Aequorin

Work on aequorin began with E. Newton Harvey in 1921.{{cite journal | vauthors = Harvey EN | title=Studies on Bioluminescence. XIII. Luminescence in the Cœlenterate | journal=Biological Bulletin | volume=41 | issue=5 | year=1921 | pages=280–287 | doi=10.2307/1536528 | jstor=1536528| s2cid=10826363 | url=https://www.journals.uchicago.edu/doi/pdf/10.2307/1536528}} Though Harvey was unable to demonstrate a classical luciferase-luciferin reaction, he showed that water could produce light from dried photocytes and that light could be produced even in the absence of oxygen. Later, Osamu Shimomura began work into the bioluminescence of Aequorea in 1961. This involved tedious harvesting of tens of thousands of jellyfish from the docks in Friday Harbor, Washington. It was determined that light could be produced from extracts with seawater, and more specifically, with calcium. It was also noted during the extraction the animal creates green light due to the presence of the green fluorescent protein, which changes the native blue light of aequorin to green.{{cite journal | vauthors = Morin JG, Hastings JW | title = Energy transfer in a bioluminescent system | journal = J. Cell. Physiol. | volume = 77 | issue = 3 | pages = 313–318 | year = 1971 | pmid = 4397528 | doi = 10.1002/jcp.1040770305 | s2cid = 42494355 }}

While the main focus of his work was on the bioluminescence,{{cite journal | vauthors = Shimomura O | title = The discovery of aequorin and green fluorescent protein | journal = J Microsc | volume = 217 | issue = Pt 1 | pages = 1–15 | year = 2005 | pmid = 15655058 | doi = 10.1111/j.0022-2720.2005.01441.x | s2cid = 36316988 }} Shimomura and two others, Martin Chalfie and Roger Tsien, were awarded the Nobel Prize in 2008 for their work on green fluorescent proteins.

Aequorin is a holoprotein composed of two distinct units, the apoprotein that is called apoaequorin, which has an approximate molecular weight of 21 kDa, and the prosthetic group coelenterazine, the luciferin.{{cite journal | vauthors = Shimomura O, Johnson FH | title = Peroxidized coelenterazine, the active group in the photoprotein aequorin | journal = PNAS USA | volume = 75 | issue = 3 | pages = 2611–2615 | year = 1978 | pmid = 275832 | pmc = 392612 | doi = 10.1073/pnas.75.6.2611 | bibcode = 1978PNAS...75.2611S | doi-access = free }} This is to say, apoaequorin is the enzyme produced in the photocytes of the animal, and coelenterazine is the substrate whose oxidation the enzyme catalyzes. When coelenterazine is bound, it is called aequorin. Notably, the protein contains three EF hand motifs that function as binding sites for Ca²⁺ ions.{{cite journal | vauthors = Charbonneau H, Walsh KA, McCann RO, Prendergast FG, Cormier MJ, Vanaman TC | title = Amino acid sequence of the calcium-dependent photoprotein aequorin | journal = Biochemistry | volume = 24 | issue = 24 | pages = 6762–6771 | year = 1985 | pmid = 2866797 | doi = 10.1021/bi00345a006 }} The protein is a member of the superfamily of the calcium-binding proteins, of which there are some 66 subfamilies.{{cite journal | vauthors = Zhou Y, Yang W, Kirberger M, Lee HW, Ayalasomayajula G, Yang JJ | title = Prediction of EF-hand calcium-binding proteins and analysis of bacterial EF-hand proteins | journal = Proteins | volume = 65 | issue = 3 | pages = 643–655 | year = 2006 | pmid = 16981205 | doi = 10.1002/prot.21139 | s2cid = 8904181 }}

