Exploration Of Selective Determination Of Some Biomolecules Based On Derivatization Technique

The components in biological samples are complicated and their dynamic abundances are extremely broad. It requires more sensitively and selectively analytic methods to determine the components in real samples. Derivatization is such a method that can not only enhance the sensitivity but also improve the selectivity of detection. It has aroused much attention in the design and synthesis of new derivatization reagents and the development of derivatization technique in the areas of life science, analytical chemistry and some other research areas.We have studied the derivatization methods for biomolecules (including thiols, protiens, peptides and etc.) by using two different derivatization reagents. Firstly, we employed4-nitro-1,8-naphthalic anhydride (NNA) as a fluorescent probe for thiols. When reacted with Cys, the4-nitro in NNA was first replaced by mercapto group followed by the displacement of mercapto group with amino group to form4-amino substitute via intramolecular nucleophilic aromatic substitution. The hydrogen bond between the sulfur atom and the carboxylic acid group in Cys could help the leaving of alkylthio group, which therfore promoted the formation of4-amino substitute. The absorption and emission maxima of the4-amino substitute were at435nm and530nm, respectively, and the solution exhibited bright green-yellow fluorescence when irradiated with UV lamp. However, since the4-alkylthio substitutes of the other thiols reaced with NNA can not transfer to the4-amino substitutes during the experimental period, the reaction solutions had absorption and emission maxima at390nm and490nm, respectively, and presented blue fluorescence when irradiated with UV lamp. Thus Cys could be recognized selectively by fluorescent and UV methods and even by naked-eye.Secondly, we used4-fluoro-7-nitrobenzo-2-oxa-l,3-diazole (NBD-F) as a probe to study its spectral responses towards thiol-containing and amino-containing compounds in different reaction meadia. Thiol group exhibited quite good reactivity in all the experimental conditions (in acidic, neutral and CTAB or SDS solutions). The thiol-substituent products of NBD-F reacted with Cys, Hcy and MEA would transfer to the corresponding amino-substituent products via intramolecular nucleophilic aromatic substitution. In addition, the rate of transfer and the stability of the products changed with the components of the media. In some specific media (such as in CTAB solution), the thiol-substituent products of NBD-F reacted with MPA and NAC would transfer to some long-wavelength but non-fluorescent products. Based on the individual sensing behavior of NBD-F towards different low-molecular-weight thiols, we could recognize these thiols (Cys, Hcy, MEA and etc.) to some extent.Thirdly, we studied the time-dependent absorption and emission spectra of NBD-F reacted with bovine serum albumin (BSA) and ovalbumin (OVA). Due to the difference in structure of these two proteins, they presented quite different reactivities with NBD-F in acidic and neutral media. The addition of anionic or cationic surfactants would change the reactivities of the protiens with NBD-F. Real biological samples such as proteins extracted from rat liver tumor tissues and their enzymic hydrolysates were analyzed via high-performance liquid chromatography coupled with laser-induced fluorescence detector (HPLC-LIF) after been labeled with NBD-F. The results indicated that the selectivity of the detection would change with the reactivities of NBD-F and proteins which were definitely influenced by the pH value of the reaction media.Finally, we designed a CE-LIF on-line protein derivatization reactor based on a hollow fiber membrane. Derivatization reagent with low molecular weight could permeate into the hollow fiber membrane to react with the active components whose molecular weights were larger than the molecular weight cutoffs (MWCO) of the membrane. The derivative products could be detected at the detection window. This device, with its merits such as small death volume of the interface, low cost and etc., has potential application in the analysis of biomacromolecules.