多肽溶解方法和步骤(Peptide dissolving protocol)
2010-05-11 11:19阅读：
没有绝对理想的一种溶剂可以做到既能溶解所有多肽,又能保持它们的完整性并且与生物学检测相一致.因此,不得不尝试一系列强溶剂直到多肽溶解.对有溶解困难的多肽,下列方法也许有所帮助.
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第一步
总的来说,先用无菌水或者稀醋酸(0.1%)将多肽溶解到一个较高浓度下作为
储存溶液,而不是直接配置到检测浓度.
等需要用的时候再把这个储备溶液用缓冲Buffer配制到需要浓度.比如一条多肽要溶解成1mg/ml的PBS
buffer中,我们需要先将多肽溶解成2mg/ml的储备溶液,然后在使用前,先移取100ul的10*PBS
buffer,再加入400ul的无菌水,最后加入500ul的2mg/ml储备液.千万不要直接用Buffer缓冲液来溶解多肽,因为很多肽在高盐浓度下溶解度是下降的,而且如果发生不溶情况,去除这些盐和有机试剂是很困难的一件事.如果多肽溶解过程中出现可见颗粒始终无法分散到水相中,可以用超声的办法来破碎.不过超声只能加速溶解,并不能起到改善溶解性的效果.
第二步
溶解前先审视一下多肽序列,如果多肽中下列氨基酸(Ala,Phe,Ile,Leu,Met,Pro,Val,Trp,Tyr,Cys)占比比较高,这条多肽基本上是难溶的.另外,要注意计算一下序列中含有多少正价基团(Lys,Arg,His和N端)和负价基团(Asp,Glu和C端),在中性条件下,最后的净价是正是负?价态为正的,在溶解时可以用稀醋酸调节pH到酸性,价态为负的,在溶解时可以用稀氨水调节pH到碱性.如果这样仍不能溶解,可以考虑冻干去除溶剂后变成用强有机溶剂来溶解.
第三步
如果你的序列在任何pH下都基本不带电荷,或者说你的序列中疏水性氨基酸含量超过50%,甚至更高,前面两步基本上是多余的,直接考虑加入少量乙腈,乙醇,DMF或者DMSO来溶解,甚至可以同盐酸胍和脲来分散多肽.这些方法溶解多肽的浓度取决于你最终生物检测需要.如果已经知道一条多肽在水相中溶解不是太好,而最终使用却必须在水相,可以考虑全部用醋酸或者DMF来溶解,然后缓慢加水来稀释.这样有机溶剂有助于多肽分散到水相中.
多肽溶液的储存问题
溶解的多肽的保质期是比较有限的,特别是含有C,M,W,N和Q的多肽.为了延长保存时间,使用无菌水,保持适当酸性(pH5-6),还有分装冷冻在-20℃或者更低是建议采用的方法。另外，一定要避免反复冻融，这个对多肽的损伤最大。
A Strategy for Dissolving Small Sets of
Peptides
The kind of individual treatment described above starts to become
impractical when handling larger numbers of peptides, say 10 or
more. Although exceptions can be found to the success of any
generalised procedure, a recommended strategy for redissolving
greater numbers of peptides with varied properties is outlined
below.
1.Add 0.1% acetic acid/water to give a target peptide concentration
of 1-5mg/mL, and sonicate.
2. To any insoluble peptides add pure acetic acid to bring the
concentration of acetic acid to 10%(v/v), and sonicate.
3.To any peptides still insoluble add acetonitrile to 20%(v/v), and
sonicate.
4.Lyophilise any remaining insoluble peptides to remove the water,
acetic acid, and acetonitrile. To the solid, add neat DMF dropwise
until the peptide dissolves. Dilute this solution slowly with water
to give approximately 10%(v/v) DMF. If the peptide precipitates at
any stage during this step, stop adding water and add a little more
DMF until the peptide redissolves. Such peptides may be too
insoluble in water to be used at concentrations equal to the others
in the set.
5.Dilute each solubilized peptide with the solvent found to be
effective for it, to bring the stock solutions to the same peptide
concentration. This simplifies calculations and subsequent
handling. Further dilutions, as needed for the bioassay, can be
made in the assay buffer e.g. as a dilution series (titration).