The crystal structure revealed that aequorin binds coelenterazine and oxygen in the form of a peroxide, coelenterazine-2-hydroperoxide.{{cite journal | vauthors = Head JF, Inouye S, Teranishi K, Shimomura O | title = The crystal structure of the photoprotein aequorin at 2.3 Å resolution | journal = Nature | volume = 405 | issue = 6784 | pages = 372–376 | year = 2000 | pmid = 10830969 | doi = 10.1038/35012659 | bibcode = 2000Natur.405..372H | s2cid = 4425033 }} The binding site for the first two calcium atoms show a 20 times greater affinity for calcium than the third site.{{cite journal | vauthors = Shimomura O | title = Luminescence of aequorin is triggered by the binding of two calcium ions | journal = Biochem. Biophys. Res. Commun. | volume = 211 | issue = 2 | pages = 359–363 | year = 1995 | pmid = 7794244 | doi = 10.1006/bbrc.1995.1821 }} However, earlier claims that only two EF-hands bind calcium{{cite journal | vauthors = Shimomura O | title = Luminescence of aequorin is triggered by the binding of two calcium ions | journal = Biochemical and Biophysical Research Communications | volume = 211 | issue = 2 | pages = 359–363 | year = 1995 | pmid = 7794244 | doi = 10.1006/bbrc.1995.1821 }} were questioned when later structures indicated that all three sites can indeed bind calcium.{{cite journal | vauthors = Deng L, Vysotski ES, Markova SV, Liu ZJ, Lee J, Rose J, Wang BC | title = All three Ca2+-binding loops of photoproteins bind calcium ions: the crystal structures of calcium-loaded apo-aequorin and apo-obelin | journal = Protein Sci. | volume = 14 | issue = 3 | pages = 663–675 | year = 2005 | pmid = 15689515 | pmc = 2279293 | doi = 10.1110/ps.041142905 }} Thus, titration studies show that all three calcium-binding sites are active but only two ions are needed to trigger the enzymatic reaction.{{cite journal | vauthors = Shimomura O, Inouye S | title = Titration of recombinant aequorin with calcium chloride | journal = Biochem. Biophys. Res. Commun. | volume = 221 | issue = 1 | pages = 77–81 | year = 1996 | pmid = 8660347 | doi = 10.1006/bbrc.1996.0548 | doi-access = free }}

Other studies have shown the presence of an internal cysteine bond that maintains the structure of aequorin.{{cite journal | vauthors = Ohmiya Y, Kurono S, Ohashi M, Fagan TF, Tsuji FI | title = Mass spectrometric evidence for a disulfide bond in aequorin regeneration | journal = FEBS Lett. | volume = 332 | issue = 3 | pages = 226–228 | year = 1993 | pmid = 8405461 | doi = 10.1016/0014-5793(93)80637-a | doi-access = free }} This has also explained the need for a thiol reagent like beta mercaptoethanol in the regeneration of the protein since such reagents weaken the sulfhydryl bonds between cysteine residues, expediting the regeneration of the aequorin.

Chemical characterization of aequorin indicates the protein is somewhat resilient to harsh treatments. Aequorin is heat resistant.{{cite journal | vauthors = Inouye S | title = Blue fluorescent protein from the calcium-sensitive photoprotein aequorin is a heat-resistant enzyme, catalyzing the oxidation of coelenterazine | journal = FEBS Lett. | volume = 577 | issue = 1–2 | pages = 105–110 | year = 2004 | pmid = 15527769 | doi = 10.1016/j.febslet.2004.09.078 | doi-access = free }} Held at 95 °C for 2 minutes the protein lost only 25% activity. Denaturants such as 6-M urea or 4-M guanidine hydrochloride did not destroy the protein.

