Citric acid is a tri-carboxylic acid alcohol. This increases the likelihood that citrate can crosslink proteins, and has been found to crosslink albumin, gliadin (as discussed before here, the immunoactive protein of wheat), and gliadin peptides. Glaidin contains multiple active sites for enzymatic transamidation, and is a target for tissue transglutaminase in humans, these 4 amino acid motifs exist at a magnitude higher density than most proteins. In celiac disease it crosslinks with transglutaminase causing autoimmunity to the transglutaminase protein. Thus it is not surprising the citrate covelantly links with gliadin peptides because itself, gliadin is rather active crosslinking substrate. My suspicion is that these crosslinking sites on gliadin evolved to prevent non-migratory animals from stock-piling wheat berries for year round consumption and favoring transmigrants who would carry their seeds in their fur to other places (e.g. ovids and caprids).
The optimal time, pH, Temperature and [Citrate] for gliadin/citrate crosslinking were: 2.5hr, pH 8, 50'C and 25%. Below 30'C, 5% Citrate rate of production formation was very slow. Below 6.9 rate of reaction dropped to less than 1/2. The optimal crosslinking occurred with wheat gliadin, but was also seen with related proteins of maize zein and soyprotein. Seed storage proteins tend to have a higher than normal nitrogen content, with higher frequency of nitrogen containing side chains, this likely explains increase crosslinking of these proteins. [See other post about its chelating properties] Citric Acid has a very low primary pKa, at 3.15, which is well below the activity range of most enzymes. Therefore is a fairly effective acidifying and buffering agent, buffering across a >3 ph range, and only small amounts should be used. As a consequence it is unlikely these reactions will interfere with the chemistry of the enzyme. If the buffer is basic without citric acid then adding the acid form can bring about the desired pH with as little citric acid as possible. An example would be a buffer that is pH 7.2 without acid and pH 6.0 with acid. Small amounts of buffer are acceptable when the enzyme is tolerant of pH changes, as citric acid has 3 pKa, these small amounts may not resist small changes but might attenuate large pH changes more effectively than most other buffers. Such a scheme is useful if the pH has a tendency to drop during the course of incubation. There are several choices of citric acid salts: monosodium citrate and disodium citrate. Monosodium and disodium citrate may be desired if there is a strong tendency for pH to change over time and outside of a desirable range. Calcium citrate is a common ingredient in canned foods, it is designed to keep the pH low and the food fresh for long periods of time, and it is added in considerably liberal amounts in things like canned tomatoes. [Take a can of tomato or tomato paste, heat it up slightly and add a teaspoon of baking soda, watch what happens. Very illuminating as to why canned foods are not recommended for people who suffer from GERD] Foods stored in citric acid tend to keep a bright color, whereas without it the foods will brown and spoil quickly. Concentrated buffer is preferred if pH tends to rise over the course of incubation (see above) alkaline pH favors protein modification, so more buffer will resist extreme deprotonation of citric carboxyl groups [IOW, avoid! the reaction described in Reddy et al (transliterated above and referenced below]. I would say, don't worry about it too much. Be aware of the signs of modification. 1 molar citrate is 192.124 which is approximately 19% weight to volume, thus .1 M solution is 1.9% well off the optimal concentration. The pH you are using is probably in the 6-ish range, which means well off the optimal pH. Biotechnol Prog. 2009 Jan-Feb;25(1):139-46. Alkali-catalyzed low temperature wet crosslinking of plant proteins using carboxylic acids. Reddy N, Li Y, Yang Y. J Nanosci Nanotechnol. 2007 Mar;7(3):742-7. Development of a novel glue consisting of naturally-derived biomolecules: citric acid and human serum albumin. Taguchi T, Saito H, Iwasashi M, Sakane M, Kakinoki S, Ochiai N, Tanaka J. BTW, good question, I didn't know about this gliadin-citrate reactivity before looking this information up. Gluten sensitivity exceeds the number of people who have bonafida celiac disease or gluten allergy. Some studies have shown that lymphocytes of neither disease group can be sensitive to gliadin in food sensitive people, but they really do not know the reason. This form of gluten sensitivity has shot up in the last few years, and it could be due to the preservatives or gliadin modification the occur in common foods. -----Original Message----- From: [email protected] [mailto:[email protected]] On Behalf Of Straube,Werner Sent: Wednesday, January 11, 2012 11:33 AM To: [email protected] Subject: Citrate Buffer - Carboxylation of proteins and peptides ? Dear all, I intend to use citrate buffer at 100 mM for some experiments, but then I stumbled over a notice from Dionex, that citrate buffer can modify proteins and peptides. However, when searching for references I was not able to find information about it. Is this really a major artefact in citrate buffer ? Does anyone have some information or references about it ? Thanks, Werner _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
