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运用荧光光谱方法研究了脱辅基的辣根过氧化物酶同工酶Ｃ［ａｐｏ－ｈｏｒｓｅｒａｄｉｓｈｐｅｒｏｘｉｄａｓｅＣ，ａｐｏ－ＨＲＰ（Ｃ）］。结果表明：（１）ａｐｏ－ＨＲＰ（Ｃ）与其他“Ｂ”类蛋白质不同，有明显的Ｔｙｒ荧光；（２）低浓度变性剂（＜２ｍｏｌ／Ｌ脲或０．２ｍｏｌ／Ｌ盐酸胍）能增强酶的Ｔｒｐ荧光，但并不改变光谱特性；（３）进一步增加变性剂浓度则使Ｔｒｐ残基暴露于水溶液中，荧光强度略有降低，荧光谱红移；（４）Ｃａ２＋络合剂ＥＤＴＡ对色氨酸荧光的影响与低浓度变性剂相同。有意思的是，ＥＤＴＡ与低浓度变性剂二者中任一个对ａｐｏ－ＨＲＰ（Ｃ）荧光发生影响后，另一个就不再起作用，由此推测ＥＤＴＡ与低浓度的变性剂可能是通过同一途径即脱钙来影响Ｔｒｐ荧光；利用荧光探针（８－ａｎｉｌｉｎｏ－１－ｎａｐｈｔａｌｅｎｅｓｕｌｆｏｎａｔｅ，ＡＮＳ）的研究结果得知，ＡＮＳ与酶结合的解离常数在无钙离子时为１５．４μｍｏｌ／Ｌ，而有钙离子时为７．３μｍｏｌ／Ｌ，表明Ｃａ２＋能增强ＡＮＳ与酶的活性中心的结合，提示钙离子在酶活性中所起的作用是通过影响血红素结合区域的构象来实现的。
Apo-horseradishperoxidase C (apo-HRP (C)] was studied by fluorescence spectroscopy. The results showed that: (1) apo-HRP (C) was different from other “B” proteins and had obvious Tyr fluorescence; (2) low concentration denaturant (<2mol / L urea or 0.2mol / L guanidine hydrochloride) (3) further increase the denaturant concentration, the Trp residue is exposed to the aqueous solution, the fluorescence intensity is slightly decreased and the fluorescence spectrum is red shifted; (4) the Ca2 + complexing agent EDTA The effect on tryptophan fluorescence is the same as for low concentration denaturants. Interestingly, the effect of either EDTA or low concentration denaturant on the fluorescence of apo-HRP (C), the other one no longer works, suggesting that EDTA and low concentrations of denaturants may be in the same pathway Decalcification to affect the Trp fluorescence; the use of fluorescent probe (8-anilino-1-naphtalenesulfonate, ANS) study found that ANS dissociation constant with the enzyme in the absence of calcium was 15.4μmol / L, and Calcium ion was 7.3μmol / L, indicating that Ca2 + can enhance the binding of ANS and enzyme activity centers, suggesting that the role of calcium ions in the enzyme activity by affecting the hemopexin binding region to achieve the conformation.