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‭The‬‭Effect‬‭of‬‭Global‬‭Warming‬‭on‬‭the‬‭Function‬‭of‬‭Enzymes‬‭in‬‭Aquatic‬‭Ectotherms‬

‭Research‬‭Question:‬‭How‬‭does‬‭analyzing‬‭the‬‭effect‬‭of‬‭global‬‭warming‬‭on‬‭enzymes’‬‭abilities‬‭to‬

‭function‬‭in‬‭aquatic‬‭ectotherms‬‭reveal‬‭what‬‭long-term‬‭issues‬‭could‬‭arise‬‭about‬‭their‬‭physical‬

‭health?‬

‭Subject:‬‭Biology‬

‭Candidate‬‭Code:‬‭KLS085‬

‭Word‬‭count:‬‭3833‬
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‭Table‬‭of‬‭Contents‬

‭Introduction‬‭-‬‭3‬

‭Background‬‭-‬‭4‬

‭Pepsin‬‭-‬‭5‬

‭Albumin‬‭-‬‭7‬

‭Methodology‬‭-‬‭8‬

‭Variables‬‭-‬‭8‬

‭Statistical‬‭Analysis‬‭-‬‭9‬

‭Materials‬‭-‬‭10‬

‭Procedure‬‭-‬‭11‬

‭Results‬‭-‬‭12‬

‭Analysis‬‭-‬‭13‬

‭Strengths‬‭and‬‭Limitations‬‭of‬‭the‬‭Experiment‬‭-‬‭15‬

‭Conclusion‬‭-‬‭16‬

‭References‬‭-‬‭19‬
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‭The‬‭Effect‬‭of‬‭Global‬‭Warming‬‭on‬‭the‬‭Function‬‭of‬‭Enzymes‬‭in‬‭Ectotherms‬

‭The‬‭world‬‭has‬‭been‬‭facing‬‭a‬‭crisis‬‭of‬‭global‬‭warming‬‭in‬‭which‬‭the‬‭Intergovernmental‬

‭Panel‬‭on‬‭Climate‬‭Change‬‭estimates‬‭the‬‭average‬‭global‬‭temperature‬‭will‬‭increase‬‭by‬‭1.5‬‭during‬

‭the‬‭remainder‬‭of‬‭the‬‭century‬‭(IPCC,‬‭2023).‬‭Additionally,‬‭higher‬‭temperatures‬‭lead‬‭to‬‭nonoptimal‬

‭effects‬‭on‬‭bodies‬‭of‬‭water.‬‭For‬‭example,‬‭hypoxia,‬‭the‬‭lack‬‭of‬‭oxygen‬‭in‬‭water,‬‭has‬‭begun‬‭to‬‭take‬

‭effect‬‭globally.‬‭In‬‭hot‬‭temperatures,‬‭oxygen‬‭is‬‭not‬‭able‬‭to‬‭completely‬‭mix‬‭with‬‭the‬‭water.‬‭This‬

‭results‬‭in‬‭a‬‭higher‬‭concentration‬‭of‬‭oxygen‬‭near‬‭the‬‭surface,‬‭and‬‭less‬‭oxygen‬‭in‬‭the‬‭deeper‬‭parts‬

‭of‬‭bodies‬‭of‬‭water‬‭(Gobler‬‭&‬‭Baumann,‬‭2016).‬‭This‬‭level‬‭of‬‭heating‬‭has‬‭provided‬‭evidence‬‭for‬

‭scientists‬‭to‬‭suggest‬‭that‬‭global‬‭warming‬‭is‬‭becoming‬‭irreversible.‬‭The‬‭gradual‬‭heating‬‭of‬‭the‬

‭Earth‬‭affects‬‭all‬‭life‬‭on‬‭Earth,‬‭thus‬‭impacting‬‭the‬‭stability‬‭and‬‭balance‬‭of‬‭food‬‭chains.‬‭The‬

‭negative‬‭effects‬‭that‬‭global‬‭warming‬‭can‬‭have‬‭on‬‭the‬‭survival‬‭of‬‭individual‬‭populations‬‭of‬

‭species‬‭can‬‭cause‬‭food‬‭chains‬‭to‬‭lose‬‭proper‬‭energy‬‭balance.‬‭In‬‭order‬‭to‬‭begin‬‭an‬‭individual‬

‭exploration‬‭into‬‭this‬‭issue,‬‭I‬‭aim‬‭to‬‭answer‬‭the‬‭following:‬‭How‬‭does‬‭analyzing‬‭the‬‭effect‬‭of‬

‭global‬‭warming‬‭on‬‭enzymes’‬‭abilities‬‭to‬‭function‬‭in‬‭aquatic‬‭ectotherms‬‭reveal‬‭what‬‭long-term‬

‭issues‬‭could‬‭arise‬‭about‬‭their‬‭physical‬‭health?‬

‭Each‬‭cell‬‭contains‬‭enzymes‬‭that‬‭carry‬‭out‬‭processes‬‭to‬‭support‬‭the‬‭main‬‭functions‬‭of‬‭life,‬

‭most‬‭importantly,‬‭metabolism.‬‭The‬‭subject‬‭of‬‭experimentation‬‭for‬‭this‬‭experiment‬‭is‬‭the‬‭enzyme‬

‭pepsin,‬‭which‬‭is‬‭vital‬‭for‬‭the‬‭digestion‬‭process,‬‭and‬‭the‬‭protein,‬‭ovalbumin.‬‭Within‬‭the‬‭gastric‬

‭juices,‬‭pepsin‬‭is‬‭the‬‭main‬‭enzyme‬‭in‬‭breaking‬‭down‬‭proteins.‬‭The‬‭aim‬‭of‬‭this‬‭experiment‬‭is‬‭to‬

‭model‬‭the‬‭breakdown‬‭of‬‭ovalbumin‬‭(shortened‬‭to‬‭just‬‭albumin)‬‭by‬‭pepsin‬‭in‬‭the‬‭stomach‬‭of‬‭fish.‬

‭Fish‬‭were‬‭chosen‬‭to‬‭base‬‭this‬‭investigation‬‭off‬‭of‬‭because‬‭of‬‭the‬‭simplicity‬‭of‬‭their‬‭digestive‬

‭systems‬‭and‬‭location‬‭on‬‭the‬‭food‬‭chain.‬‭Most‬‭fish‬‭species;‬‭such‬‭as‬‭the‬‭Salmo,‬‭Clarias,‬‭or‬

‭Oreochromis‬‭genera‬‭(Kiew‬‭&‬‭Don,‬‭2012);‬‭are‬‭low‬‭on‬‭the‬‭food‬‭chain,‬‭thus‬‭making‬‭them‬‭a‬
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‭common‬‭source‬‭of‬‭nutrients‬‭for‬‭many‬‭organisms.‬‭For‬‭this‬‭reason,‬‭the‬‭overall‬‭survival‬‭of‬‭fish‬‭can‬

‭impact‬‭the‬‭dietary‬‭health‬‭of‬‭these‬‭organisms.‬

‭By‬‭focusing‬‭on‬‭one‬‭specific‬‭enzyme,‬‭pepsin,‬‭and‬‭one‬‭specific‬‭substrate,‬‭albumin,‬‭much‬

‭can‬‭be‬‭revealed‬‭and‬‭analyzed‬‭from‬‭the‬‭results‬‭in‬‭regards‬‭to‬‭how‬‭global‬‭warming‬‭may‬‭affect‬‭the‬

‭long-term‬‭functioning‬‭of‬‭these‬‭enzymes.‬‭The‬‭knowledge‬‭gained‬‭from‬‭this‬‭experiment‬‭may‬‭reveal‬

‭what‬‭issues‬‭fish‬‭face‬‭in‬‭their‬‭digestive‬‭systems‬‭and‬‭what‬‭can‬‭happen‬‭to‬‭the‬‭diets‬‭of‬‭organisms‬‭as‬

‭a‬‭result.‬

‭Background‬

‭The‬‭differences‬‭in‬‭physiological‬‭natures‬‭between‬‭ectotherms‬‭like‬‭fish‬‭and‬‭endotherms‬

