RNase A 核糖核酸酶A

RNase A 核糖核酸酶A
干燥失重：≤5.0%
性状：白色粉末。一种水解核糖核酸的酶，对湿敏感，极易溶于水。酶反应：专一地催化核糖核酸的核糖部分3′和5′磷酸二酯键的裂开，形成具有2′,3′-环一磷酸衍生物寡聚核苷酸。水溶液在25℃以下相当稳定，最大稳定范围pH2-4.5。最适pH7.7。最适温度65℃。等电点(pl)7.8。抑制剂有0.0005mol/L镁(Mg2+)、脱氧核糖核酸(竞争性抑制剂)、变性脱氧核糖核酸(比天然的更有效)
用途：生化研究。测定核酸的结构，脱氧核糖核酸和核糖核酸的降解
保存：-20℃
有效期：3年

核糖核酸酶A是内切核糖核酸酶，可特异地攻击RNA上嘧啶残基的3'端，切割与相邻核苷酸形成的磷酸二酯键。反应终产物是嘧啶3'磷酸及末端带嘧啶3'磷酸的寡核苷酸。无辅因子及二价阳离子存在时，核糖核酸酶A的作用可被胎盘RNA酶抑制剂(B1ackburn et al．1977)或氧钒—核糖核苷复合物(Puskas et al．1982)所抑制。说明：生化研究；测定核酸的结构；核糖核酸(RNA)序列分析； 水解蛋白样品中的RNA；纯化DNA。

制备方法：
 

将RNase A粉末溶解在0.01M NaAc  PH5.2中，使终浓度为10mg/mL，于100℃沸水浴中加热15min，室温下缓慢冷却 。用0.1体积的1M Tris-HCl  调PH值至7.4左右，分装-20℃保存。当浓缩的RNase A溶液在中性PH条件下加热到100℃时，RNase A 产生沉淀。（注：引自分子克隆实验指南第三版 1647页）

 

Product Description
RNase A is an endoribonuclease that attacks at the 3’ phosphate of a pyrimidine nucleotide. The sequence of pG-pG-pC-pA-pG will be cleaved to give pG-pG-pCp and A-pG. The highest activity is exhibited with single stranded RNA. RNase A is a single chain polypeptide containing 4 disulfide bridges. In contrast to RNase B, it is not a glycoprotein. 

RNase A can be inhibited by alkylation of His12 or His119, which are present in the active site of the enzyme. Activators of RNase A include potassium and sodium salts. Molecular mass:～13.7 kDa (amino acid sequence) Isoelectric point: pI = 9.6

Preparation

An RNase A solution can be made free of DNase by boiling. According to a literature method, prepare a 10 mg/mL stock solution in 10 mM sodium acetate buffer, pH 5.2. Heat to 100℃ for 15 minutes, allow to cool to room temperature, and then adjust to pH 7.4 using 0.1 volume of 1 M Tris-HCl, pH 7. 4. Aliquot and store at –20 ℃. If RNase A is boiled at a neutral pH, precipitation will occur. When boiled at the lower pH, some precipitation may occur because of protein impurities that are present. 


Stability
Store at Rnase A at –20 ℃. Stock solutions stored in frozen aliquots remain active for at least 6 months. RNase A is a very stable enzyme and solutions have been repo rted to withstand temperatures up to 100 ℃. At 100 ℃, an RNase A solution is most stable between pH 2.0 and 4.5. 

Procedure 
A major application for RNase A is the removal of RNA from preparations of plasmid DNA. For this application, DNase free RNase A is used at a final concentration of 10μg/mL.