蛋白质的沉淀与变性反应

蛋白质的沉淀与变性反应 實驗 3 蛋白質的沉澱與變性反應 目的 (1)瞭解蛋白質的沉澱反應、變性作用和凝固作用的原理及它們的相互關係。 (2)學習鹽析和透析等生物化學的操作技術。 原理 在水溶液中,蛋白質分子的表面,由於形成水化層和雙電層而成為穩定 的膠體顆粒,所以蛋白質溶液和其他親水膠體溶液相類似。但是,蛋白質膠 體顆粒的穩定性是有條件的,相對的。在一定的物理化學因素影響下,蛋白 質顆粒失去電荷,脫水,甚至變性,則以固態形式從溶液中析出,這個過程 稱為蛋白質的沉澱反應。這種反應可分為以下兩種類型: 一、可逆澱汲反應 在發生沉澱反應時,蛋白質雖已沉澱析出,但它的分子內部結構並未發 生顯著變化,基本上保持原有的性質,沉澱因素除去後,能再溶於原來的溶 劑中。這種作用稱為可逆沉澱反應,又叫作不變性沉澱反應。屬於這一類的 反應有鹽析作用; 在低溫下,乙醇、丙酮對蛋白質的短時間作用以及利用等 電點的沉澱等。 二、不可逆沉澱反應 welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineering 1 GB150-98 2 GB50128-2005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ T4735-1997 steel welded atmospheric pressure vessel 4 GB50231-2009 mechanical equipment installation engineering construction and acceptance specification for 5 GB50275-98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-2000 construction installation engineering construction standard 7, HG ... Pressure gauges, using an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan. Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the opening and provisional protective measures have been taken, the responsible unit fined 200 Yuan, construction person in charge a fine of 50 Yuan. (8) no permit to work and the job in accordance with the provisions, on the job unit fined 300 yuan, the unit will charge a fine of 100 Yuan. 7.5.3 accident penalties (1) injuries fatalities directly punished 1~2 million. Direct responsibility for the accident and have the corresponding responsibility of leadership, such as concerning administrative sanctions, should be brought to the company or the relevant Department. (2) personal injury accident occurred, the direct punishment 0.5-10,000 yuan, responsible for the direct responsibility for the accident and the responsibility of leadership, such as concerning administrative sanctions should be dealt with by the personnel Department of the company. (3) personal injury 在發生沉澱反應時,蛋白質分子內部結構、空間構象遭到破壞,失去原 來的天然性質,這時蛋白質已發生變性。這種變性蛋白質的沉澱不能再溶解 於原來溶劑中的作用叫作不可逆沉澱反應。重金屬鹽、植物鹼試劑、過酸、 過鹼、加熱、震盪、超聲波,有機溶劑等都能使蛋白質發生不可逆沉澱反應。 試劑和器材 一、試劑 1,蛋白質溶液 取5m1雞蛋蛋白蛋清,用蒸餾水稀釋至100ml,攪拌均勻後用4-8層紗 布過濾,新鮮配製。 2,蛋白質氯化鈉溶液 取20ml蛋清,加蒸餾水200ml和飽和氯化鈉溶液100ml,充分攪勻 後,以紗布濾去不溶物(加入氯化鈉的目的是溶解球蛋白)。 硫酸銨粉末,飽和硫酸銨溶液,3%硝酸銀,0.5% 醋酸鉛,10% 三氯醋 酸,濃鹽酸,濃硫酸,濃硝酸,5% 磺基水楊酸(sulfosalicyclic acid),0.1% 硫酸銅,飽和硫酸銅溶液,0.1% 醋酸,10% 醋酸,飽和氯化鈉溶液,10% 氫氧化鈉溶液。 二、器材 試管,試管架,小玻璃漏斗,濾紙,玻璃紙,玻璃棒,500ml 燒杯,10 ml量筒。 welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineering 1 GB150-98 2 GB50128-2005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ T4735-1997 steel welded atmospheric pressure vessel 4 GB50231-2009 mechanical equipment installation engineering construction and acceptance specification for 5 GB50275-98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-2000 construction installation engineering construction standard 7, HG ... Pressure gauges, using an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan. Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the opening and provisional protective measures have been taken, the responsible unit fined 200 Yuan, construction person in charge a fine of 50 Yuan. (8) no permit to work and the job in accordance with the provisions, on the job unit fined 300 yuan, the unit will charge a fine of 100 Yuan. 7.5.3 accident penalties (1) injuries fatalities directly punished 1~2 million. Direct responsibility for the accident and have the corresponding responsibility of leadership, such as concerning administrative sanctions, should be brought to the company or the relevant Department. (2) personal injury accident occurred, the direct punishment 0.5-10,000 yuan, responsible for the direct responsibility for the accident and the responsibility of leadership, such as concerning administrative sanctions should be dealt with by the personnel Department of the company. (3) personal injury 操作 一、蛋白質的可逆沉澱反應 蛋白質的鹽析作用 用大量中性鹽使蛋白質從溶液中沉澱析出的過程稱為蛋白質的鹽析作用。蛋白質是親水膠體,在高濃度的中性鹽影響下,蛋白質分子被鹽脫去水化層,同時蛋白質分子所帶的電荷被中和,結果蛋白質的膠體穩定性遭受破壞而沉澱析出。析出的蛋白質仍保持其天然蛋白質的性質。減低鹽的濃度時,還能溶解。 沉澱不同的蛋白質所需中性鹽的濃度不同,而鹽類不同也有差異。例如:向含有白蛋白和球蛋白的雞蛋清溶液中加硫酸鎂或氯化鈉至飽和,則球蛋白沉澱析出。加硫酸錢至飽和,則白蛋白沉澱析出。另外,在等電點時,白蛋白可被飽和硫酸鎂或氯化鈉或半飽和的硫酸銨溶液沉澱析出。所以在不同條件下,用不同濃度的鹽類可將各種蛋白質從混合溶液中分別沉澱析出,該法稱為蛋白質的分級鹽析。目前在醃的生產和製備、科學研究工作和臨床化驗等工作中廣泛應用。 取一支試管加入3m1蛋白質氯化鈉溶液和3m1飽和硫酸銨溶液,混勻,靜置約10 min,球蛋白則沉澱析出,過濾後向濾液中加入硫酸銨粉末,邊加邊用玻璃棒攪拌,直至粉末不再溶解,達到飽和為止。析出的沉澱為白蛋白。靜置,倒去上部清液,白蛋白沉澱,取出部分加水稀釋,觀察它是否溶解,留存部分作透析用。 二、蛋白質的不可逆沉激反應 welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineering 1 GB150-98 2 GB50128-2005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ T4735-1997 steel welded atmospheric pressure vessel 4 GB50231-2009 mechanical equipment installation engineering construction and acceptance specification for 5 GB50275-98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-2000 construction installation engineering construction standard 7, HG ... Pressure gauges, using an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan. Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the opening and provisional protective measures have been taken, the responsible unit fined 200 Yuan, construction person in charge a fine of 50 Yuan. (8) no permit to work and the job in accordance with the provisions, on the job unit fined 300 yuan, the unit will charge a fine of 100 Yuan. 7.5.3 accident penalties (1) injuries fatalities directly punished 1~2 million. Direct responsibility for the accident and have the corresponding responsibility of leadership, such as concerning administrative sanctions, should be brought to the company or the relevant Department. (2) personal injury accident occurred, the direct punishment 0.5-10,000 yuan, responsible for the direct responsibility for the accident and the responsibility of leadership, such as concerning administrative sanctions should be dealt with by the personnel Department of the company. (3) personal injury 1,重金屬沉澱蛋白質 重金屬鹽類易與蛋白質結合成穩定的沉澱而析出。蛋白質在水溶液中是酸鹼兩性電解質,在鹼性溶液中(對蛋白質的等電點而言),蛋白質分子帶負電荷,能與帶正電荷的金屬離子結合成蛋白質鹽。在有機體內,蛋白質常以其可溶性的鈉鹽或鉀鹽的形式存在,當加入汞,鉛、銅、銀等重金屬鹽時,則蛋白質形成不溶性的鹽類而沉澱。經過這種處理後的蛋白質沉澱不再溶解於水中,說明它已發生了變性。