‭like‬‭mammals‬‭must‬‭first‬‭be‬‭discussed.‬‭These‬‭groups‬‭have‬‭different‬‭conditions‬‭for‬‭proper‬‭growth.‬

‭With‬‭that‬‭being‬‭said,‬‭endotherms‬‭and‬‭ectotherms‬‭share‬‭the‬‭same‬‭categorization‬‭as‬‭mesophiles.‬

‭Mesophilic‬‭organisms‬‭grow‬‭and‬‭develop‬‭ideally‬‭in‬‭temperatures‬‭ranging‬‭from‬‭25-40°C‬‭(Barik,‬

‭2020).‬‭Anything‬‭above‬‭this‬‭range‬‭poses‬‭physiological‬‭problems,‬‭of‬‭which‬‭this‬‭paper‬‭will‬‭focus‬

‭on‬‭enzyme‬‭denaturation.‬‭However,‬‭the‬‭main‬‭difference‬‭between‬‭endotherms‬‭and‬‭ectotherms‬‭is‬

‭the‬‭categorization‬‭of‬‭how‬‭they‬‭maintain‬‭their‬‭body‬‭temperature.‬‭Ectotherms‬‭regulate‬‭their‬

‭internal‬‭temperatures‬‭by‬‭being‬‭dependent‬‭on‬‭the‬‭external‬‭temperatures‬‭of‬‭their‬‭surroundings.‬‭On‬

‭the‬‭other‬‭hand,‬‭endotherms‬‭typically‬‭maintain‬‭an‬‭internal‬‭body‬‭temperature‬‭of‬‭35-40°C.‬

‭Evaluating‬‭the‬‭vulnerability‬‭of‬‭species‬‭is‬‭a‬‭complex‬‭process‬‭because‬‭this‬‭is‬‭determined‬‭by‬

‭various‬‭sources‬‭of‬‭biological‬‭information.‬‭However,‬‭ectotherms‬‭are‬‭typically‬‭more‬‭resistant‬

‭against‬‭relatively‬‭rapid‬‭temperature‬‭changes‬‭within‬‭their‬‭environments.‬‭Endotherms,‬‭however,‬

‭are‬‭not‬‭as‬‭resistant.‬‭Most‬‭do‬‭not‬‭have‬‭the‬‭same‬‭physiological‬‭abilities‬‭in‬‭place‬‭to‬‭shift‬‭their‬

‭‘comfort’‬‭temperature‬‭zone‬‭to‬‭acclimate‬‭to‬‭rapidly‬‭changing‬‭temperatures.‬‭According‬‭to‬‭Huey‬‭et‬

‭al.‬‭(2012),‬‭“Torpor‬‭may‬‭be‬‭an‬‭endotherm's‬‭primary‬‭way‬‭of‬‭dealing‬‭with‬‭stressfully‬‭warm‬
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‭environments,‬‭even‬‭in‬‭the‬‭tropics.”‬‭Essentially,‬‭if‬‭endotherms,‬‭such‬‭as‬‭humans,‬‭cannot‬‭tolerate‬

‭chronic‬‭increases‬‭in‬‭temperature,‬‭they‬‭will‬‭be‬‭forced‬‭to‬‭undergo‬‭long‬‭states‬‭of‬‭lethargy.‬‭This‬‭is‬

‭detrimental‬‭to‬‭the‬‭growth‬‭of‬‭mammals,‬‭causing‬‭lack‬‭of‬‭energy‬‭to‬‭complete‬‭tasks.‬‭Most‬‭mammals‬

‭are‬‭not‬‭biologically‬‭programmed‬‭to‬‭be‬‭able‬‭to‬‭withstand‬‭frequent‬‭heat‬‭waves,‬‭as‬‭they‬‭render‬‭their‬

‭biological‬‭systems‬‭to‬‭slow‬‭down‬‭significantly.‬‭Because‬‭most‬‭endotherms‬‭are‬‭such‬‭complex‬

‭organisms,‬‭simple‬‭aquatic‬‭life‬‭was‬‭used‬‭to‬‭base‬‭my‬‭findings‬‭on.‬‭Because‬‭fish‬‭have‬‭inverse‬

‭temperature‬‭maintaining‬‭systems‬‭of‬‭mammals,‬‭it‬‭might‬‭be‬‭possible‬‭to‬‭say‬‭that‬‭the‬‭results‬‭gained‬

‭from‬‭this‬‭finding‬‭could‬‭present‬‭differently‬‭in‬‭the‬‭bodies‬‭of‬‭larger‬‭mammals‬‭like‬‭humans.‬

‭Pepsin‬

‭Pepsin‬‭is‬‭a‬‭naturally‬‭occurring‬‭enzyme‬‭located‬‭in‬‭the‬‭gastrointestinal‬‭tract,‬‭more‬

‭specifically‬‭in‬‭the‬‭duodenum‬‭and‬‭stomach.‬‭This‬‭enzyme‬‭breaks‬‭down‬‭the‬‭primary‬‭bonds‬‭in‬

‭protein‬‭molecules‬‭called‬‭peptide‬‭bonds‬‭(Heda‬‭et‬‭al.,‬‭2022).‬‭Pepsin‬‭cuts‬‭the‬‭peptide‬‭bonds‬‭at‬‭the‬

‭amino-terminal‬‭side‬‭of‬‭the‬‭cyclic‬‭amino‬‭acid‬‭residues‬‭,‬‭producing‬‭proteoses‬‭and‬‭peptones‬

‭(Moraes‬‭&‬‭de‬‭Almeida,‬‭2020).‬‭At‬‭a‬‭temperature‬‭range‬‭of‬‭40-60°C,‬‭pepsin‬‭is‬‭at‬‭its‬‭optimal‬

‭temperature‬‭to‬‭break‬‭down‬‭substrates‬‭at‬‭the‬‭highest‬‭rate‬‭(See‬‭Figure‬‭1).‬‭Like‬‭any‬‭other‬‭enzyme,‬

‭when‬‭denatured,‬‭the‬‭structure‬‭of‬‭an‬‭enzyme‬‭is‬‭compromised,‬‭as‬‭the‬‭high-order‬‭structure‬‭is‬

‭unfolded.‬‭In‬‭this‬‭way,‬‭substrates‬‭are‬‭not‬‭able‬‭to‬‭bond‬‭to‬‭the‬‭activation‬‭site‬‭of‬‭the‬‭enzyme‬‭.‬‭The‬

‭concept‬‭of‬‭protein-unfolding‬‭in‬‭the‬‭presence‬‭of‬‭a‬‭stressor,‬‭like‬‭high‬‭temperatures,‬‭is‬‭a‬

‭characteristic‬‭of‬‭all‬‭life‬‭on‬‭earth.‬‭However,‬‭in‬‭the‬‭case‬‭of‬‭pepsin,‬‭it‬‭is‬‭unlikely‬‭that‬‭high‬

‭temperatures‬‭will‬‭have‬‭an‬‭effect‬‭on‬‭pepsin’s‬‭inactivity.‬‭Most‬‭likely,‬‭pepsin‬‭will‬‭have‬‭increased‬

‭activities‬‭of‬‭breakdown‬‭at‬‭higher‬‭temperatures,‬‭according‬‭to‬‭the‬‭literature.‬
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‭Figure‬‭1‬

‭Note.‬‭The‬‭effect‬‭of‬‭temperature‬‭on‬‭pepsin‬‭activity‬‭(Sholeh‬‭et‬‭al.,‬‭2020).‬

‭A‬‭lack‬‭of‬‭pepsin‬‭in‬‭the‬‭gastrointestinal‬‭tract‬‭can‬‭cause‬‭a‬‭host‬‭of‬‭physical‬‭health‬‭problems‬

‭for‬‭humans‬‭and‬‭animals‬‭alike.‬‭A‬‭common‬‭physiopathological‬‭issue‬‭that‬‭can‬‭arise‬‭is‬‭the‬

‭occurrence‬‭of‬‭hypochlorhydria,‬‭the‬‭deficiency‬‭of‬‭gastric‬‭acid‬‭in‬‭the‬‭stomach.‬‭Typically,‬‭this‬

