Vibrational Spectroscopy as a Promising Tool to Study Enzyme-Carrier Interactions: A Review

Abstract

The high selectivity and specificity together with the catalytic activity of enzymes lead them to be a key tool in biotechnology industries. The enzyme immobilization on a carrier material facilitates the reuse of the enzyme and improves its stability. This article is a comprehensive review that reports papers in which different enzymes have been immobilized on distinct carrier materials and enzyme-carrier interactions were evaluated by infrared and/or Raman spectroscopy. Vibrational spectroscopy was used to inspect the spectral bands of different classes of enzymes before and after their immobilization on carriers. In fact, the characteristic spectral bands that prove the interaction enzyme-carrier are related with the amide functional groups of enzymes. In addition, those interactions are characterized by the shift, broadening, or increment on intensity of specific bands in reference to the spectrum of their carrier material. In this review, 20 of these studies used infrared spectroscopy and only three used Raman spectroscopy. The use of both vibrational spectroscopy techniques is increasingly due to their proved capacity to characterize the enzyme-carrier interactions without damaging them.     

Two-colour operation of a Free-Electron Laser and applications in the mid-infrared

The two-colour operation of a Free-Electron Laser (FEL) has been demonstrated with the “CLIO” infrared facility. A two section undulator allows the production of picosecond laser pulses at two different wavelengths and simultaneously, in a wavelength range from to , and with a wavelength separation of two colours of up to . The time overlap, between both colours, has been measured on a picosecond time scale and on a microsecond time scale. An initial pump-probe application experiment has been performed with the two colours: stimulated emission has been measured in a 3-level Quantum-Well system. This is the best demonstration of the stability and reliability of the two-colour laser operation. Received: 4 November 1997 / Revised in final form: 31 March 1998 / Accepted: 10 April 1998     

Histidine affinity chromatography of homo‐oligonucleotides. Role of multiple interactions on retention

The recent application of histidine–agarose affinity supports in plasmid purification takes advantage of the biorecognition of nucleic acid bases by the histidine ligand. This consideration prompted the need for better understanding the interactions involved in affinity chromatography of plasmid DNA with the histidine–agarose support. In this work, we used synthetic homo‐deoxyoligonucleotides with different sizes (1–30 nucleotides long), to explore the effect of several conditions like hydrophobic character of the individual bases, presence of secondary structures, temperature, pH and salt concentration on the mechanism of retention of nucleic acids to histidine–agarose support. One of the most striking results shows that histidine interacts preferentially with guanine, and the presence of secondary structures on polyA and polyG oligonucleotides has a significant influence on retention. Otherwise, the temperature manipulation has not shown a direct influence on oligonucleotide retention, only inducing conformational changes on secondary structures. Overall, the results obtained provide valuable information for the future development and implementation of histidine and other amino acids as ligands in chromatography for the purification of plasmid DNA and other nucleic acids, by improving the knowledge of the interactions involved as well as of the parameters influencing the retention. Copyright © 2009 John Wiley & Sons, Ltd.     

The role of probe–probe interactions on the hybridization of double-stranded DNA targets onto DNA-modified magnetic microparticles

In this work, we have studied the effect of different probe lengths and surface densities on the hybridization of a 181-bp polymerase chain reaction product to probes tethered onto magnetic microparticles. Hybridization was shown to be favored by longer probes but only at probe surface densities where probe-to-probe interactions are absent. From these results, a simple rule was inferred for determining maximum surface densities above which hybridization signals decreased. According to this rule, if the average surface area occupied by an immobilized probe (Σ) is larger than the projected surface area of each tethered probe molecule (S _ss ), hybridization efficiency increases with surface density, whereas the reverse occurs when Σ −  S _ss  < 0.     

Sub-100 nm IR spectromicroscopy of living cells

We have performed IR spectromicroscopy of cells immersed in liquid water, with a lateral resolution better than 100 nm. Here, we use the motion of an atomic force microscope tip, probing the local transient deformation induced by an IR pulsed laser tuned at a sample absorbing wavelength. By Fourier analysis of the vibration of the cantilever tip, we can discriminate frequencies that are characteristic of the object, thus eliminating the influence of the water absorption. This opens the door of chemical imaging of living species in vivo, with spatial resolution of the order of the size of cell components.     

Long polaron lifetime in InAs/GaAs self-assembled quantum dots

We have investigated the polaron dynamics in n-doped InAs/GaAs self-assembled quantum dots by pump-probe midinfrared spectroscopy. A long T1 polaron decay time is measured at both low temperature and room temperature, with values around 70 and 37 ps, respectively. The decay time decreases for energies closer to the optical phonon energy. The relaxation is explained by the strong coupling for the electron-phonon interaction and by the finite lifetime of the optical phonons. We show that, even for a large detuning of 19 meV from the LO photon energy in GaAs, the carrier relaxation remains phonon assisted.     

Long-acting androgens: Analytical and preparative HPLC of testosterone esters

Procedures are described for the normal phase and reverse phase HPLC analysis of testosterone esters having diverse side chains: examples are given of the gram-scale purification of these compounds using two commercial preparative liquid chromatography and an economical and efficient laboratory-assembled preparative liquid chromatograph.     

Baryon pairs production ine + e − annihilation at\sqrt s = 2.4 GeV

Search for baryon pairs production ine ⁺ e ^? annihilation at \(\sqrt s = 2386 MeV\) is reported. The data relate to a luminosity of 161 nb^?1 collected by the DM2 experiment at DCI, the Orsay colliding ring. First measurements of directe ⁺ e ^? annihilation into \(\Lambda \bar \Lambda \) and ofe ⁺ e ^?→ \(p\bar p\) at this energy are presented. First observation of a goode ⁺ e ^?→ \(n\bar n\) candidate is reported and upper limits are given fore ⁺ e ^?→ \(n\bar n, \Lambda \bar \sum ^0 + c.c.\) and \(\Sigma ^0 \bar \Sigma ^0 \) .     

Hydrophobic interaction chromatography of homo-oligonucleotides on derivatized Sepharose CL-6B. Application of the solvophobic theory

This work describes the hydrophobic interaction chromatography of homo-deoxyoligonucleotides polyA, polyT and polyU with sizes up to 30 bases on a Sepharose gel derivatized with the 1,4-butanediol diglycidyl ether. The oligonucleotides interacted differently with the column according to the molecular mass, the hydrophobic character of the individual bases, the secondary structure of the molecule and the concentration of ammonium sulphate in the eluent. The retention factor, k', was determined from the chromatographic profiles at different concentrations of ammonium sulphate. A linear relationship between log k' and the concentration of ammonium sulphate in the eluent was found for all oligonucleotides at the higher concentrations (> 1.0 M) of ammonium sulphate. The slope of these plots, termed the hydrophobic interaction parameter, was found to be an increasing function of the number of nucleotides. The same plots reveal that polyA molecules with high molecular mass have lower retention factors when compared with polyT, an observation that was not expected since the hydrophobicity of adenine is higher than that of thymine. This behaviour was due to the existence of secondary structures in polyA, which decrease the exposed hydrophobic area of the molecule.