Dilution of a relatively insoluble peptide with buffer at this step
may successfully avoid precipitation because it is now at a low
concentration (below its solubility limit).
6.Except after addition of DMF, all solutions can be easily
lyophilised to return the peptide to a form suitable for long term
storage, if required.
This is only one of a large number of possible procedures. The one
chosen depends on the assay system, and the need for a particular
buffer or peptide concentration. Contact Mimotopes for free
technical advice if you wish to use a particular buffer not
mentioned here.
A Strategy for Dissolving Large Sets of Peptides
Peptide sets such as a General net (Gnet), Replacement net (Rnet)
etc. for mass screening are supplied as lyophilised solids in
polypropylene tubes. In these cases, the only practical method may
be to apply one solvent to all peptides and use the solutions
obtained without trying to optimize for each peptide. For example,
for T cell determinant mapping, we have found that a solvent
comprising 0.1M HEPES buffer pH7.4 in a 40% acetonitrile/water
solution gives effective solutions of most peptides and is nontoxic
when diluted 20-fold or more in an assay. In this case, sonication
of the tubes is also advisable to maximise the dissolution of the
peptides. If acceptable in the assay system at the intended working
concentration, a good general solvent such as DMF or DMSO could
also be used as the first reagent added to all peptides in the
set.
On special request, peptide sets can be shipped in the form of
frozen solutions, avoiding the problems of redissolving the
peptides. Shipping frozen materials adds substantially to the cost
of the peptides and can create problems if the peptides are delayed
in transit.
Chemical Changes in Your Peptides
Peptides vary in stability, and a peptide as supplied may soon be
degraded if care is not taken to ensure proper storage. In addition
to the risk of degradation from proteolytic enzymes, other chemical
changes can occur. The short section below is meant to help with
situations which will commonly arise.
1. Oxidation
A characteristic of cysteine- and methionine-containing peptides is
the tendency of these residues to oxidise. Susceptibility to
oxidation is sequence-dependent and sometimes even minimal exposure
to air of peptides containing these amino acids can lead to
oxidation. If you wish to avoid oxidation, always work with
degassed or deoxygenated solvents and solutions. If possible,
maintain peptide solutions at acidic pH (<7). Rate of oxidation
increases with pH, so even if the peptide is in the fully reduced
form initially, some oxidation will occur if the peptide is
maintained under neutral or basic conditions.
Normally, single peptides are consigned in the fully reduced form.
If handled properly, they will remain so, but if you need to carry
out reduction of a peptide the procedures are as follows:
1. Reduction of
oxidised cysteine. Dissolve the peptide in 0.1M ammonium
bicarbonate containing dithiothreitol (approximately 10-50 fold
molar excess) and hold for 4h at room temperature. This procedure
is a reasonable starting point but certain sequences may require
more forcing conditions of temperature and time. After reduction,
lyophilise the solution, or de-salt using size exclusion gel
chromatography (e.g. Pharmacia Sephadex G-25) or reverse- phase
chromatography (e.g. Millipore/Waters Sep-Pak). To prevent
re-oxidation, follow the handling procedures mentioned above and
store the peptide powder under nitrogen gas and in a freezer.
2. Reduction of
oxidised methionine by the method of Houghten and Li
[2]. Dissolve the peptide in 10% acetic acid in water,
and add N-methylmercaptoacetamide to 10%(v/v). Incubate at 37
degrees C for 24h or more, then lyophilise or de- salt as for
cysteine-containing peptides.
2. Other reactions
Peptides have a variety of reactive side chains, and side reactions
can occur under both acidic and basic conditions. For example, if
glutamine or glutamic acid occurs at the N- terminus of a peptide,
cyclisation to form pyroglutamate is likely under acidic conditions
(10% acetic acid). Mild basic solutions (0.1M ammonium bicarbonate)
will lead to imide formation in asparagine-containing peptides.
When dissolving single peptides, avoid conditions known to promote
side reactions with the residues present.
Degradation due to microbial growth should not occur provided
sterile distilled water or buffers are used, and solutions are
frozen for storage. Sterilizing filtration of the peptide solution
is another option, which also removes traces of insoluble or
particulate materials. If filtration is chosen, ensure that the
filters used are resistant to the solvent in which the peptide is
dissovled, and have low peptide-binding properties.