‭disease‬‭is‬‭caused‬‭by‬‭other‬‭origins‬‭such‬‭as‬‭the‬‭chronic‬‭use‬‭of‬‭antacids‬‭to‬‭treat‬‭heartburn‬‭or‬‭acid‬

‭reflux‬‭(Kines‬‭&‬‭Krupczak,‬‭2016).‬‭However,‬‭the‬‭same‬‭principles‬‭of‬‭how‬‭this‬‭can‬‭lead‬‭to‬

‭hypochlorhydria,‬‭can‬‭be‬‭applied‬‭to‬‭how‬‭a‬‭continuous‬‭increase‬‭in‬‭temperatures‬‭can‬‭lead‬‭to‬‭the‬

‭same‬‭condition.‬‭Through‬‭an‬‭increase‬‭in‬‭temperature,‬‭pepsin‬‭is‬‭denatured‬‭as‬‭its‬‭effective‬‭form’s‬

‭concentration‬‭in‬‭the‬‭stomach‬‭is‬‭decreased.‬‭Essentially,‬‭this‬‭produces‬‭the‬‭same‬‭effect‬‭as‬‭if‬‭pepsin‬

‭concentration‬‭was‬‭low‬‭in‬‭the‬‭stomach.‬‭Low‬‭concentration‬‭of‬‭functioning‬‭pepsin‬‭is‬‭dangerous‬‭to‬

‭the‬‭dietary‬‭health‬‭of‬‭organisms.‬‭One‬‭specific‬‭example,‬‭in‬‭regards‬‭to‬‭the‬‭bodies‬‭of‬‭fish,‬‭is‬‭how‬‭an‬

‭insufficient‬‭amount‬‭of‬‭pepsin‬‭can‬‭also‬‭lead‬‭to‬‭vitamin‬‭B12‬‭deficiencies.‬‭Fish‬‭are‬‭incredibly‬‭high‬

‭in‬‭B12‬‭concentration.‬‭This‬‭high‬‭concentration‬‭is‬‭most‬‭likely‬‭due‬‭to‬‭proper‬‭functioning‬‭of‬
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‭enzymes‬‭such‬‭as‬‭pepsin‬‭to‬‭be‬‭able‬‭to‬‭break‬‭down‬‭proteins,‬‭releasing‬‭B12‬‭into‬‭the‬‭body‬‭system.‬‭If‬

‭B12‬‭cannot‬‭be‬‭properly‬‭produced‬‭by‬‭pepsin,‬‭then‬‭organisms‬‭that‬‭feed‬‭off‬‭of‬‭fish‬‭will‬‭face‬‭effects‬

‭of‬‭a‬‭lack‬‭of‬‭B12‬‭from‬‭less‬‭nutrient-dense‬‭fish‬‭(Lyon‬‭et‬‭al.,‬‭2020).‬

‭Albumin‬

‭Albumin‬‭comes‬‭in‬‭a‬‭few‬‭different‬‭forms‬‭including‬‭ovalbumin‬‭and‬‭serum‬‭albumin‬

‭(Raoufinia‬‭et‬‭al.,‬‭2016).‬‭Ovalbumin‬‭is‬‭a‬‭protein‬‭found‬‭in‬‭egg‬‭whites.‬‭The‬‭subject‬‭of‬‭focus‬‭for‬‭this‬

‭investigation‬‭is‬‭ovalbumin.‬‭This‬‭substrate‬‭was‬‭chosen‬‭for‬‭its‬‭simplicity.‬

‭Figure‬‭2‬

‭Note.‬‭Albumin‬‭structure‬‭(Raoufinia‬‭et‬‭al.,‬‭2016).‬
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‭Methodology‬

‭In‬‭this‬‭experiment,‬‭small‬‭tubes‬‭will‬‭be‬‭filled‬‭with‬‭solidified‬‭albumin‬‭protein.‬‭These‬‭pieces‬

‭will‬‭be‬‭placed‬‭into‬‭pepsin‬‭solutions‬‭in‬‭beakers‬‭that‬‭sit‬‭in‬‭water‬‭baths‬‭of‬‭varying‬‭temperatures.‬

‭This‬‭method‬‭was‬‭chosen‬‭because‬‭of‬‭its‬‭simplicity,‬‭but‬‭also‬‭its‬‭similarity‬‭to‬‭the‬‭process‬‭of‬‭enzyme‬

‭activity‬‭in‬‭all‬‭organisms.‬‭After‬‭a‬‭set‬‭amount‬‭of‬‭time,‬‭the‬‭length‬‭of‬‭albumin‬‭still‬‭remaining‬‭in‬‭the‬

‭tubes‬‭will‬‭be‬‭measured.‬

‭Hypothesis‬

‭It‬‭is‬‭predicted‬‭that‬‭the‬‭pieces‬‭of‬‭encased‬‭albumin‬‭will‬‭be‬‭more‬‭broken‬‭down‬‭in‬‭pepsin‬

‭solutions‬‭that‬‭are‬‭higher‬‭than‬‭40°C.‬‭This‬‭is‬‭because‬‭the‬‭ideal‬‭temperature‬‭for‬‭pepsin‬‭activation‬‭is‬

‭around‬‭40°C‬‭(Mazumder‬‭et‬‭al.,‬‭2018).‬‭As‬‭a‬‭result,‬‭the‬‭enzyme’s‬‭ability‬‭to‬‭function‬‭is‬‭affected‬‭in‬

‭that‬‭the‬‭enzymes‬‭are‬‭denatured.‬‭At‬‭40°C,‬‭pepsin‬‭becomes‬‭denatured‬‭and‬‭is‬‭not‬‭able‬‭to‬‭properly‬

‭digest‬‭proteins.‬

‭The‬‭null‬‭hypothesis‬‭for‬‭this‬‭experiment‬‭is‬‭that‬‭the‬‭data‬‭collected‬‭will‬‭show‬‭no‬‭significant‬

‭difference‬‭in‬‭the‬‭decrease‬‭in‬‭lengths‬‭between‬‭the‬‭varying‬‭temperatures.‬‭The‬‭alternative‬

‭hypothesis‬‭for‬‭this‬‭experiment‬‭is‬‭that‬‭the‬‭albumin‬‭pieces‬‭are‬‭more‬‭broken‬‭down‬‭in‬‭temperatures‬

‭that‬‭are‬‭higher.‬‭This‬‭hypothesis‬‭would‬‭support‬‭that‬‭global‬‭warming‬‭does‬‭not‬‭have‬‭a‬‭strong‬‭impact‬

‭on‬‭the‬‭denaturation‬‭of‬‭pepsin‬‭in‬‭ectotherms.‬

‭Variables‬

‭Independent‬‭Variables:‬

‭Temperature‬‭was‬‭used‬‭as‬‭the‬‭independent‬‭variable‬‭in‬‭this‬‭experiment.‬‭Each‬‭of‬‭the‬‭five‬

‭water‬‭baths‬‭were‬‭set‬‭to‬‭either‬‭20°C,‬‭26℃,‬‭35℃,‬‭40℃,‬‭or‬‭45℃.‬‭These‬‭were‬‭chosen‬‭to‬‭simulate‬‭a‬

‭control‬‭temperature,‬‭normal‬‭temperatures‬‭of‬‭water‬‭that‬‭fish‬‭survive‬‭in,‬‭and‬‭high‬‭temperatures‬

‭produced‬‭by‬‭a‬‭warming‬‭of‬‭waters.‬‭Additionally,‬‭these‬‭temperatures‬‭were‬‭chosen‬‭because‬‭of‬‭their‬
 ‭9‬

‭relationship‬‭to‬‭the‬‭range‬‭of‬‭temperatures‬‭that‬‭pepsin‬‭exhibited‬‭the‬‭highest‬‭activity‬‭at,‬‭according‬

‭to‬‭previous‬‭data.‬‭The‬‭albumin‬‭pieces‬‭subjected‬‭to‬‭pepsin‬‭solution‬‭that‬‭was‬‭at‬‭20°C‬‭are‬‭expected‬

‭to‬‭show‬‭no‬‭difference‬‭in‬‭length‬‭because‬‭it‬‭is‬‭well‬‭outside‬‭of‬‭the‬‭optimal‬‭temperature‬‭range.‬‭Thus,‬

