Characterization of ß-trypsin at acid pH by differential scanning calorimetry
Characterization of ß-trypsin at acid pH by differential scanning calorimetry
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DOI：
10.1590/s0100-879x2003001200003
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发表时间：
2003-12-01
影响因子：
2.3
通讯作者：
Santoro, M.M.
中科院分区：
文献类型：
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作者：
Bittar, E.R.;Caldeira, F.R.;Santoro, M.M.
胰蛋白酶是一种丝氨酸蛋白酶，具有223个氨基酸残基的多肽链，含有6个二硫桥。它是一种球状蛋白，二级结构以反平行的β -片和螺旋结构为主，具有两个结构域。我们使用微量热法（差示扫描量热法）技术评估β -胰蛋白酶在酸性pH范围内的稳定性。蛋白质浓度在0.05 ~ 2.30 mg/ml范围内变化。缓冲溶液为50.0 mM β -丙氨酸和20.0 mM CaCl2， pH值（2.0 ~ 4.2）不同，山梨醇（1.0、2.0 M）、尿素（0.5 M）或盐酸胍（0.5、1.0 M）浓度不同。这些数据表明，在pH、山梨醇、尿素和盐酸胍作为干扰剂的所有实验条件下，我们研究的是相同的蛋白质构象转变。在3.2 ~ 4.2的pH范围内，范霍夫比值(δ tah （cal)/ δ tah (vH)）为1.0 ~ 0.5，表明蛋白质聚集。相反，δ tah (cal)/ δ tah （vH）比值在2.0 ~ 3.2的pH范围内等于1，表明该蛋白以单体形式展开。在pH 3.00时，β -胰蛋白酶展开时Tm = 54°c, δ tah = 101.8 kcal/mol，蛋白质（cCp）的天然形式和未展开形式之间的热容变化估计为2.50 +/- 0.07 kcal mol(-1) K-1。在298 K下计算得到β -胰蛋白酶的稳定性，pH值为3.00时δ tag (D) = 5.7 kcal/mol， pH值为7.00时δ tag (D) = 15.2 kcal/mol，其值在小球状蛋白的预期范围内。
Trypsin is a serino-protease with a polypeptide chain of 223 amino acid residues and contains six disulfide bridges. It is a globular protein with a predominance of antiparallel beta-sheet and helix in its secondary structure and has two domains with similar structures. We assessed the stability of beta-trypsin in the acid pH range using microcalorimetric (differential scanning calorimetry) techniques. Protein concentrations varied in the range of 0.05 to 2.30 mg/ml. Buffer solutions of 50.0 mM beta-alanine and 20.0 mM CaCl2 at different pH values (from 2.0 to 4.2) and concentrations of sorbitol (1.0 and 2.0 M), urea (0.5 M) or guanidinium hydrochloride (0.5 and 1.0 M) were used. The data suggest that we are studying the same conformational transition of the protein in all experimental situations using pH, sorbitol, urea and guanidinium hydrochloride as perturbing agents. The observed van't Hoff ratios (DeltaH(cal)/DeltaH(vH)) of 1.0 to 0.5 in the pH range of 3.2 to 4.2 suggest protein aggregation. In contrast, DeltaH(cal)/DeltaH(vH) ratios equal to one in the pH range of 2.0 to 3.2 suggest that the protein unfolds as a monomer. At pH 3.00, beta-trypsin unfolded with Tm = 54degreesC and DeltaH = 101.8 kcal/mol, and the change in heat capacity between the native and unfolded forms of the protein (cCp) was estimated to be 2.50 +/- 0.07 kcal mol(-1) K-1. The stability of beta-trypsin calculated at 298 K was DeltaG(D) = 5.7 kcal/mol at pH 3.00 and DeltaG(D) = 15.2 kcal/mol at pH 7.00, values in the range expected for a small globular protein.