The N- and C-terminal blocked hexapeptide Ac-Leu-Ala-His-Tyr-Asn-Lys-amide (LAHYNK) representing the 80–85 fragment of histone H2B was synthesized and its interactions with Cu(II) and Ni(II) ions were studied by potentiometric, UV-Vis, CD, EPR, and NMR spectroscopic techniques in solution. Our data reveal that the imidazole N(3) nitrogen atom is the primary ligating group for both metal ions. Sequential amide groups deprotonation and subsequent coordination to metal ions indicated an {Nimidazole, 3Namide} coordination mode above pH∼9, in all cases. In the case of Cu(II)-peptide system, the almost exclusive formation of the predominant species CuL in neutral media accounting for almost 98% of the total metal ion concentration at pH 7.3 strongly indicates that at physiological pH values the sequence -LAHYNK- of histone H2B provides very efficient binding sites for metal ions. The imidazole pyrrole N(1) ionization (but not coordination) was also detected in species
CuH−4L present in solution above pH ∼ 11.
Cheese production is relied upon the action of rennet (a mixture of chymosin and pepsin) onto casein micelles of milk. For the first time, the monitoring of this interaction with electrochemical impedance spectroscopy (EIS) was used to develop a faradic impedimetric biosensor for the assessment of the clotting activity of rennet, using hexacyanoferrate(II)/(III) couple as a redox probe. Gold electrodes were modified with self-assembled monolayers of different thiols (thioctic acid, dithiobis-N-succinimidyl propionate, and cysteamine), and (artificial) casein micelles were immobilized on the modified gold surfaces. The proposed method is based on the measurement of charge-transfer resistance (R(ct)) changes attributed to the degradation of the negatively charged immobilized casein micelles by rennet to neutral biostructures. This action results in the increase of the flux of the redox probe, which exists in the bulk solution, to the surface of the electrode and, consequently, in the decrease of R(ct). Experimental parameters such as the micelle loading, the reaction time, the concentration of rennet, and the working pH, were optimized. Besides EIS measurements, cyclic voltammetry, FT-IR, and atomic force microscopy (AFM) experiments were also performed before and after the interaction of the immobilized micelles with rennet. Finally, the proposed biosensors were successfully tried for various commercial samples.
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