‭this‬‭temperature‬‭was‬‭chosen‬‭as‬‭the‬‭control‬‭group.‬‭Next,‬‭26°C‬‭and‬‭35°C‬‭were‬‭chosen‬‭because‬

‭they‬‭were‬‭also‬‭outside‬‭the‬‭optimal‬‭temperature‬‭range,‬‭but‬‭not‬‭far‬‭enough‬‭outside‬‭for‬‭pepsin‬‭to‬

‭exhibit‬‭a‬‭complete‬‭lack‬‭of‬‭function.‬‭Finally,‬‭40°C‬‭and‬‭45°C‬‭were‬‭chosen‬‭because‬‭they‬‭fall‬‭within‬

‭the‬‭temperature‬‭range,‬‭and‬‭the‬‭albumin‬‭pieces‬‭are‬‭expected‬‭to‬‭show‬‭higher‬‭decreases‬‭in‬‭length.‬

‭Controlled‬‭Variables:‬

‭The‬‭first‬‭component‬‭that‬‭was‬‭controlled‬‭in‬‭this‬‭experiment‬‭was‬‭the‬‭concentration‬‭of‬

‭pepsin.‬‭Each‬‭beaker‬‭in‬‭the‬‭hot‬‭baths‬‭contained‬‭a‬‭1%‬‭pepsin‬‭solution.‬‭If‬‭the‬‭pepsin‬‭concentration‬

‭varied‬‭from‬‭beaker‬‭to‬‭beaker,‬‭the‬‭reaction‬‭rates‬‭of‬‭the‬‭beakers‬‭with‬‭a‬‭higher‬‭concentration‬‭of‬

‭pepsin‬‭would‬‭increase‬‭because‬‭there‬‭are‬‭more‬‭enzymes‬‭for‬‭the‬‭substrate‬‭to‬‭react‬‭with.‬‭The‬

‭second‬‭component‬‭that‬‭was‬‭important‬‭to‬‭control‬‭was‬‭the‬‭pH‬‭level‬‭of‬‭the‬‭pepsin‬‭solution.‬‭Pepsin’s‬

‭ideal‬‭pH‬‭is‬‭around‬‭2.‬‭In‬‭order‬‭to‬‭keep‬‭the‬‭pH‬‭at‬‭an‬‭ideal‬‭level,‬‭potassium‬‭biphthalate‬‭was‬‭added‬

‭in‬‭order‬‭to‬‭create‬‭an‬‭acidic‬‭system‬‭for‬‭the‬‭pepsin.‬‭pH‬‭is‬‭important‬‭to‬‭keep‬‭controlled‬‭because‬‭it‬

‭has‬‭an‬‭effect‬‭on‬‭the‬‭denaturation‬‭of‬‭pepsin.‬‭Once‬‭the‬‭system‬‭that‬‭pepsin‬‭is‬‭in‬‭reaches‬‭above‬‭a‬‭pH‬

‭of‬‭6,‬‭pepsin‬‭becomes‬‭denatured.‬‭Additionally,‬‭pH‬‭had‬‭to‬‭be‬‭kept‬‭controlled‬‭because‬‭then‬‭the‬

‭experiment‬‭would‬‭have‬‭to‬‭be‬‭classified‬‭as‬‭a‬‭two‬‭factor‬‭test.‬

‭Statistical‬‭Analysis‬

‭A‬‭one-factor‬‭ANOVA‬‭test‬‭will‬‭be‬‭used‬‭to‬‭compare‬‭variances‬‭between‬‭the‬‭groups‬‭of‬

‭albumin‬‭pieces‬‭from‬‭each‬‭temperature‬‭variation.‬‭Xccel‬‭software‬‭will‬‭be‬‭used‬‭to‬‭calculate‬‭the‬

‭f-statistic,‬‭p-value,‬‭and‬‭other‬‭significant‬‭analysis‬‭values‬‭for‬‭the‬‭data.‬‭The‬‭alpha-value‬‭for‬‭this‬

‭experiment‬‭is‬‭0.05.‬‭The‬‭p-value‬‭will‬‭be‬‭compared‬‭to‬‭this‬‭value,‬‭and‬‭the‬‭null‬‭hypothesis‬‭will‬‭be‬
 ‭10‬

‭rejected‬‭if‬‭the‬‭p-value‬‭is‬‭lower‬‭than‬‭the‬‭alpha-value.‬‭In‬‭order‬‭to‬‭calculate‬‭an‬‭ANOVA‬‭test,‬‭there‬

‭needs‬‭to‬‭be‬‭a‬‭standard‬‭deviation‬‭that‬‭is‬‭not‬‭0.‬‭This‬‭experiment‬‭should‬‭most‬‭likely‬‭not‬‭yield‬

‭standard‬‭deviations‬‭of‬‭0.‬

‭Ethical‬‭Considerations‬

‭Although‬‭the‬‭nature‬‭of‬‭animals‬‭were‬‭studied,‬‭no‬‭animals‬‭were‬‭experimented‬‭upon.‬‭All‬

‭data‬‭and‬‭research‬‭of‬‭fish‬‭was‬‭from‬‭secondary‬‭research.‬

‭Materials‬

‭5‬‭hot‬‭water‬‭baths‬

‭Distilled‬‭water‬

‭13g‬‭albumin‬‭powder‬

‭100mL‬‭beaker‬

‭Stirring‬‭rod‬

‭Hot‬‭plate‬‭stirrer‬

‭2‬‭pH‬‭buffer‬‭(potassium‬‭biphthalate)‬‭capsules‬

‭1‬‭large‬‭beaker‬

‭10-12‬‭pipettes‬

‭1‬‭medium‬‭beaker‬

‭Hot‬‭plate‬

‭8g‬‭pepsin‬‭powder‬

‭pH‬‭strips‬‭and‬‭tester‬

‭Scissors‬

‭25‬‭small‬‭beakers‬
 ‭11‬

‭Procedure‬

‭1.‬ ‭Filled‬‭each‬‭hot‬‭water‬‭bath‬‭with‬‭water‬‭to‬‭about‬‭halfway‬‭full.‬‭Prepared‬‭each‬‭of‬‭the‬‭hot‬

‭water‬‭baths‬‭by‬‭setting‬‭them‬‭according‬‭to‬‭the‬‭temperatures‬‭listed‬‭in‬‭the‬‭table‬‭below.‬

‭2.‬ ‭Made‬‭an‬‭87%‬‭albumin‬‭solution‬‭by‬‭mixing‬‭87‬‭mL‬‭of‬‭distilled‬‭water‬‭with‬‭13‬‭g‬‭of‬‭albumin‬

‭powder‬‭in‬‭a‬‭100‬‭mL‬‭beaker.‬‭Stirred‬‭with‬‭a‬‭stirring‬‭rod‬‭until‬‭it‬‭reached‬‭a‬‭thick‬‭consistency.‬

‭(Note:‬‭Needed‬‭to‬‭place‬‭on‬‭a‬‭hotplate‬‭stirrer‬‭on‬‭low‬‭heat‬‭in‬‭order‬‭to‬‭completely‬‭dissolve‬

‭the‬‭albumin‬‭in‬‭the‬‭water.)‬‭Set‬‭off‬‭to‬‭the‬‭side.‬

‭3.‬ ‭Prepared‬‭the‬‭2‬‭pH‬‭buffer‬‭solution‬‭by‬‭mixing‬‭800‬‭mL‬‭of‬‭distilled‬‭water‬‭with‬‭8‬‭capsules.‬

‭Set‬‭off‬‭to‬‭the‬‭side‬‭in‬‭a‬‭large‬‭beaker.‬

‭4.‬ ‭Using‬‭the‬‭pipettes,‬‭took‬‭in‬‭just‬‭enough‬‭of‬‭the‬‭albumin‬‭solution‬‭so‬‭that‬‭just‬‭the‬‭narrow‬

‭ends‬‭filled‬‭all‬‭the‬‭way.‬

‭5.‬ ‭Placed‬‭a‬‭medium-sized‬‭beaker‬‭filled‬‭with‬‭distilled‬‭water‬‭onto‬‭a‬‭hot‬‭plate‬‭and‬‭boiled.‬