重金屬鹽類沉澱蛋白質的反應通常很完全,特別是在鹼金屬鹽類存在時。因此,生化分析中,常用重金屬鹽除去體液中的蛋白質;臨床上用蛋白質解除重金屬鹽的食物性中毒。但應注意,使用醋酸鉛或硫酸銅沉澱蛋白質時,試劑不可加過量,否則可使沉澱出的蛋白質重新溶解。 取2支試管,各加入約1m1蛋白質溶液,分別加入3 % 硝酸銀3-4滴,0.5% 醋酸鉛1-3滴和0.1%硫酸銅3-4滴,觀察沉澱的生成。第一、二支試管再分別加入過量的醋酸鉛和飽和硫酸銅溶液,觀察沉澱的再溶解。 2,有機酸沉澱蛋白質 有機酸能使蛋白質沉澱。三氯醋酸和磺基水楊酸最有效,能將血清等生物體液中的蛋白質完全除去,因此得到廣泛使用。 取兩支試管,各加入蛋白質溶液約0.5 m1,然後分別滴加10% 三氯醋酸和5%磺基水楊酸溶液各數滴,觀察蛋白質的沉澱。 welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineering 1 GB150-98 2 GB50128-2005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ T4735-1997 steel welded atmospheric pressure vessel 4 GB50231-2009 mechanical equipment installation engineering construction and acceptance specification for 5 GB50275-98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-2000 construction installation engineering construction standard 7, HG ... Pressure gauges, using an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan. Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the opening and provisional protective measures have been taken, the responsible unit fined 200 Yuan, construction person in charge a fine of 50 Yuan. (8) no permit to work and the job in accordance with the provisions, on the job unit fined 300 yuan, the unit will charge a fine of 100 Yuan. 7.5.3 accident penalties (1) injuries fatalities directly punished 1~2 million. Direct responsibility for the accident and have the corresponding responsibility of leadership, such as concerning administrative sanctions, should be brought to the company or the relevant Department. (2) personal injury accident occurred, the direct punishment 0.5-10,000 yuan, responsible for the direct responsibility for the accident and the responsibility of leadership, such as concerning administrative sanctions should be dealt with by the personnel Department of the company. (3) personal injury 3,無機酸沉澱蛋白質 濃無機酸(除磷酸外)都能使蛋白質發生不可逆的沉澱反應。這種沉澱作用可能是蛋白質顆粒脫水的結果。過量的無機酸(硝酸除外)可使沉澱出的蛋白質重新溶解。臨床診斷上,常利用硝酸沉澱蛋白質的反應,檢查尿中蛋白質的存在。 取3支試管,分別加入濃鹽酸15滴,濃硫酸、濃硝酸10滴。小心地向3支試管中,沿管壁加入蛋白質溶液6滴,不要搖動,觀察各管內兩液界面處有白色環狀蛋白質沉澱出現。然後,搖動每個試管。蛋白質沉澱應在過量的鹽酸及硫酸中溶 解。在含硝酸的試管中,雖經振盪,蛋白質沉澱也不溶解。 4,加熱沉澱蛋白質 幾乎所有的蛋白質都因加熱變性而凝固,變成不可逆的不溶狀態。鹽類和氫離子濃度對蛋白質加熱凝固有重要影響。少量鹽類促進蛋白質的加熱凝固。當蛋白質處於等電點時,加熱凝固最完全、最迅速。在酸性或鹼性溶液中,蛋白質分子帶有正電荷或負電荷,雖加熱蛋白質也不會凝固。若同時有足量的中性鹽存在,則蛋白質可因加熱而凝固。 取5支試管。編號,按下表加入有關試劑(單位:滴): 蛋白質溶液 0.1% 醋酸 10% 醋酸 飽和NaCl 10% NaOH 蒸餾水 試劑 管號 1 10 - - - - 7 welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineering 1 GB150-98 2 GB50128-2005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ T4735-1997 steel welded atmospheric pressure vessel 4 GB50231-2009 mechanical equipment installation engineering construction and acceptance specification for 5 GB50275-98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-2000 construction installation engineering construction standard 7, HG ... Pressure gauges, using an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan. Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the opening and provisional protective measures have been taken, the responsible unit fined 200 Yuan, construction person in charge a fine of 50 Yuan. (8) no permit to work and the job in accordance with the provisions, on the job unit fined 300 yuan, the unit will charge a fine of 100 Yuan. 7.5.3 accident penalties (1) injuries fatalities directly punished 1~2 million. Direct responsibility for the accident and have the corresponding responsibility of leadership, such as concerning administrative sanctions, should be brought to the company or the relevant Department. (2) personal injury accident occurred, the direct punishment 0.5-10,000 yuan, responsible for the direct responsibility for the accident and the responsibility of leadership, such as concerning administrative sanctions should be dealt with by the personnel Department of the company. (3) personal injury 2 10 5 - - - 2 3 10 - 5 - - 2 4 10 - 5 2 - - 5 10 - - - 2 5 將各管混勻,觀察記錄各管現象後,放人沸水浴中加熱10 min,注意觀察比較各管的沉澱情況。然後將第3,4,5號管分別用10% NaOH或10% 醋酸中和,觀察並解釋實驗結果。 將3,4,5號管繼續分別加入過量的酸或鹼,觀察它們發生的現象。然後,用過量的酸或鹼中和第3,5號管,沸水浴加熱10 min,觀察沉澱變化,檢查這種沉澱是否溶於過量的酸或鹼中,並解釋實驗結果。 問題: 1,為什麼蛋清可用作鉛中毒或汞中毒的解毒劑? 2,蛋白質分子中的哪些基團可以與 (1)重金屬離子作用而使蛋白質沉澱? (2)有機酸、無機酸作用而使蛋白質沉澱? 3,高濃度的硫酸銨對蛋白質溶解度有何影響,為什麼? 4,在蛋白質可逆沉澱反應的實驗中,為何要用蛋白質氯化鈉溶液? welding procedures of steel pressure vessel NDT 9 JB/T4744-2000 steel pressure vessel products mechanical properties test of welded plate II, mechanical equipment installation engineering 1 GB150-98 2 GB50128-2005 vertical cylindrical steel pressure vessel steel welded specification for construction and acceptance of oil tank 3 JB/ T4735-1997 steel welded atmospheric pressure vessel 4 GB50231-2009 mechanical equipment installation engineering construction and acceptance specification for 5 GB50275-98 compressor, fan and pump installation engineering construction and acceptance specification for lifting 6 HG20201-2000 construction installation engineering construction standard 7, HG ... Pressure gauges, using an installed spark arrestor for acetylene cylinders, illegal carrying, using gas cylinders, each operator fined 20 Yuan. Gas bottle without the hot sun exposure measures, responsibilities of team a fine of 50 Yuan. (7) at height and the opening and provisional protective measures have been taken, the responsible unit fined 200 Yuan, construction person in charge a fine of 50 Yuan. (8) no permit to work and the job in accordance with the provisions, on the job unit fined 300 yuan, the unit will charge a fine of 100 Yuan. 7.5.3 accident penalties (1) injuries fatalities directly punished 1~2 million. Direct responsibility for the accident and have the corresponding responsibility of leadership, such as concerning administrative sanctions, should be brought to the company or the relevant Department. (2) personal injury accident occurred, the direct punishment 0.5-10,000 yuan, responsible for the direct responsibility for the accident and the responsibility of leadership, such as concerning administrative sanctions should be dealt with by the personnel Department of the company. (3) personal injury