RESUMO
The influence of l-homoarginine on the heat-induced aggregation of three model proteins, i.e. porcine, mink, and human growth hormones was investigated by circular dichroism spectroscopy. It was found that the effect of l-homoarginine as an analogue of arginine depends on the concentration of the additive as well as the protein itself. l-Homoarginine increased the onset temperature of heat-induced aggregation of both porcine and mink growth hormones. However, the formation of human growth hormone aggregates was increased at low concentrations of l-homoarginine. Only at higher concentrations of the additive was the onset temperature of human growth hormone aggregation found to increase. Additional experiments of human growth hormone melting in the presence of histidine, lysine, and sodium chloride were performed. The effect of lysine was similar as in the presence of l-homoarginine. It follows that in protein formulations low concentrations of amino acids should be used with some precaution. At low concentration of additive, depending on the charge of both protein and amino acid used, the promotion of aggregation of unfolding intermediates may occur.
Assuntos
Arginina/química , Hormônio do Crescimento/química , Homoarginina/química , Hormônio do Crescimento Humano/química , Animais , Arginina/análogos & derivados , Dicroísmo Circular , Histidina/química , Temperatura Alta , Humanos , Lisina/química , Vison , Cloreto de Sódio/química , SuínosRESUMO
Circular dichroism spectroscopy was used to study the effect of l-arginine on the temperature related unfolding and aggregation of three growth hormones, i.e. human, porcine and mink growth hormones, and human interferon-α2b. (L)-arginine can stabilize some proteins and suppress their aggregation as it was exemplified by porcine and mink growth hormones. For some other proteins, on the contrary, the effect of arginine can be negative. Even at low concentrations the amino acid is able to promote the aggregation as it was demonstrated by the experiments with human growth hormone and interferon-α2b. (L)-arginine seems not to be a universal excipient for preventing the temperature related aggregation of proteins in contrast to its widespread application in the refolding process.
Assuntos
Arginina/farmacologia , Multimerização Proteica/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Dicroísmo Circular , Hormônio do Crescimento/química , Hormônio do Crescimento/efeitos dos fármacos , Temperatura Alta , Hormônio do Crescimento Humano/química , Hormônio do Crescimento Humano/efeitos dos fármacos , Humanos , Técnicas In Vitro , Interferon alfa-2 , Interferon-alfa/química , Interferon-alfa/efeitos dos fármacos , Vison , Nefelometria e Turbidimetria , Dobramento de Proteína/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Proteínas Recombinantes , SuínosRESUMO
The influence of L-arginine on the temperature-induced aggregation of porcine and mink growth hormones was studied by fluorescence spectroscopy. It was found that L-arginine suppresses the heat-induced aggregation. Moreover, the analysis of L-arginine interaction with the native proteins by fluorescence spectroscopy and circular dichroism spectroscopy revealed no significant changes in their native structure. On the basis of the results, L-arginine could be considered as a potential additive for the prevention of storage and temperature-related denaturation and aggregation of veterinary growth hormones.
Assuntos
Arginina/química , Hormônio do Crescimento/química , Desnaturação Proteica , Animais , Dicroísmo Circular/métodos , Temperatura Alta , Vison , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , SuínosRESUMO
Cyclodextrins with different ring size and ring substituents were tested for recombinant mink and porcine growth hormones aggregation suppression in the refolding process from Escherichia coli inclusion bodies. Methyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin show a positive effect on the aggregation suppression of both proteins. The influence of different methyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin concentrations on the renaturation yield of both growth hormones was investigated. Moreover, methyl-beta-cyclodextrin and 2-hydroxypropyl-beta-cyclodextrin suppress not only folding-related, but also temperature-related aggregates formation of both proteins. Circular dichroism experiments (monitoring of protein solution turbidity by registering high tension voltage) showed that the onset temperature of aggregation of both growth hormones increased with increasing 2-hydroxypropyl-beta-cyclodextrin concentration. In conclusion, cyclodextrins have perspectives in biotechnology of veterinary growth hormones not only for protein production, but also for its storage.