‭Once‬‭boiling,‬‭placed‬‭each‬‭pipette‬‭into‬‭the‬‭beaker.‬‭Removed‬‭each‬‭pipette‬‭once‬‭the‬

‭albumin‬‭turned‬‭white.‬

‭6.‬ ‭Allowed‬‭each‬‭pipette‬‭to‬‭cool‬‭for‬‭a‬‭few‬‭minutes.‬

‭7.‬ ‭Weighed‬‭out‬‭8‬‭grams‬‭of‬‭pepsin‬‭powder‬‭to‬‭make‬‭the‬‭1%‬‭pepsin‬‭solution.‬‭Added‬‭to‬‭the‬

‭buffer‬‭solution‬‭and‬‭stirred‬‭with‬‭a‬‭clean‬‭stirring‬‭rod.‬‭(Made‬‭sure‬‭the‬‭pH‬‭of‬‭this‬‭solution‬‭is‬

‭at‬‭2‬‭by‬‭using‬‭pH‬‭strips.)‬

‭8.‬ ‭Cut‬‭each‬‭pipette‬‭into‬‭pieces‬‭three‬‭centimeters‬‭in‬‭length.‬‭Ensured‬‭that‬‭each‬‭piece‬‭is‬

‭completely‬‭filled‬‭with‬‭the‬‭solidified‬‭albumin.‬‭(Resulted‬‭in‬‭about‬‭30‬‭3-cm‬‭pieces.)‬

‭9.‬ ‭Poured‬‭30‬‭ml‬‭of‬‭the‬‭1%‬‭pepsin‬‭and‬‭buffer‬‭solution‬‭into‬‭all‬‭of‬‭the‬‭small‬‭beakers.‬

‭10.‬‭Placed‬‭five‬‭of‬‭the‬‭small‬‭beakers‬‭into‬‭each‬‭of‬‭the‬‭hot‬‭water‬‭baths.‬‭(Removed‬‭water‬‭from‬

‭the‬‭hot‬‭water‬‭bath‬‭as‬‭needed‬‭if‬‭it‬‭started‬‭to‬‭overflow‬‭from‬‭the‬‭addition‬‭of‬‭the‬‭small‬
 ‭12‬

‭beakers.)‬‭Also,‬‭I‬‭made‬‭sure‬‭to‬‭measure‬‭the‬‭pH‬‭once‬‭again‬‭in‬‭case‬‭any‬‭water‬‭from‬‭the‬

‭water‬‭baths‬‭overflowed‬‭into‬‭the‬‭beakers.‬‭This‬‭would‬‭cause‬‭the‬‭pH‬‭to‬‭increase‬‭which‬‭is‬‭an‬

‭undesired‬‭effect.‬

‭11.‬‭Placed‬‭1‬‭albumin‬‭piece‬‭into‬‭each‬‭small‬‭beaker.‬

‭12.‬‭Allowed‬‭the‬‭heated‬‭baths‬‭to‬‭run‬‭for‬‭24‬‭hours.‬‭(Initially,‬‭each‬‭water‬‭bath‬‭ran‬‭for‬‭10‬

‭minutes,‬‭but‬‭upon‬‭noticing‬‭that‬‭there‬‭was‬‭no‬‭change‬‭in‬‭any‬‭of‬‭albumin‬‭pieces,‬‭the‬‭time‬

‭was‬‭gradually‬‭increased‬‭as‬‭needed.‬‭Eventually,‬‭I‬‭realized‬‭it‬‭was‬‭optimal‬‭for‬‭the‬‭water‬

‭baths‬‭to‬‭run‬‭for‬‭24‬‭hours.)‬‭Then,‬‭measured‬‭the‬‭change‬‭in‬‭length‬‭for‬‭each‬‭albumin‬‭piece.‬

‭The‬‭data‬‭is‬‭recorded‬‭in‬‭the‬‭table.‬

‭Results‬

‭Quantitative‬‭Findings‬

‭Table‬‭1‬‭:‬‭Change‬‭in‬‭lengths‬‭(mm)‬‭of‬‭albumin‬‭pieces‬‭after‬‭soaking‬‭in‬‭pepsin‬‭solution‬‭for‬‭24‬

‭hours‬‭in‬‭varying‬‭temperatures‬

‭Temperature‬ ‭Decrease‬‭in‬‭length‬‭of‬‭each‬‭#‬‭trial‬‭(mm)‬
‭of‬‭Hot‬‭Bath‬
‭(℃)‬ ‭1‬ ‭2‬ ‭3‬ ‭4‬ ‭5‬

‭20‬ ‭1‬ ‭0‬ ‭0‬ ‭0‬ ‭1‬

‭26‬ ‭2‬ ‭2‬ ‭4‬ ‭3‬ ‭1‬

‭35‬ ‭2‬ ‭2‬ ‭3‬ ‭3‬ ‭2‬

‭40‬ ‭5‬ ‭4‬ ‭4‬ ‭4‬ ‭1‬

‭43‬ ‭9‬ ‭2‬ ‭4‬ ‭2.5‬ ‭6‬

‭Table‬‭2:‬‭Processed‬‭data‬‭of‬‭change‬‭in‬‭lengths‬‭of‬‭albumin‬‭pieces‬‭after‬‭soaking‬‭in‬‭pepsin‬

‭solution‬‭for‬‭24‬‭hours‬
 ‭13‬

‭Temperature‬‭of‬ ‭Mean‬‭decrease‬‭in‬‭length‬‭of‬‭each‬‭#‬ ‭Variance‬‭of‬‭decrease‬‭in‬‭length‬‭(mm)‬

‭Hot‬‭Bath‬‭(℃)‬ ‭trial‬‭(mm)‬‭±‬‭standard‬‭deviation‬

‭20‬ ‭0.4‬‭±‬‭0.548‬ ‭0.3‬

‭26‬ ‭2.4‬‭±‬‭1.14‬ ‭1.3‬

‭35‬ ‭2.4‬‭±‬‭0.548‬ ‭0.3‬

‭40‬ ‭3.6‬‭±‬‭1.52‬ ‭2.3‬

‭43‬ ‭4.7‬‭±‬‭2.86‬ ‭8.2‬

‭Table‬‭3:‬‭Single‬‭Factor‬‭ANOVA‬‭Test‬‭comparing‬‭the‬‭differences‬‭in‬‭changes‬‭in‬‭length‬‭of‬

‭resulting‬‭from‬‭each‬‭temperature‬

‭ ource‬‭of‬
S
‭Variation‬ ‭SS‬ ‭df‬ ‭MS‬ ‭F‬ ‭P-value‬ ‭F‬‭crit.‬
‭ etween‬
B
‭Groups‬ ‭51.4‬ ‭4‬ ‭12.85‬ ‭5.181452‬ ‭0.004968‬ ‭2.866081‬
‭ ithin‬
W
‭Groups‬ ‭49.6‬ ‭20‬ ‭2.48‬

‭Total‬ ‭101‬ ‭24‬

‭Analysis‬

‭The‬‭purpose‬‭of‬‭the‬‭Single‬‭Factor‬‭ANOVA‬‭test‬‭was‬‭to‬‭determine‬‭differences‬‭between‬‭the‬

‭results‬‭from‬‭each‬‭of‬‭the‬‭different‬‭temperatures.‬‭The‬‭𝛼-value‬‭assigned‬‭to‬‭this‬‭data‬‭was‬‭0.05.‬

‭Because‬‭the‬‭p-value‬‭is‬‭less‬‭than‬‭the‬‭𝛼-value,‬‭the‬‭null‬‭hypothesis‬‭is‬‭rejected,‬‭and‬‭the‬‭alternative‬

‭hypothesis‬‭is‬‭accepted.‬‭This‬‭means‬‭that‬‭there‬‭is‬‭a‬‭significant‬‭difference‬‭between‬‭the‬‭mean‬‭lengths‬

‭of‬‭each‬‭group.‬‭The‬‭ANOVA‬‭test‬‭showed‬‭that‬‭there‬‭was‬‭a‬‭difference‬‭in‬‭the‬‭decrease‬‭in‬‭lengths‬