Aequorin is presumably encoded in the genome of Aequorea. At least four copies of the gene were recovered as cDNA from the animal.{{cite journal | vauthors = Prasher D, McCann RO, Cormier MJ | title = Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein | journal = Biochem. Biophys. Res. Commun. | volume = 126 | issue = 3 | pages = 1259–68 | year = 1985 | pmid = 2579647 | doi = 10.1016/0006-291X(85)90321-3 }}{{cite journal | vauthors = Inouye S, Noguchi M, Sakaki Y, Takagi Y, Miyata T, Iwanaga S, Miyata T, Tsuji FI | title = Cloning and sequence analysis of cDNA for the luminescent protein aequorin | journal = Proc. Natl. Acad. Sci. U.S.A. | volume = 82 | issue = 10 | pages = 3154–58 | year = 1985 | pmid = 3858813 | pmc = 397733 | doi = 10.1073/pnas.82.10.3154 | bibcode = 1985PNAS...82.3154I | doi-access = free }} Because the genome has not been sequenced, it is unclear if the cDNA variants can account for all of the isoforms of the protein.{{cite journal | vauthors = Masuda H, Takenaka Y, Shikamoto Y, Kagawa M, Mizuno H, Tsuji FI | title = Chromatography of isoforms of recombinant apoaequorin and method for the preparation of aequorin | journal = Protein Expr. Purif. | volume = 31 | issue = 2 | pages = 181–187 | year = 2003 | pmid = 14550635 | doi = 10.1016/s1046-5928(03)00186-4 }}

Early studies of the bioluminescence of Aequorea by E. Newton Harvey had noted that the bioluminescence appears as a ring around the bell, and occurs even in the absence of air.{{cite journal | vauthors = Harvey EN | title = Oxygen and Luminescence, with a Description of Methods for Removing Oxygen from Cells and Fluids | journal = Biological Bulletin | volume = 51 | issue = 2 | pages = 89–97 | year = 1926 | doi=10.2307/1536540| jstor = 1536540 | url = https://www.biodiversitylibrary.org/part/10295 }} This was remarkable because most bioluminescence reactions require oxygen, and led to the idea that the animals somehow store oxygen.{{cite book | vauthors = Harvey EN | title = Bioluminescence | publisher = Academic Press | date = 1952}} It was later discovered that the apoprotein can stably bind coelenterazine-2-hydroperoxide, and oxygen is required for the regeneration to this active form of aequorin.{{cite journal | vauthors = Shimomura O, Johnson FH | title = Regeneration of the photoprotein aequorin | journal = Nature | volume = 256 | issue = 5514 | pages = 236–238 | year = 1975 | pmid = 239351 | doi = 10.1038/256236a0 | bibcode = 1975Natur.256..236S | s2cid = 4176627 }} However, in the presence of calcium ions, the protein undergoes a conformational change and converts its prosthetic group, coelenterazine-2-hydroperoxide, into excited coelenteramide and CO₂.{{cite journal | vauthors = Shimomura O, Johnson FH, Morise H | title = Mechanism of the luminescent intramolecular reaction of aequorin | journal = Biochemistry | volume = 13 | issue = 16 | pages = 3278–3286 | year = 1974 | pmid = 4152180 | doi = 10.1021/bi00713a016 }} As the excited coelenteramide relaxes to the ground state, blue light (wavelength of 465 nm) is emitted. Before coelenteramide is exchanged out, the entire protein is still fluorescent blue.{{cite journal | vauthors = Shimomura O, Johnson FH | title = Calcium binding, quantum yield, and emitting molecule in aequorin bioluminescence | journal = Nature | volume = 227 | issue = 5265 | pages = 1356–1357 | year = 1970 | pmid = 4393938 | doi = 10.1038/2271356a0 | bibcode = 1970Natur.227.1356S | s2cid = 4284185 }}{{cite journal | vauthors = Inouye S, Sasaki S | title = Blue fluorescent protein from the calcium-sensitive photoprotein aequorin: catalytic properties for the oxidation of coelenterazine as an oxygenase | journal = FEBS Lett. | volume = 580 | issue = 8 | pages = 1977–1982 | year = 2006 | pmid = 16545379 | doi = 10.1016/j.febslet.2006.02.065 | doi-access = free }} because of the connection between bioluminescence and fluorescence, this property was ultimately important in the discovery of the luciferin coelenterazine.{{cite journal | vauthors = Shimomura O, Johnson FH | title = Chemical nature of bioluminescence systems in coelenterates | journal = Proceedings of the National Academy of Sciences | volume = 72 | issue = 4 | pages = 1546–1549 | year = 1975 | pmid = 236561 | pmc = 432574 | doi = 10.1073/pnas.72.4.1546 | bibcode = 1975PNAS...72.1546S | doi-access = free }}

