Phillips R S, Marmorstein R Q
Department of Chemistry, School of Chemical Sciences, University of Georgia, Athens 30602.
Arch Biochem Biophys. 1988 Apr;262(1):337-44. doi: 10.1016/0003-9861(88)90196-8.
The binding of 6-nitro-L-tryptophan to trp aporepressor and human serum albumin has been examined by visible difference spectroscopy and circular dichroism. 6-Nitro-L-tryptophan, prepared by nitration of L-tryptophan with nitric acid in glacial acetic acid, exhibits a visible and near-uv absorption spectrum with lambda max at about 330 nm (epsilon = 7 X 10(3) M-1 cm-1) and a shoulder near 380 nm in H2O. In the presence of trp aporepressor, the visible absorption intensity is sharply diminished. Visible difference spectral titration data give KD = 1.27 X 10(-4) M and n = 0.95 per subunit at 25 degrees C. While 6-nitro-L-tryptophan exhibits no significant circular dichroism between 300 and 500 nm, the complex with trp aporepressor exhibits strong circular dichroism signals, with a negative maximum at 386 nm (delta epsilon = -7.5 M-1 cm-1) and a positive maximum at 310 nm (delta epsilon = +6 M-1 cm-1). Circular dichroism titration data give KD = 1.69 X 10(-4) M and n = 0.90 per subunit at 25 degrees C. The KD values determined spectroscopically are in excellent agreement with that determined by equilibrium dialysis, KD = 1.5 X 10(-4) M at 25 degrees C. In the presence of human serum albumin, the spectrum of 6-nitro-L-tryptophan exhibits a blue shift and an increase in absorption intensity; similar changes are observed in solvents of low dielectric contrast such as 80% aqueous dioxane. Visible difference spectral titration data give KD = 8.0 X 10(-5) M and n = 0.95 for human serum albumin. The complex of 6-nitro-L-tryptophan with human serum albumin exhibits a strong positive circular dichroism maximum at 380 nm (delta epsilon = +9.8 M-1 cm-1) with a shoulder at 310-320 nm. Circular dichroism titration data give KD = 6.4 X 10(-5) M and n = 0.83, in good agreement with the visible difference spectral results. Taken together, our results demonstrate the utility of 6-nitro-L-tryptophan as a spectroscopic probe for tryptophan-binding proteins.
通过可见差分光谱法和圆二色性研究了6-硝基-L-色氨酸与色氨酸脱辅基阻遏蛋白及人血清白蛋白的结合情况。6-硝基-L-色氨酸由L-色氨酸在冰醋酸中用硝酸硝化制备，在水中呈现可见和近紫外吸收光谱，最大吸收波长λmax约为330nm（ε = 7×10³ M⁻¹ cm⁻¹），在380nm附近有一个肩峰。在色氨酸脱辅基阻遏蛋白存在下，可见吸收强度急剧降低。可见差分光谱滴定数据表明，在25℃时，解离常数KD = 1.27×10⁻⁴ M，每个亚基的结合数n = 0.95。虽然6-硝基-L-色氨酸在300至500nm之间没有明显的圆二色性，但与色氨酸脱辅基阻遏蛋白形成的复合物呈现出很强的圆二色性信号，在386nm处有一个负的最大值（Δε = -7.5 M⁻¹ cm⁻¹），在310nm处有一个正的最大值（Δε = +6 M⁻¹ cm⁻¹）。圆二色性滴定数据表明，在25℃时，KD = 1.69×10⁻⁴ M，每个亚基的结合数n = 0.90。通过光谱法测定的KD值与通过平衡透析法测定的结果非常吻合，在25℃时KD = 1.5×10⁻⁴ M。在人血清白蛋白存在下，6-硝基-L-色氨酸的光谱出现蓝移且吸收强度增加；在低介电常数的溶剂如80%的二氧六环水溶液中也观察到类似变化。可见差分光谱滴定数据表明，与人血清白蛋白结合时，KD = 8.0×10⁻⁵ M，n = 0.95。6-硝基-L-色氨酸与人血清白蛋白形成的复合物在380nm处有一个很强的正圆二色性最大值（Δε = +9.8 M⁻¹ cm⁻¹），在310 - 320nm处有一个肩峰。圆二色性滴定数据给出KD = 6.4×10⁻⁵ M，n = 0.83，与可见差分光谱结果吻合良好。综上所述，我们的结果证明了6-硝基-L-色氨酸作为色氨酸结合蛋白的光谱探针的实用性。
