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二硫键广泛存在与蛋白结构中，对稳定蛋白结构具有非常重要的意义，二硫键一般是通过序列中的2个Cys的巯基，经氧化形成。
 

形成二硫键的方法很多：空气氧化法，DMSO氧化法，过氧化氢氧化法等。
 

二硫键的合成过程，  可以通过Ellman检测以及HPLC检测方法对其反应进程进行监测。  
   

如果多肽中只含有1对Cys，那二硫键的形成是简单的。多肽经固相或液相合成，然后在pH8-9的溶液中进行氧化。      
 

当需要形成2对或2对以上的二硫键时，合成过程则相对复杂。尽管二硫键的形成通常是在合成方案的最后阶段完成，但有时引入预先形成的二硫化物是有利于连合或延长肽链的。通常采用的巯基保护基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多种基团。我们分别列出两种以2-Cl树脂和Rink树脂为载体合成的多肽上多对二硫键形成路线：
 

二硫键反应条件选择    
 

 二硫键即为蛋白质或多肽分子中两个不同位点Cys的巯基（-SH）被氧化形成的S-S共价键。 一条肽链上不同位置的氨基酸之间形成的二硫键，可以将肽链折叠成特定的空间结构。多肽分 子通常分子量较大，空间结构复杂，结构中形成二硫键时要求两个半胱氨酸在空间距离上接近。 此外，多肽结构中还原态的巯基化学性质活泼，容易发生其他的副反应，而且肽链上其他侧链 也可能会发生一系列修饰，因此，肽链进行修饰所选取的氧化剂和氧化条件是反应的关键因素， 反应机理也比较复杂，既可能是自由基反应，也可能是离子反应。      

反应条件有多种选择，比如空气氧化，DMSO氧化等温和的氧化过程，也可以采用H2O2，I2， 汞盐等激烈的反应条件。
 

空气氧化法： 空气氧化法形成二硫键是多肽合成中最经典的方法，通常是将巯基处于还原态的多肽溶于水中，在近中性或弱碱性条件下（PH值6.5-10），反应24小时以上。为了降低分子之间二硫键形成的可能，该方法通常需要在低浓度条件下进行。
 

碘氧化法：将多肽溶于25%的甲醇水溶液或30%的醋酸水溶液中，逐滴滴加10-15mol/L的碘进行氧化，反应15-40min。当肽链中含有对碘比较敏感的Tyr、Trp、Met和His的残基时，氧化条件要控制的更精确，氧化完后，立即加入维生素C或硫代硫酸钠除去过量的碘。 当序列中有两对或多对二硫键需要成环时，通常有两种情况：
 

自然随机成环:       序列中的Cys之间随机成环，与一对二硫键成环条件相似；
 

定点成环：       定点成环即序列中的Cys按照设计要求形成二硫键，反应过程相对复杂。在 固相合成多肽之前，需要提前设计几对二硫键形成的顺序和方法路线，选择不同的侧链 巯基保护基，利用其性质差异，分步氧化形成两对或多对二硫键。       通常采用的巯基保护 基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多种基团。

Definition

Intermedin (IMD) is a novel member of the calcitonin gene-related peptide (CGRP) family which also includes adrenomedullin (AM).

Discovery

In early 2004, a novel peptide was independently identified by two separate groups. Roh and colleagues initially identified human IMD from an expressed sequence tag and used phylogenetic profiling to identify the peptide in other mammals and nonvertebrates. Immunohistochemical analysis of peptide expression revealed that high levels of the peptide and/or preprohormone were present in cells within the intermediate lobe of the pituitary; hence, the group called the peptide intermedin1. At the same time, Takei et al. were examining a rat cDNA library for potential mammalian homologs of the five identified puffer fish adrenomedullin (AM) family members. They identified a 148-amino acid preprohormone that was processed into a 47-amino acid, mature peptide with 33% sequence homology to AM and 71% homology to puffer fish AM2 and thus referred to the peptide as mammalian AM22.

Structural Characteristics

The human IMD gene is believed to encode a prepropeptide of 148 amino acids with a signal peptide for secretion at the N terminus, which may generate a 47-amino acid mature peptide (IMD1–47) i.e. IMDL and a shorter 40-amino acid one (IMD8–47) i.e.  IMDS by proteolytic cleavage at the N-terminal proximate basic residues followed by an amidated C terminus. Intermedins has a stretch of 47 residues at the C terminus is flanked by dibasic proteolytic cleavage sites at the N terminus and an a-amidation donor residue at the C terminus. The putative mature region of intermedin shares 28% sequence identity with ADM and 20% with CGRP. Importantly, Intermedins adopts an N-terminal disulfide- bonded loop leading into an a-helix, followed by a disordered structure that is shared by all calcitonin/CGRP family peptides. Analysis of orthologous intermedins indicate that the positions of N-terminal dibasic cleavage sites vary by a few amino acids among different species, whereas an arginine residue seven amino acids downstream of the dibasic cleavage motif of human intermedin is conserved in all species, suggesting that the mature intermedin from human and other species could be a 40-amino acid peptide3.