Since the emitted light can be easily detected with a luminometer, aequorin has become a useful tool in molecular biology for the measurement of intracellular Ca²⁺ levels.{{cite journal | vauthors = Shimomura O, Inouye S, Musicki B, Kishi Y | title = Recombinant aequorin and recombinant semi-synthetic aequorins. Cellular Ca2+ ion indicators | journal = Biochem. J. | volume = 270 | issue = 2 | pages = 309–312 | year = 1990 | pmid = 2400391 | pmc = 1131721 | doi = 10.1042/bj2700309}} The early successful purification of aequorin led to the first experiments involving the injection of the protein into the tissues of living animals to visualize the physiological release of calcium in the muscle fibers of a barnacle.{{cite journal | vauthors = Ridgway EB, Ashley CC | title = Calcium transients in single muscle fibers | journal = Biochem. Biophys. Res. Commun. | volume = 29 | issue = 2 | pages = 229–234 | year = 1967 | pmid = 4383681 | doi = 10.1016/0006-291x(67)90592-x }} Since then, the protein has been widely used in many model biological systems, including zebrafish,{{cite journal | vauthors = Cheung CY, Webb SE, Meng A, Miller AL | title = Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development | journal = Int. J. Dev. Biol. | volume = 50 | issue = 6 | pages = 561–569 | year = 2006 | pmid = 16741871 | doi = 10.1387/ijdb.062151cc | url = http://www.ijdb.ehu.es/web/descarga/paper/062151cc | doi-access = free }} rats, mice, and cultured cells.{{cite journal | vauthors = Rembold CM, Kendall JM, Campbell AK | title = Measurement of changes in sarcoplasmic reticulum [Ca2+] in rat tail artery with targeted apoaequorin delivered by an adenoviral vector | journal = Cell Calcium | volume = 21 | issue = 1 | pages = 69–79 | date = January 1997 | pmid = 9056079 | doi = 10.1016/s0143-4160(97)90098-1 }}{{cite journal | vauthors = Yamano K, Mori K, Nakano R, Kusunoki M, Inoue M, Satoh M | title = Identification of the functional expression of adenosine A3 receptor in pancreas using transgenic mice expressing jellyfish apoaequorin | journal = Transgenic Res. | volume = 16 | issue = 4 | pages = 429–435 | year = 2007 | pmid = 17387626 | doi = 10.1007/s11248-007-9084-0 | s2cid = 19339429 }}{{cite journal | vauthors = Sheu YA, Kricka LJ, Pritchett DB | title = Measurement of intracellular calcium using bioluminescent aequorin expressed in human cells | journal = Anal. Biochem. | volume = 209 | issue = 2 | pages = 343–347 | year = 1993 | pmid = 8470808 | doi = 10.1006/abio.1993.1132 | doi-access = free }}{{cite journal|vauthors=Mithöfer A, Mazars C |title=Aequorin-based measurements of intracellular Ca²⁺ signatures in plant cells |journal=Biol. Proced. Online |volume=4 |pages=105–118 |year=2002 |pmid=12734562 |pmc=145563 |doi=10.1251/bpo40 |url=http://www.biologicalprocedures.com/bpo/arts/1/40/m40.htm |url-status=dead |archive-url=https://web.archive.org/web/20050728155815/http://www.biologicalprocedures.com/bpo/arts/1/40/m40.htm |archive-date=2005-07-28 }}

