Aqueous solutions nitroxide spin labels

The interactions between a 22-amino acid long peptide (called N-peptide) and a nascent mRNA hairpin element (called boxB) are of a crucial function in transcription anti-termination. Qin and coworkers have studied this N-peptide/boxB complex by monitoring the nanosecond dynamics of nitroxide spin labels attached to different positions of the N-peptide [90]. Analysis of CW EPR spectra recorded in viscous aqueous solutions at 5°C indicates that the bound N-peptide exists in a d)mamic equilibrium between two different conformational states, with the... 

Polarimetry of the brucine salts of L-guluronic acid and D-mannuronic acid released from alginates by acid hydrolysis has been used to measure the ratio of these acids in the native alginate. Nitroxide spin-labels have been investigated as molecular probes to determine the microviscosity of aqueous solutions of carbohydrates such as alginate. ... 

Consider a spin-labeled residue that is on the exterior of the protein and buried within the membrane. On exposure to O2, the residue in the membrane will exhibit an enhanced relaxation rate. In contrast, CROX will not affect the relaxation rate. Following the same reasoning, a residue exposed to the aqueous solution, say, in a loop region, will show sensitivity to CROX. A residue buried within the protein will show sensitivity to neither. Mobility may also be used to distinguish whether a nitroxide side chain is buried within the protein or is present on the protein exterior. 

Measuring in frozen solution is desired in order to avoid the averaging out of the dipole-dipole interactions and, in particular, the strong decrease in transverse relaxation time T2 that is induced even by moderate spin label d3mamics. Additionally, the proper choice of temperature is important in pulsed EPR to optimize relaxation rates. T = 50 K is ideal for DEER at nitroxides in aqueous solution, so liquid helium cooling is advantageous [85]. 

The stereospecificity of the interactions of several spin-labelled substrates with cyclohexa- and cyclohepta-amyloses, as models for chymotrypsin, has been studied. Complexes of the cycloamyloses with 2,2,6,6-tetramethyl-4-oxy-pyridyl-1-oxide in aqueous solution were examined by e.s.r. spectroscopy the nitroxide function moved to a relatively hydrophobic environment on binding to cyclohepta-amylose, and lost some freedom of rotation on binding to both cycloamyloses. The dissociation constant for the cyclohexa-amylose complex of the nitroxide is greater than that for the cyclohepta-amylose complex, consistent with measurements made on molecular models. In the hydrolysis of the asymmetric compound 3-carboxy-2,2,5,5-tetramethylpyrrolidyl-l-oxide 3-nitro-phenyl ester, catalysed by cyclohexa-amylose, enantiomeric specificity was observed in the acylation step but not in formation of the Michaelis complex , or on hydrolysis of the acylated cycloamylose intermediate. No differences were found in the e.s.r. spectra of solutions of the trapped acylcyclohexa-amylose intermediates derived from ( + )- and ( )-forms of the asymmetric nitroxide. The nitroxide function is less free to rotate in the acylcycloamylose intermediate than in the Michaelis complex and is not included in the cycloamylose cavity. 

A second example is a 220 GHz EPR study of an oligonucleotide with a newly synthesized cytosine spin-label. The spectrum from a monomer in an aqueous solution exhibited a three-line pattern characteristic of a nitroxide in a fast motion regime. The anisotropic diffusion tensor estimated from this fast motion spectrum was consistent with the expected rapid motion of the probe molecule around its tether. 

The 4-pyridone (151), an analogue of maltol, has been isolated from the reaction of lactose or maltose with a hot aqueous solution of methylammonium acetate. The heterocyclic derivative (152) and the 1,2,4-triazine derivatives (153) have been obtained from 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bromide by standard routes. Bischler-Napieralski cyclization of the corresponding iV-acetyl-tryptamines and dehydrogenation (Pd-BaSO ) gave the )S-D-glucopyranosyl-carboline (154) and the related a-L-arabinopyranosylcarboline. The spin-labelled nitroxide derivative (155) has been prepared by reaction of the nicotinamide derivative with 2,3,5-tri-0-benzoyl-j8-D-ribofuranosyl bromide. ... 

Figure 15.8 Examples of spectral integration and normalization. Spectra shown were obtained with nitroxide label 14 (Fig. 15.3C). Acquisition parameters are listed in Table 15.1, except that number of scans = 4 and number of points = 1024. (A) Spectrum of an aqueous sample of a 23-nt RNA, together with its 1st and 2nd integrals. (B) Spectral comparison between a 23-nt RNA (40 gM, dotted line) and a 49-nt RNA (30 jiM, sobd Une). Comparison of the normalized spectra is not skewed by the different amount of labeled RNAs used in the measurement, and reports different nitroxide behavior due primarily to the difference in RNA size. (C) An example of spin counting. The calibration curve was generated by linear fitting (solid Une) of data points (sobd square) obtained using tempol solutions of various concentrations. Using this calibration curve, the sample measured in (A) was found to contain 37.5 gM of spins ( sample = 2.5). Based on an RNA concentration of 40 jiM, the nitroxide labeling efficiency was determined to be 93.6%.

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