‭between‬‭each‬‭temperature‬‭group,‬‭and‬‭analyzing‬‭the‬‭mean‬‭values‬‭in‬‭Table‬‭2‬‭led‬‭to‬‭the‬‭finding‬‭that‬
 ‭14‬

‭as‬‭temperature‬‭increases,‬‭the‬‭change‬‭in‬‭length‬‭also‬‭increased,‬‭showing‬‭a‬‭proportional‬

‭relationship.‬‭On‬‭average,‬‭the‬‭decrease‬‭in‬‭lengths‬‭of‬‭the‬‭albumin‬‭pieces‬‭in‬‭each‬‭temperature‬‭were‬

‭differentiated‬‭by‬‭about‬‭1‬‭mm.‬‭So,‬‭approximately,‬‭in‬‭each‬‭water‬‭bath,‬‭the‬‭pepsin‬‭had‬‭a‬‭higher‬‭rate‬

‭of‬‭reaction‬‭and‬‭was‬‭able‬‭to‬‭break‬‭down‬‭more‬‭albumin‬‭within‬‭24‬‭hours.‬‭In‬‭this‬‭experiment,‬

‭temperature‬‭increased‬‭pepsin’s‬‭ability‬‭to‬‭function.‬‭This‬‭finding‬‭was‬‭expected,‬‭as‬‭at‬‭the‬‭highest‬

‭temperature,‬‭pepsin‬‭was‬‭able‬‭to‬‭break‬‭down‬‭more‬‭albumin‬‭within‬‭the‬‭24‬‭hours‬‭than‬‭at‬‭any‬‭of‬‭the‬

‭other‬‭temperatures.‬‭This‬‭relationship‬‭was‬‭what‬‭had‬‭been‬‭predicted‬‭prior‬‭to‬‭running‬‭the‬

‭experiment,‬‭according‬‭the‬‭research‬‭that‬‭was‬‭conducted.‬‭The‬‭lowest‬‭temperature,‬‭20°C‬‭makes‬

‭sense,‬‭as‬‭pepsin‬‭is‬‭minimally‬‭effective‬‭at‬‭temperatures‬‭at‬‭or‬‭below‬‭20°C.‬‭According‬‭to‬‭the‬‭data,‬‭3‬

‭out‬‭of‬‭the‬‭five‬‭trials‬‭exhibited‬‭no‬‭change‬‭in‬‭length‬‭when‬‭submerged‬‭in‬‭the‬‭pepsin‬‭solution‬‭at‬

‭20°C.‬

‭This‬‭experiment‬‭was‬‭meant‬‭to‬‭simulate‬‭the‬‭global‬‭warming‬‭of‬‭waters‬‭as‬‭closely‬‭as‬

‭possible‬‭with‬‭the‬‭limited‬‭resources‬‭at‬‭hand.‬‭Any‬‭increase‬‭in‬‭heat‬‭would‬‭most‬‭likely‬‭increase‬

‭pepsin's‬‭ability‬‭to‬‭properly‬‭break‬‭the‬‭peptide‬‭bonds‬‭in‬‭the‬‭albumin.‬‭Even‬‭at‬‭the‬‭most‬‭extreme‬

‭temperature,‬‭43°C,‬‭the‬‭temperature‬‭not‬‭only‬‭failed‬‭to‬‭denature‬‭the‬‭pepsin‬‭molecules,‬‭but‬‭also‬

‭aided‬‭the‬‭power‬‭of‬‭the‬‭enzyme‬‭to‬‭break‬‭the‬‭bonds.‬

‭The‬‭higher‬‭decrease‬‭in‬‭length‬‭of‬‭each‬‭of‬‭the‬‭albumin‬‭pieces‬‭in‬‭each‬‭level‬‭of‬‭temperature‬

‭shows‬‭that‬‭a‬‭higher‬‭temperature‬‭increased‬‭pepsin‬‭activity.‬‭Through‬‭previous‬‭research,‬‭it‬‭was‬

‭predicted‬‭that‬‭as‬‭the‬‭temperature‬‭of‬‭the‬‭solution‬‭that‬‭pepsin‬‭was‬‭dissolved‬‭in‬‭increased,‬‭so‬‭would‬

‭pepsin‬‭activity.‬‭This‬‭is‬‭because‬‭pepsin‬‭has‬‭an‬‭ideal‬‭temperature‬‭that‬‭is‬‭best‬‭for‬‭its‬‭function.‬‭At‬‭a‬

‭certain‬‭point,‬‭the‬‭enzyme‬‭becomes‬‭denatured‬‭and‬‭its‬‭ability‬‭to‬‭function‬‭is‬‭compromised.‬‭This‬

‭level‬‭of‬‭denaturation,‬‭however,‬‭occurs‬‭at‬‭extremely‬‭high‬‭temperatures‬‭(>60°C).‬
 ‭15‬

‭In‬‭order‬‭to‬‭verify‬‭the‬‭results‬‭gained‬‭in‬‭this‬‭experiment,‬‭it‬‭is‬‭important‬‭to‬‭review‬‭similar‬

‭experiments‬‭and‬‭studies.‬‭In‬‭the‬‭research‬‭that‬‭was‬‭done‬‭in‬‭the‬‭background‬‭of‬‭this‬‭paper,‬‭it‬‭was‬

‭assessed‬‭that‬‭pepsin‬‭would‬‭perform‬‭well‬‭in‬‭its‬‭breakdown‬‭abilities‬‭at‬‭temperatures‬‭above‬‭40°C.‬

‭In‬‭a‬‭study‬‭done‬‭in‬‭Malaysia,‬‭pepsin‬‭activity‬‭in‬‭fish‬‭was‬‭measured‬‭by‬‭altering‬‭the‬‭temperature‬‭of‬

‭the‬‭surroundings.‬‭An‬‭additional‬‭factor‬‭was‬‭tested,‬‭which‬‭was‬‭diet.‬‭With‬‭that‬‭being‬‭said,‬

‭temperature‬‭proved‬‭to‬‭show‬‭an‬‭impact‬‭on‬‭the‬‭digestion‬‭of‬‭the‬‭fish‬‭tested.‬‭Fish‬‭that‬‭were‬

‭subjected‬‭to‬‭water‬‭temperatures‬‭at‬‭a‬‭high‬‭temperature‬‭(30°C)‬‭had‬‭digestive‬‭tract‬‭masses‬‭that‬‭were‬

‭less‬‭in‬‭weight‬‭and‬‭relative‬‭gut‬‭lengths‬‭that‬‭were‬‭shorter‬‭than‬‭the‬‭fish‬‭that‬‭were‬‭subjected‬‭to‬‭lower‬

‭temperatures‬‭(Mazumder‬‭et‬‭al.,‬‭2018).‬‭This‬‭means‬‭that‬‭over‬‭time,‬‭more‬‭food‬‭was‬‭digested‬‭in‬

‭their‬‭systems,‬‭resulting‬‭in‬‭smaller‬‭digestive‬‭tracts.‬‭This‬‭then‬‭suggests‬‭that‬‭pepsin‬‭activity‬‭levels‬

‭in‬‭these‬‭fish‬‭were‬‭higher‬‭than‬‭any‬‭of‬‭the‬‭other‬‭fish‬‭tested‬‭upon.‬‭In‬‭this‬‭study,‬‭pepsin‬‭activities‬

‭were‬‭also‬‭higher‬‭at‬‭high‬‭temperatures.‬‭Although‬‭the‬‭results‬‭yielded‬‭from‬‭this‬‭Malaysian‬‭study‬

‭were‬‭slightly‬‭different‬‭from‬‭the‬‭results‬‭yielded‬‭from‬‭study‬‭of‬‭this‬‭paper,‬‭it‬‭can‬‭still‬‭be‬‭concluded‬

‭that‬‭pepsin‬‭functions‬‭best‬‭and‬‭breaks‬‭down‬‭substrate‬‭at‬‭a‬‭higher‬‭rate‬‭at‬‭higher‬‭temperatures.‬

‭Strengths‬‭and‬‭Limitations‬‭of‬‭the‬‭Experiment‬

‭A‬‭variety‬‭of‬‭temperatures‬‭were‬‭used‬‭to‬‭model‬‭realistic‬‭temperatures‬‭that‬‭are‬‭seen‬‭with‬‭the‬