Cultured cells expressing the aequorin gene can effectively synthesize apoaequorin; however, recombinant expression yields only the apoprotein. Therefore it is necessary to add coelenterazine into the culture medium of the cells to obtain a functional protein and thus use its blue light emission to measure Ca²⁺ concentration. Coelenterazine is a hydrophobic molecule, and therefore is easily taken up across plant and fungal cell walls, as well as the plasma membrane of higher eukaryotes, making aequorin suitable as a Ca²⁺ reporter in plants, fungi, and mammalian cells.{{cite journal | vauthors = Blinks JR, Wier WG, Hess P, Prendergast FG | title = Measurement of Ca²⁺ concentrations in living cells | journal = Prog Biophys Mol Biol | volume = 40 | issue = 1–2 | pages = 1–114 | year = 1982 | pmid = 6758036 | doi = 10.1016/0079-6107(82)90011-6 | doi-access = free }}{{cite journal | vauthors = Montero M, Brini M, Marsault R, Alvarez J, Sitia R, Pozzan T, Rizzuto R | title = Monitoring dynamic changes in free Ca²⁺ concentration in the endoplasmic reticulum of intact cells | journal = EMBO J | volume = 14 | issue = 22 | pages = 5467–5475 | year = 1995 | pmid = 8521803 | pmc = 394660 | doi = 10.1002/j.1460-2075.1995.tb00233.x }}

Aequorin has a number of advantages over other Ca²⁺ indicators. Because the protein is large, it has a low leakage rate from cells compared to lipophilic dyes such as DiI. It lacks phenomena of intracellular compartmentalization or sequestration as is often seen for Voltage-sensitive dyes, and does not disrupt cell functions or embryo development. Moreover, the light emitted by the oxidation of coelenterazine does not depend on any optical excitation, so problems with auto-fluorescence are eliminated.{{cite journal | vauthors = Kendall JM, Badminton MN, Sala-Newby GB, Campbell AK, Rembold CM | title = Recombinant apoaequorin acting as a pseudo-luciferase reports micromolar changes in the endoplasmic reticulum free Ca²⁺ of intact cells | journal = Biochem J | volume = 318 | issue = 2 | pages = 383–387 | year = 1996 | pmid = 8809023 | pmc = 1217633 | doi=10.1042/bj3180383}} The primary limitation of aequorin is that the prosthetic group coelenterazine is irreversibly consumed to produce light, and requires continuous addition of coelenterazine into the media. Such issues led to developments of other genetically encoded calcium sensors including the calmodulin-based sensor cameleon,{{cite journal | vauthors = Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY | title = Fluorescent indicators for Ca²⁺ based on green fluorescent proteins and calmodulin | journal = Nature | volume = 388 | issue = 6645 | pages = 882–887 | year = 1997 | pmid = 9278050 | doi = 10.1038/42264 | bibcode = 1997Natur.388..882M | s2cid = 13745050 | doi-access = free }} developed by Roger Tsien and the troponin-based sensor, TN-XXL, developed by Oliver Griesbeck.{{cite journal | vauthors = Heim N, Griesbeck O | title = Genetically encoded indicators of cellular calcium dynamics based on troponin C and green fluorescent protein | journal = J Biol Chem | volume = 279 | issue = 14 | pages = 14280–14286 | year = 2004 | pmid = 14742421 | doi = 10.1074/jbc.M312751200 | doi-access = free }}

