Gardlik S, Barber M J, Rajagopalan K V
Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
Arch Biochem Biophys. 1987 Dec;259(2):363-71. doi: 10.1016/0003-9861(87)90502-9.
A previously unidentified fraction lacking xanthine:O2 activity has been isolated during affinity chromatography of bovine milk xanthine oxidase preparations on Sepharose 4B/folate gel. Unlike active, desulfo, or demolybdo forms of xanthine oxidase, this form, which typically comprises about 5% of an unfractionated enzyme solution, passes through the affinity column without binding to it, and is thus easily separated from the other species. The absorption spectrum of this fraction is very similar to that of the active form, but has a 7% lower extinction at 450 nm. Analysis of the fraction has shown that it is a dimer of normal size, but that it does not contain molybdenum or molybdopterin (MPT). The "MPT-free" xanthine oxidase contains 90-96% of the Fe found in active xanthine oxidase, and 100% of the expected sulfide. EPR and absorption difference spectroscopy indicate that the MPT-free fraction is missing approximately half of its Fe/S I centers. The presence of a new EPR signal suggests that an altered Fe/S center may account for the nearly normal Fe and sulfide content. Microwave power saturation parameters for the Fe/S II and Fe/S I centers in the MPT-free fraction are normal, with P1/2 equal to 1000 and 60 mW, respectively. The new EPR signal shows intermediate saturation behavior with a P1/2 = 200 mW. The circular dichroism spectrum of the MPT-free fraction shows distinct differences from that of active enzyme. The NADH:methylene blue activity of the MPT-free fraction is the same as that of active xanthine oxidase which exhibits xanthine:O2 activity, but NADH:cytochrome c and NADH:DCIP activities are diminished by 54 and 37%, respectively.
在牛乳腺黄嘌呤氧化酶制剂于琼脂糖4B/叶酸凝胶上进行亲和层析期间，分离出了一种先前未鉴定的、缺乏黄嘌呤：O₂活性的组分。与黄嘌呤氧化酶的活性形式、脱硫形式或脱钼形式不同，这种形式（通常占未分级酶溶液的约5%）通过亲和柱时不与之结合，因此很容易与其他形式分离。该组分的吸收光谱与活性形式非常相似，但在450 nm处的消光值低7%。对该组分的分析表明，它是正常大小的二聚体，但不含钼或钼蝶呤（MPT）。“无MPT”黄嘌呤氧化酶所含的铁为活性黄嘌呤氧化酶中铁含量的90 - 96%，且硫含量为预期的100%。电子顺磁共振（EPR）和吸收差光谱表明，无MPT组分大约缺失了一半的铁硫I中心。新EPR信号的存在表明，改变的铁硫中心可能解释了近乎正常的铁和硫含量。无MPT组分中铁硫II和铁硫I中心的微波功率饱和参数正常，P1/2分别等于1000和60 mW。新的EPR信号表现出中间饱和行为，P1/2 = 200 mW。无MPT组分的圆二色光谱与活性酶的光谱有明显差异。无MPT组分的NADH：亚甲蓝活性与具有黄嘌呤：O₂活性的活性黄嘌呤氧化酶相同，但NADH：细胞色素c和NADH：2,6 - 二氯靛酚（DCIP）活性分别降低了54%和37%。
