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Effect of guanidine hydrochloride(cas 50-01-1) concentration on the refolding of denatured bovine carbonic acid dehydratase (CAB) by different additives

In this paper, the effects of different additives on the renaturation of denatured bovine carbonic acid dehydratase(CAB) were studied. The effects of various factors (additive concentration, pH value, temperature and concentration of additive at lower concentration of guanidine hydrochloride(cas 50-01-1)) on the refolding yield were investigated. The kinetic process of amide compound assisted CAB refolding was studied by using the relevant kinetic model. The mechanism of acetamide promoting CAB refolding was discussed. The possibility of refolding CAB on other compounds was tested.

The results showed that acetamide, urea, thiourea, acetone, β-CD, CTAB + β-CD and Tween 40 + β-CD could effectively degrade denatured CAB. Β-CD, CTAB + β-CD and Tween 40 + β-CD increased with the increase of concentration, while acetamide, urea and thiourea had the best concentration to denature the denatured yield of denatured CAB. Amide, urea, thiourea, acetone and other additives do not change the pH value of the refolding solution, can be in a wide pH range (5.5 ~ 8.5) to promote CAB refolding; additive addition to change the CAB refolding yield does not follow temperature changes in the law, urea, acetamide at 37 ℃, thiourea at 45 ℃ when the maximum refolding yield.

The kinetic process of CAB refolding with amide compounds was studied by using the third - order reaction kinetic equation. The results show that the calculated values are in good agreement with the experimental data. The amide compound is similar to guanidine hydrochloride(50-01-1). The refolding rate constant KN is greater than that of the absence of additive, but decreases with the increase of the additive concentration. The aggregation rate constant KA is the smallest at the optimum additive concentration, KN /KA value of the largest, the highest rate of refolding r value. By reducing the concentration of guanidine hydrochloride in the refolding solution and increasing the concentration of the additive, acetamide and urea can still achieve the same renaturation effect on the denatured CAB. Thiourea must be effective in promoting the repressibility of CAB in the presence of guanidine hydrochloride(50-01-1). Because high concentrations of acetamide can cause protein irreversibility, acetamide can be used as an effective additive for adjuvant protein refolding, but can not be used as a denaturant for renatured proteins. In order to further elucidate the mechanism of action of acetamide-assisted denatured CAB refolding, similar compounds such as formamide, propionamide, N, N-dimethylformamide, N, N-dimethyl. The effects of acetoamides on the renaturation of denatured CAB have been studied and found that they are effective in promoting CAB refolding, both in terms of their kinetic properties of assisted CAB refolding, or with pH and temperature, so that the amide group on the refolding of protein has a greater contribution to the role.

 

 

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