‭high‬‭heat‬‭in‬‭the‬‭age‬‭of‬‭global‬‭warming.‬‭43°C‬‭is‬‭an‬‭abnormally‬‭high‬‭temperature‬‭that‬‭is‬‭present‬‭in‬

‭the‬‭occurrence‬‭of‬‭frequent‬‭heat‬‭waves.‬‭Although‬‭the‬‭temperature‬‭seems‬‭extreme,‬‭it‬‭is‬‭important‬

‭to‬‭note‬‭extreme‬‭temperatures‬‭because‬‭of‬‭how‬‭they‬‭are‬‭becoming‬‭increasingly‬‭more‬‭frequent‬‭in‬

‭the‬‭hotter‬‭months.‬‭Thus,‬‭it‬‭is‬‭important‬‭to‬‭review‬‭how‬‭these‬‭high‬‭temperatures‬‭can‬‭affect‬‭the‬

‭performance‬‭of‬‭pepsin‬‭with‬‭the‬‭increasing‬‭frequency‬‭of‬‭these‬‭high‬‭temperatures.‬

‭This‬‭experiment‬‭modeled‬‭global‬‭warming‬‭on‬‭the‬‭simplest‬‭scale.‬‭In‬‭order‬‭to‬‭simulate‬

‭global‬‭warming‬‭on‬‭a‬‭more‬‭accurate‬‭scale,‬‭more‬‭advanced‬‭technology‬‭and‬‭resources‬‭would‬‭need‬
 ‭16‬

‭to‬‭be‬‭used.‬‭I‬‭did‬‭not‬‭have‬‭access‬‭to‬‭these‬‭kinds‬‭of‬‭resources.‬‭Although‬‭similar‬‭results‬‭would‬‭have‬

‭been‬‭gained‬‭if‬‭this‬‭change‬‭was‬‭added,‬‭my‬‭results‬‭would‬‭have‬‭been‬‭more‬‭reliable‬‭and‬‭relevant‬‭to‬

‭the‬‭issue‬‭of‬‭global‬‭warming.‬‭Although‬‭pepsin‬‭was‬‭already‬‭in‬‭its‬‭active‬‭form,‬‭a‬‭more‬‭closely‬

‭simulated‬‭environment‬‭to‬‭that‬‭of‬‭an‬‭organism’s‬‭stomach‬‭might‬‭have‬‭provided‬‭more‬‭significance‬

‭to‬‭my‬‭data.‬‭Because‬‭the‬‭data‬‭gained‬‭from‬‭this‬‭experiment‬‭was‬‭yielded‬‭from‬‭a‬‭much‬‭simpler‬

‭experiment‬‭than‬‭that‬‭of‬‭the‬‭experiments‬‭I‬‭researched‬‭in‬‭addition,‬‭my‬‭results‬‭seem‬‭less‬‭significant.‬

‭In‬‭order‬‭to‬‭more‬‭closely‬‭model‬‭the‬‭conditions‬‭of‬‭the‬‭stomach,‬‭HCl‬‭could‬‭have‬‭been‬‭implemented‬

‭in‬‭the‬‭solution‬‭instead‬‭of‬‭the‬‭pH‬‭buffer‬‭capsules.‬‭HCl‬‭is‬‭the‬‭origin‬‭of‬‭the‬‭pH‬‭of‬‭2‬‭in‬‭the‬‭stomach.‬

‭Additionally,‬‭a‬‭different‬‭method‬‭for‬‭measuring‬‭the‬‭rate‬‭of‬‭breakdown‬‭of‬‭the‬‭albumin‬‭could‬‭have‬

‭been‬‭employed.‬‭A‬‭larger‬‭surface‬‭area‬‭of‬‭the‬‭albumin‬‭pieces‬‭may‬‭have‬‭been‬‭helpful‬‭as‬‭more‬

‭albumin‬‭would‬‭have‬‭been‬‭broken‬‭down.‬‭With‬‭increased‬‭surface‬‭area,‬‭there‬‭are‬‭more‬‭particles‬‭for‬

‭the‬‭pepsin‬‭to‬‭bind‬‭with.‬‭Thus,‬‭I‬‭would‬‭have‬‭gained‬‭more‬‭variance‬‭between‬‭each‬‭group‬‭because‬

‭the‬‭decrease‬‭in‬‭albumin‬‭left‬‭over‬‭from‬‭each‬‭group‬‭would‬‭have‬‭increased‬‭as‬‭a‬‭whole.‬

‭Conclusion‬

‭Although‬‭the‬‭results‬‭of‬‭this‬‭experiment‬‭supported‬‭the‬‭hypothesis‬‭that‬‭increased‬

‭temperatures‬‭does‬‭not‬‭denature‬‭enzymes‬‭in‬‭the‬‭bodies‬‭of‬‭ectotherms,‬‭rendering‬‭them‬

‭dysfunctional,‬‭it‬‭is‬‭important‬‭to‬‭note‬‭some‬‭possible‬‭solutions‬‭for‬‭this‬‭potential‬‭problem‬‭that‬‭other‬

‭life‬‭forms‬‭may‬‭come‬‭across.‬‭Humans,‬‭as‬‭a‬‭major‬‭example,‬‭are‬‭not‬‭ectotherms,‬‭and‬‭their‬‭internal‬

‭body‬‭temperatures‬‭are‬‭not‬‭dependent‬‭on‬‭the‬‭temperature‬‭of‬‭their‬‭surroundings,‬‭increases‬‭in‬‭heat‬

‭can‬‭prove‬‭to‬‭be‬‭harmful.‬‭In‬‭regards‬‭to‬‭the‬‭question‬‭of‬‭whether‬‭more‬‭thermostable‬‭organisms‬‭can‬

‭exist,‬‭it‬‭is‬‭not‬‭probable.‬‭By‬‭attempting‬‭to‬‭mutate‬‭the‬‭DNA‬‭which‬‭codes‬‭for‬‭amino‬‭acids‬‭and‬‭the‬

‭structures‬‭that‬‭form‬‭proteins,‬‭its‬‭stability‬‭and‬‭functionality‬‭is‬‭impacted.‬‭The‬‭way‬‭a‬‭protein‬‭folds‬
 ‭17‬

‭is‬‭important,‬‭especially‬‭with‬‭enzymes.‬‭A‬‭more‬‭likely‬‭resolution‬‭is‬‭the‬‭‘spontaneous‬‭generation‬‭of‬

‭a‬‭phenotype’‬‭(Barik,‬‭2020).‬‭Under‬‭this‬‭theory,‬‭organisms‬‭are‬‭subjected‬‭to‬‭gradual‬‭temperature‬

‭changes,‬‭allowing‬‭evolution‬‭to‬‭take‬‭control‬‭of‬‭altering‬‭the‬‭thermostability‬‭of‬‭enzymes.‬‭The‬

‭underlying‬‭issue,‬‭however,‬‭rests‬‭on‬‭a‬‭small‬‭margin‬‭of‬‭temperature‬‭increase‬‭from‬‭the‬‭optimal‬

‭growth‬‭temperature‬‭range.‬‭Mesophiles‬‭can‬‭tolerate‬‭a‬‭few‬‭degrees‬‭higher‬‭than‬‭the‬‭maximum‬

‭optimal‬‭temperature,‬‭but‬‭only‬‭if‬‭the‬‭rate‬‭at‬‭which‬‭global‬‭warming‬‭progresses‬‭is‬‭slow‬‭enough.‬

‭According‬‭to‬‭recent‬‭data,‬‭the‬‭global‬‭temperature‬‭has‬‭risen‬‭to‬‭about‬‭0.08°C‬‭each‬‭decade‬‭(Lindsey‬

‭&‬‭Dahlman,‬‭2023).‬‭Additionally,‬‭it‬‭is‬‭also‬‭predicted‬‭that‬‭throughout‬‭the‬‭remainder‬‭of‬‭the‬‭century,‬

‭global‬‭temperature‬‭will‬‭rise‬‭from‬‭1-4°C.‬‭It‬‭can‬‭then‬‭be‬‭predicted‬‭that‬‭this‬‭rate‬‭will‬‭either‬‭continue‬