Apoaequorin is an ingredient in Prevagen, which is marketed by Quincy Bioscience as a memory supplement. In 2017, the US Federal Trade Commission (FTC) charged the maker with falsely advertising that the product improves memory, provides cognitive benefits, and is "clinically shown" to work.{{cite news |last=Hamilton |first=Martha M. |title=Does the supplement Prevagen improve memory? A court case is asking that question. |url=https://www.washingtonpost.com/health/prevagen-memory-loss-does-it-work/2021/09/10/53e5d3e8-f3a6-11eb-a49b-d96f2dac0942_story.html |date=September 11, 2021 |newspaper=The Washington Post |accessdate=September 11, 2021 }} {{Cite journal |last=Moran |first=Daniel L. |last2=Underwood |first2=Mark Y. |last3=Gabourie |first3=Taylor A. |last4=Lerner |first4=Kenneth C. |date=2016 |title=Effects of a Supplement Containing Apoaequorin on Verbal Learning in Older Adults in the Community |url=https://pubmed.ncbi.nlm.nih.gov/26878676/ |journal=Adv Mind Body Med |volume=30 |issue=1 |pages=4-11 |pmid=26878676 |via=National Library of Medicine}}According to the FTC, "the marketers of Prevagen preyed on the fears of older consumers experiencing age-related memory loss". Quincy said that it would fight the charges.{{cite news |title=Jellyfish Memory Supplement Prevagen Is a Hoax, FTC Says | first = Maggie | last = Fox | name-list-style = vanc | date = January 9, 2017 |work=NBC News |url=https://www.nbcnews.com/health/health-care/jellyfish-memory-supplement-prevagen-hoax-ftc-says-n704886 |access-date=January 9, 2017}}{{cite news |title=Schneiderman slams Prevagen as a 'clear-cut fraud' in lawsuit | first = David K. | last = Li | name-list-style = vanc | date = January 9, 2017 | work = New York Post | url = https://nypost.com/2017/01/09/schneiderman-slams-prevagen-as-a-clear-cut-fraud-in-lawsuit/ | access-date = January 9, 2017 }}{{cite web | title = Prevagen's Fishy Memory Claims Under Fire by Federal Regulators | date = January 9, 2017 | publisher = Truth in Advertising | url = https://www.truthinadvertising.org/prevagens-fishy-memory-claims-fire-federal-regulators/ | access-date = January 9, 2017 }}

Prior to the suit, a clinical trial run by researchers employed by Quincy Bioscience "found no overall benefit compared to a placebo for its primary endpoints involving memory and cognition", while the company's advertising misleadingly cited a few contested subgroup analyses that showed slight improvements.University of California Berkeley School of Public Health, Health After 50, "Forget Jellyfish Protein", Winter, 2017–18, p. 6{{cite web |title=Prevagen: How Can This Memory Supplement Flunk Its One Trial and Still Be Advertised as Effective? |url=https://cspinet.org/news/prevagen-how-can-memory-supplement-flunk-its-one-trial-and-still-be-advertised-effective |publisher=Center for Science in the Public Interest |access-date=September 20, 2018 |date=November 20, 2017}}

The suit (Spath, et al. v. Quincy Bioscience Holding Company, Inc., et al., Case No. 18-cv-12416, D. NJ.) was dismissed in the District court, but an appeal seeking to overturn the dismissal was filed. The suit was consolidated with another against Quincy Pharmaceuticals, Vanderwerff v. Quincy Bioscience (Case No. 17-cv-784, D. NJ), which was the lead case.[https://www.truthinadvertising.org/quincy-biosciences-prevagen-supplement/ "Quincy Bioscience's Prevagen Supplement October 2018"], Truth in Advertising, October 2018. Retrieved November 14, 2018.

On February 21, 2019, the United States Court of Appeals for the Second Circuit ruled that the FTC and the state of New York could proceed with their lawsuit against Quincy Bioscience for its claims that Prevagen can improve memory. The order came less than two weeks after the parties argued the case before a three-judge panel of the circuit, where company lawyers admitted they did not "dispute that if you look across the entire 211 people who completed the study there was no statistically significant difference". The court vigorously dismissed allegations by the company lawyers that the FTC pursued its action for political reasons.[https://www.truthinadvertising.org/wp-content/uploads/2019/02/Prevagen-_2nd-Cir-Summary-Order.pdf FTC vs. Quincy Bioscience Holding Company], United States Court of Appeals for the Second Circuit, Case 17-3745, Document 257, February 21, 2019. Retrieved March 26, 2019.[https://www.truthinadvertising.org/prevagen-is-going-to-the-dogs/ "Prevagen Is Going to the Dogs"], Truth in Advertising, February 22, 2019. Retrieved March 26, 2019.

