliphatic amino acids. In particular, the analyses of the tyrosine doublet (830–850 cm⁻¹) and of the tryptophan vibration at 880 cm⁻¹ indicated that these residues are predominantly ‘exposed’ on the surface of the molecules.

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Accurate discriminating nerve fibers is a prerequisite for right suturing nerves in nerve transfer operation. Various methods have been developed for identification of motor and sensory fibers, but no simple method meets the requirements in clinic. In this study, a surface-enhanced Raman scattering (SERS) lever strategy is designed and developed to detect Acetylcholinesterase (AchE) ultrasensitively, in which using produced thiocholine with weak intrinsic Raman activity (four ounces) to adjust absorbance of Rhodamine B with strong intrinsic Raman activity (thousand catties) on SERS-active substrates is to increase the sensitivity. Employing a miniaturized SERS substrate, SERS-active microneedles, is to decrease the volume of enzymolysis systems. Adopting an internal reference is to increase the repeatability of collected signal. The ultrasensitive AchE detection method discriminate samples with four times of difference in enzyme activity between 1–1 × 10⁻⁴ U/mL in about 10 min of enzymolysis time. AchE amounts in 2-mm-long segments of ventral and dorsal roots were about 0.00025–0.001 U and 0.01–0.02 U, respectively. The developed method would be a reliable method met the requirements of identifying motor and sensory fibers in clinic.
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Less common techniques include microcalorimetry [31] and potentiometric pH-stat method [32]. One particular highlight concerns Raman spectroscopy which can be used for cholinesterase detection [33,34]. This method bears certain relevance in light of its simplicity of experiment and sample preparation.
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^∗: Permanent address: Institute of Biophysics, Semmelweis Medical University, Budapest, Hungary.

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A comparative Raman spectroscopic study of cholinesterases

Abstract

J Mol Biol

Methods Enzymol

FEBS Letters

J Biol Chem

J Biol Chem

Biochem Biophys Acta

J Biol Chem

J Biol Chem

J Biol Chem

J Mol Biol

J Chromatog

Biochem Pharmacol

J Biol Chem

Arch Biochem Biophys

Analyt Biochemistry

Biophys J

Arch Biochem Biophys

J Mol Biol

Biochim Biophys Acta

Biophys J

Biochim Biophys Acta

Spectrochim Acta

Biophys J

Biochim Biophys Acta

Biochim Biophys Acta

Biochim Biophys Acta

Biochim Biophys Acta

Biochim Biophys Acta

Arch Biochem

Raman and infrared study of acetylcholine and postsynaptic membranes

The molecular forms of cholinesterases in vertebrates

Annu Rev Neurosci