Mode of action

Intermedin activates the cAMP-dependent Pathway via the CGRP Receptor— A study shows that treatment of human neuroblastoma SK-N-MC cells and rat L6 skeletal myoblast cells, known to express different levels of calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein (RAMPs), with synthetic intermedin peptides resulted in increased cAMP production, whereas  treatment with the  truncated amidated intermedin fragment (intermedin (IMD)- (17–47)), or a 31-amino acid peptide from the preproregion of human intermedin (prointermedin-(55–85)) had no effect in either cell line. This implied that a-amidation and residues 8–16 of intermedin are important for intermedin bioactivity. Further it was also shown that the stimulatory effect of intermedin was suppressed by cotreatment with a calcitonin gene-related peptide (CGRP) receptor antagonist (CGRP-(8–37)) in L6 cells3.

Functions

Intermedin/Adrenomedullin-2 elevate blood pressure and inhibit food and water intake - Intermedin is homologous to adrenomedullin (AM) and can activate both the AM and calcitonin gene-related peptide (CGRP) receptors. It has been reported that administration of IMD into the lateral cerebroventricle (i.c.v.) of rats caused significant, long-lasting elevations in mean arterial pressure and heart rate. These elevations are similar to the effects of CGRP and significantly greater than the effects of AM. IMD-induced elevations in mean arterial pressure were inhibited by i.c.v. administration of phentolamine indicating that IMD activates the sympathetic nervous system. I.c.v administration of IMD also inhibited food and water intake. The effects on feeding are likely related to activation of the CGRP receptor and are independent of the effects on water intake, which are likely through the AM receptor. A study indicate that IMD has potent actions within the CNS that may be a result of the combined activation of both the AM and CGRP receptors4.

Expression of the Counter-Regulatory Peptide Intermedin is Augmented in the Presence of Oxidative Stress in Hypertrophied Cardiomyocytes - A study was done using the spontaneously hypertensive rat (SHR) and normotensive Wistar Kyoto (WKY), to examine the presence of myocardial oxidative stress, concentric hypertrophy expression of IMD, AM and receptor components. It was found that in left and right ventricular cardiomyocytes from SHR vs. WKY cell width (26% left, 15% right) and mRNA expression of hypertrophic markers ANP (2.7 fold left, 2.7 fold right) and BNP (2.2 fold left, 2.0 fold right) were enhanced. In left ventricular cardiomyocytes only oxidative stress was indicated by increased membrane protein carbonyl content (71%) and augmented production of O2- anion (64%) and IMD (6.8 fold), RAMP1 (2.5 fold) and RAMP3 (2.0 fold) mRNA was increased while AM and RAMP2 mRNA was not altered and abundance of RAMP1 (by 48%), RAMP2 (by 41%) and RAMP3 (by 90%) monomers in cell membranes was decreased. This implied that robust augmentation of IMD expression in hypertrophied left ventricular cardiomyocytes indicates a prominent role for this counter regulatory peptide in the adaptation of the SHR myocardium to the stresses imposed by chronic hypertension5.

References

1.     Roh J, Chang CL, Bhalla A, Klein C, and Hsu SYT (2004). Intermedin is a calcitonin/calcitonin gene-related peptide family peptide acting through the calcitonin receptor-like receptor/receptor activity-modifying protein receptor complex. J. Biol. Chem., 279, 7264–7274.

2.     Takei Y, Inoue K, Ogoshi M, Kawahara T, Bannai H, and Miyano S (2004). Novel fish-derived adrenomedullin in mammals: structure and possible function. Peptides, 25, 1643- 1656.

3.     Jaesook Roh, Chia Lin Chang, Alka Bhalla, Cynthia Klein, and Sheau Yu Teddy Hsu (2004). Intermedin Is a Calcitonin/Calcitonin Gene-related Peptide Family Peptide Acting through the Calcitonin Receptor-liken Receptor/Receptor Activity-modifying Protein Receptor Complexes. J. Bio. Chem., 279 (20), 7264–7274.

4.     Taylor MM, Bagley SL, Samson WK (2004). Intermedin/Adrenomedullin-2 acts within the central nervous system to elevate blood pressure and inhibit food and water intake. Am. J Physiol. Regul. Integr. Comp. Physiol., 288, R919-R927.

5.     David Bell, Y Zhao, Francis PG. McCoy, Adrian Devine and Barbara J. McDermott (2008). Expression of the Counter-Regulatory Peptide Intermedin is augmented in the Presence of Oxidative Stress in Hypertrophied Cardiomyocytes. Cell. Physiol. Biochem., 21, 409-420.