On March 23, 2020, a federal magistrate judge in the United States District Court for the Southern District of Florida entered a report and recommendations certifying a nationwide class action for the class of consumers who purchased Prevagen over the previous four years.{{cite news |first=Michael A. |last=Mora |title=Federal Magistrate Judge Recommends Certifying Nationwide Prevagen Class Action in Florida |url=https://www.law.com/dailybusinessreview/2020/03/20/032020classactionprevagen/ |work=Daily Business Review |publisher=law.com |date=March 24, 2020 |access-date=March 24, 2020}} The trial in the case was set for October 2020.{{Cite web|url=https://drive.google.com/file/d/1OOYG9hDSMic38NxVh7MFNXD27Q--Ncch/view?usp=embed_facebook|title=Report and Recommendations on Plaintiff's Motion for Class Certification|publisher=United States District Court, Southern District of Florida, Miami Division|website=Google Docs|date=March 19, 2020|access-date=October 24, 2020}}

{{asof|2020|9|21|df=US}}, Quincy Bioscience agreed to settle the claims that it misrepresented its Prevagen products as supporting brain health and helping with memory loss. Under the terms of the settlement, eligible purchasers applying by October 26, 2020, for purchases made from 2007 through July 31, 2020, could recover refunds of up to $70.

{{cite web|url= https://topclassactions.com/lawsuit-settlements/closed-settlements/prevagen-brain-health-supplement-class-action-settlement/|website= Top Class Actions|title= Prevagen Brain Health Supplement Class Action Settlement|date= September 21, 2020}}

Dr. Harriet Hall, writing for Science-Based Medicine, noted that the Quincy-sponsored study (known as "Madison Memory Study") was negative, but that the company utilized p-hacking to find favorable results. She wrote that their cited safety studies were all rat studies and their claim that apoaequorin crosses the blood–brain barrier was based solely on a dog study{{Cite journal |last=Milgram |first=Norton W. |last2=Landsberg |first2=Gary |last3=Merrick |first3=David |last4=Underwood |first4=Mark Y. |date=2015 |title=A novel mechanism for cognitive enhancement in aged dogs with the use of a calcium-buffering protein {{!}} Journal of Veterinary Behavior |url=https://www.sciencedirect.com/science/article/pii/S1558787815000258 |journal=Journal of Veterinary Behavior |volume=10 |issue=3 |pages=217-222 |doi=10.1016/j.jveb.2015.02.003 |issn=1558-7878 |pmid=26092396 |via=Elsevier Science Direct}}.{{cite web |last1=Hall |first1=Harriet |title=Reader's Digest Promotes Prevagen |url=https://sciencebasedmedicine.org/readers-digest-promotes-prevagen/ |website=Science-Based Medicine |date=4 December 2018 |access-date=5 December 2018 |archive-url=https://archive.today/20181205003824/https://sciencebasedmedicine.org/readers-digest-promotes-prevagen/ |archive-date=5 December 2018 |url-status=live |df=dmy-all }} The American Pharmacists Association warns that Apoaequorin "is unlikely to be absorbed to a significant degree; instead it degrades into amino acids".{{cite web |last1=Hume |first1=Anne |title=Apoaequorin for memory enhancement? |url=https://www.pharmacist.com/apoaequorin-memory-enhancement |publisher=Pharmacist.com |access-date=5 December 2018 |archive-url=https://archive.today/20181205002300/https://www.pharmacist.com/apoaequorin-memory-enhancement |archive-date=5 December 2018 |url-status=live |df=dmy-all }}

{{Reflist|colwidth=30em}}

• [http://ca.expasy.org/uniprot/P07164 Swiss-Prot Aequorin entry] {{Webarchive|url=https://web.archive.org/web/20070323182127/http://ca.expasy.org/uniprot/P07164 |date=2007-03-23 }}

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Category:Bioluminescence

Category:Cnidarian proteins

Category:EC 1.13.12

Category:Protein methods