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巨大螺旋藻(Spirulina maxima)藻胆蛋白的特点及稳定性研究
陈建中
Subtype硕士
Thesis Advisor张宪孔
1990
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline水生生物学
Abstract巨大螺旋藻(Spirulina maxima)的水溶性粗提物经硫酸铵沉淀后用羟基磷灰石和Bio-geli-p300进行两次柱层析可得两种藻胆蛋白,即藻蓝蛋白(C-PC)用别藻蓝蛋白(AP C)。它们的纯度(在可见光部分的最大吸收与280nm处吸收之比)可分别达到6.2和6.35。将已纯化后的APC再经过一次羟基磷灰石柱层析,可分离出别藻蓝素B(APC-B)。C-PC、APC和APC-B在可见光区的最大吸收峰分别为620nm、650nm和667nm(肩峰621nm)。其室温荧光发射峰分别为642nm、657nm和676nm。纯化后的C-PC和APC在聚丙烯酰胺凝胶电泳上仅见一条色带,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE),C-PC和APC均为α和β两个亚单位。C-PCAPC的亚单位分子量分别为:α,20.0KD和23.0KD;β,18.5KD和21.0KD。依次推算该藻的C-PC和APC的最低分子量应为43.0KD和39.5KD。它与钝顶螺旋藻基本相似而又稍高于盐泽螺旋藻。经等电聚焦测定其C-PC、APC和APC-B物等电点分别为4.9、5.3和5.5。三种藻胆蛋白的氨基酸组成以酸性氨基和疏水性氨基酸为主。C-PC和APDC均含有所测定的17种氨基酸,而APC-B只测出14种氨基酸。研究了温度、pH、光和伴溶物对巨大螺旋藻的两种主要藻胆蛋白稳定性的影响。藻胆蛋白对温度的冲击(加热5min)有一定的忍耐度。在低于60 ℃温度下吸收峰高有所下降,但未见因蛋白质变性而产生沉淀。70 ℃有相当量的蛋白变性沉淀物,同时,藻胆蛋白的发色团的构型也由直线型变为环型。在pH4.5-9.0范围内,藻胆蛋白保持相对稳定。藻蓝蛋白在pH > 10.5或<2.0时,吸收峰分别向短波光区和长波光区移动。这主要应该是由于聚集状态改变引起的。APC在pH > 9.0或<4.5时,吸收峰从650nm移到625nm,即从三聚体变为单聚体形式。在pH > 13.5或<1.0的极端pH条件下,藻胆蛋白因变性而产生少量沉淀。光对藻胆蛋白的影响明显,接触光照藻胆蛋白迅速褪色,看来主要是发色团的光氧化作用所致。在可见光下,藻胆蛋白的发色团的A环断裂从而使藻胆蛋白褪色。加入适量的NaCl和NgSO_4会使光稳定性有所增加,这可能是由于Mg~(2+)、Na~+降低了发色团的光氧化作用。紫外照射,既引起蛋白质的变性,又产生光氧化作用,因此,在照射第四天后几乎完全褪色。紫外光照射后导致藻胆蛋白溶液在近紫外区358nm处形成一个明显的吸收峰585nm出现荧光发射峰,造成这一变化的原因尚待进一步研究。
Other AbstractPhycobiliproteins from Spirulina maxima were separated on a hydroxylaptite column and Bio-gelP300 columinto three biliproteins C-PC and APC and APC-B. The absorption and fluorescence maxima of the C-PC APC resemble those reported for other blue-green algae, while APC-B pssesses an absorption maxima at 667 nm with a shulder at 621 nm and room fluorescence emission at 676 nm. Electrophoresis of the biliproteins after treated with SDS indicating that each one included two subunits. The molecular weigthts for the α and β subunits C-PC are 20.0 KD and 23.0 KD respectively and 18.5 KD, 21.0KD for these respective subunits in APC. Acording to these data, the minimum molecular, are similar to those in spirulina plantensis and greater than those in spiulina subsalsa var. Isoelectric points of C-PC, APC and APC-B are 4.9, 5.3, and 5.5 respectively. The amino acid composition are not significantly different from that reported for other blue-green algae. All biliproteins from Spirulina maxima are relatively rich in aliphatic and biliproteins from Spirulina maxima are relatively rich in aliphatic and acidic amino acids. Stability of biliproteins denpend upon a number of factors including pH, temperature, ionic strength and light. C-PC and APC remained stable when less than 60 ℃ for 5 min except that some degression of the absorption intensity. At 70 ℃ the biliproteins were denatured and precipited. These denatured biliproteins are so similar in their properties to free bile pigments that their chromophores are also expected to assume preferentially cyclohelical conformations. Both of biliproteins retained relatively stable with a broad pH range from 4.5 to 9.0. When pH > 10.5 or < 2.0, the absorption maxima were respectively red-shifted or blue-shifted, probably resulting from its dissociation. At pH values greater than 9.5 or less than 4.5, the shoulder peak of APC at 625nm was almost the same but the peak at 650nm was completely disappeared, indicating that the peak at 650 nm was completely disappeared, indicating that the trimetric (αβ)_3 was dissociated into monomers (αβ). The biliproteins from Spiruina maxima are also sensitive to illumination. Under visiable light, the photo breaching of the C-PC and /or APC probably is due to photooxydation of bilinbound to the protein complexes. However, ultraviolet light cause not only photooxydation but denaturation of the pigments also. It is interesting that a new absorption pak at 358 nm and fluorescence emission peak at 585 nm appeared from APC under ultroviolet light. Which will be further investigated.(αβ)
Pages32
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12808
Collection学位论文
Recommended Citation
GB/T 7714
陈建中. 巨大螺旋藻(Spirulina maxima)藻胆蛋白的特点及稳定性研究[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,1990.
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