Abstract Dunaliella salina, a single-celled marine alga with extreme salt tolerance, is an important model organism for studying fundamental extremophile survival mechanisms and their potential practical applications. In this study, two-dimensional differential in-gel electrophoresis (2D-DIGE) was used to investigate the expression of halotolerant proteins under high (3 M NaCl) and low (0.75 M NaCl) salt concentrations. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) and bioinformatics were used to identify and characterize the differences among proteins. 2D-DIGE analysis revealed 141 protein spots that were significantly differentially expressed between the two salinities. Twenty-four differentially expressed protein spots were successfully identified by MALDI-TOF/TOF MS, including proteins in the following important categories: molecular chaperones, proteins involved in photosynthesis, proteins involved in respiration and proteins involved in amino acid synthesis. Expression levels of these proteins changed in response to the stress conditions, which suggests that they may be involved in the maintenance of intracellular osmotic pressure, cellular stress responses, physiological changes in metabolism, continuation of photosynthetic activity and other aspects of salt stress. The findings of this study enhance our understanding of the function and mechanisms of various proteins in salt stress.

Neste artigo, foi desenvolvido um novo método fluorométrico, simples, sensível e seletivo, para a medição de tripsina em amostras biológicas. O método foi baseado na medição da supressão da intensidade da fluorescência de agregados de nanopartículas de ouro estabilizados com albumina sérica bovina (BSA), pela proteólise enzimática. A curva de calibração para tripsina foi atingida no intervalo de concentração de 1-60 nmol L-1 com coeficiente de correlação 0,995 e um limite de detecção de 0,6 nmol L-1. O método foi também usado satisfatoriamente para avaliação da atividade da tripsina e os resultados mostraram que os valores das constantes de Michaelis-Menten (Km) e catalítica (Kcat), de tripsina para estes substratos de nanoagreagados BSA-Au, foram 1,6×10-5 mol L-1 e 3,8 s-1 a 37 ºC, respectivamente. Este biossensor enzimático é de interesse considerável devido à simplicidade do procedimento, e o método estabelecido tem grande potencial na detecção de outras proteases em diagnósticos clínicos de várias doenças.

A novel, simple, sensitive, and selective fluorometric method was developed for measuring trypsin in biological samples in this article. The method was based upon measuring the quenching of the fluorescence intensity of the bovine serum albumin (BSA) stabilized Au nanoclusters by enzymatic proteolysis. The calibration plot for trypsin was achieved over the concentration range 1-60 nmol L-1 with a correlation coefficient of 0.995 and a limit of detection of 0.6 nmol L-1. The method was also used satisfactorily for the assessment of the trypsin activity and the results showed that the Michaelis-Menten (Km) and catalytic (Kcat) constant values of trypsin for BSA-Au nanoclusters substrate were 1.6×10-5 mol L-1 and 3.8 s-1 at 37 ºC, respectively. This enzyme biosensor is of considerable interest due its promise for simple procedure and the established method has great potential in detection of other proteases in clinical diagnostics of various diseases.