‭or‬‭increase‬‭throughout‬‭the‬‭remainder‬‭of‬‭the‬‭century.‬‭This‬‭places‬‭the‬‭adaptive‬‭abilities‬‭of‬‭many‬

‭mesophiles‬‭at‬‭a‬‭high‬‭disadvantage,‬‭leaving‬‭their‬‭physiological‬‭systems‬‭inadequate‬‭time‬‭to‬

‭acclimate‬‭to‬‭changing‬‭temperatures‬‭in‬‭their‬‭environments.‬

‭Knowing‬‭that‬‭ectotherms‬‭are‬‭less‬‭vulnerable‬‭to‬‭rapid‬‭increases‬‭in‬‭temperatures‬‭over‬

‭future‬‭decades‬‭than‬‭endotherms,‬‭these‬‭findings‬‭may‬‭be‬‭applied‬‭to‬‭what‬‭the‬‭future‬‭of‬‭endotherms‬

‭looks‬‭like.‬‭A‬‭question‬‭that‬‭arises‬‭from‬‭these‬‭findings‬‭is‬‭if‬‭there‬‭is‬‭an‬‭inverse‬‭relationship‬‭between‬

‭ectotherms‬‭and‬‭endotherms‬‭in‬‭terms‬‭of‬‭their‬‭enzymes’‬‭abilities‬‭to‬‭function.‬‭From‬‭the‬‭data‬

‭collected‬‭in‬‭this‬‭experiment,‬‭as‬‭well‬‭as‬‭the‬‭research‬‭conducted‬‭to‬‭confirm‬‭such‬‭findings,‬‭it‬‭could‬

‭be‬‭predicted‬‭that‬‭endotherms‬‭would‬‭experience‬‭an‬‭inverted‬‭version‬‭of‬‭these‬‭effects‬‭in‬‭the‬‭long‬

‭term.‬‭Since‬‭ectotherms‬‭regulate‬‭their‬‭internal‬‭body‬‭temperatures‬‭opposite‬‭of‬‭endotherms,‬‭it‬‭can‬

‭be‬‭hypothesized‬‭that‬‭endothermic‬‭organisms‬‭may‬‭experience‬‭lower‬‭pepsin‬‭activity‬‭rates‬‭at‬‭higher‬

‭temperatures.‬‭To‬‭provide‬‭evidence‬‭for‬‭this‬‭claim,‬‭however,‬‭would‬‭be‬‭difficult‬‭as‬‭endotherms‬‭are‬

‭often‬‭more‬‭complex‬‭than‬‭ectotherms.‬‭Fish‬‭were‬‭more‬‭resistant‬‭to‬‭long-term‬‭increases‬‭in‬

‭temperature‬‭because‬‭they‬‭have‬‭the‬‭evolutionary‬‭adaptations‬‭necessary‬‭for‬‭sustaining‬‭their‬
 ‭18‬

‭internal‬‭temperatures‬‭(Clark‬‭et‬‭al.,‬‭2013).‬‭Because‬‭there‬‭is‬‭a‬‭significant‬‭lack‬‭of‬‭data‬‭on‬‭the‬

‭temperature‬‭physiology‬‭of‬‭endotherms,‬‭it‬‭would‬‭be‬‭difficult‬‭to‬‭determine‬‭whether‬‭this‬‭claim‬‭is‬

‭plausible.‬‭Another‬‭source‬‭of‬‭further‬‭investigation‬‭is‬‭whether‬‭mammals‬‭and‬‭other‬‭endotherms‬‭can‬

‭undergo‬‭adaptations‬‭that‬‭ectotherms‬‭such‬‭as‬‭fish‬‭have‬‭undergone‬‭in‬‭order‬‭to‬‭acclimate‬‭better‬‭to‬

‭heating‬‭environments.‬‭While‬‭this‬‭investigation‬‭also‬‭requires‬‭extensive‬‭amounts‬‭of‬‭research‬‭and‬

‭data‬‭collection‬‭as‬‭well,‬‭it‬‭is‬‭a‬‭valuable‬‭route‬‭of‬‭investigation‬‭that‬‭could‬‭reveal‬‭just‬‭how‬

‭threatening‬‭global‬‭warming‬‭is‬‭to‬‭the‬‭survival‬‭of‬‭organisms‬‭not‬‭currently‬‭equipped‬‭to‬‭handle‬

‭rising‬‭high‬‭temperatures.‬‭The‬‭findings‬‭gained‬‭from‬‭this‬‭experiment‬‭are‬‭only‬‭a‬‭small‬‭piece‬‭of‬

‭what‬‭could‬‭help‬‭biologists,‬‭environmentalists,‬‭and‬‭many‬‭other‬‭scientists‬‭to‬‭solve‬‭a‬‭much‬‭larger‬

‭problem.‬‭The‬‭simple‬‭acts‬‭of‬‭asking‬‭questions‬‭and‬‭gaining‬‭answers‬‭can‬‭bring‬‭more‬‭awareness‬‭to‬

‭the‬‭issue‬‭of‬‭global‬‭warming,‬‭giving‬‭rise‬‭to‬‭more‬‭important‬‭questions‬‭and‬‭discoveries.‬

‭The‬‭study‬‭of‬‭how‬‭the‬‭smallest‬‭structures‬‭in‬‭the‬‭bodies‬‭of‬‭organisms‬‭react‬‭to‬‭climate‬

‭change‬‭can‬‭open‬‭up‬‭a‬‭field‬‭dedicated‬‭to‬‭discovering‬‭how‬‭various‬‭other‬‭types‬‭of‬‭animals‬‭can‬

‭evolve‬‭to‬‭combat‬‭a‬‭new‬‭normal.‬‭With‬‭global‬‭warming‬‭rapidly‬‭becoming‬‭irreversible,‬‭simply‬

‭understanding‬‭the‬‭evolutionary‬‭abilities‬‭of‬‭organisms‬‭and‬‭how‬‭genomes‬‭will‬‭acclimate‬‭can‬‭allow‬

‭the‬‭survival‬‭through‬‭global‬‭warming‬‭to‬‭be‬‭probable.‬
 ‭19‬

‭References‬

‭Alfonso,‬‭S.,‬‭Gesto,‬‭M.,‬‭&‬‭Sadoul,‬‭B.‬‭(2020).‬‭Temperature‬‭Increase‬‭and‬‭Its‬‭Effects‬‭on‬‭Fish‬‭Stress‬

‭Physiology‬‭in‬‭the‬‭Context‬‭of‬‭Global‬‭Warming.‬‭Journal‬‭of‬‭Fish‬‭Biology‬‭,‬‭98‬‭(6).‬

‭https://doi.org/10.1111/jfb.14599‬

‭Barik,‬‭S.‬‭(2020).‬‭Evolution‬‭of‬‭Protein‬‭Structure‬‭and‬‭Stability‬‭in‬‭Global‬‭Warming.‬‭International‬

‭Journal‬‭of‬‭Molecular‬‭Sciences‬‭,‬‭21‬‭(24),‬‭9662.‬‭https://doi.org/10.3390/ijms21249662‬

‭Clark,‬‭T.‬‭D.,‬‭Sandblom,‬‭E.,‬‭&‬‭Jutfelt,‬‭F.‬‭(2013,‬‭August).‬‭Aerobic‬‭scope‬‭measurements‬‭of‬‭fishes‬

‭in‬‭an‬‭era‬‭of‬‭climate‬‭change:‬‭respirometry,‬‭relevance‬‭and‬‭recommendations.‬‭The‬‭Journal‬

‭of‬‭Experimental‬‭Biology‬‭,‬‭216(15),‬‭2771–2782.‬‭https://doi.org/10.1242/jeb.084251‬

‭Gobler,‬‭C.‬‭J.,‬‭&‬‭Baumann,‬‭H.‬‭(2016).‬‭Hypoxia‬‭and‬‭acidification‬‭in‬‭ocean‬‭ecosystems:‬‭coupled‬

‭dynamics‬‭and‬‭effects‬‭on‬‭marine‬‭life.‬‭Biology‬‭Letters‬‭,‬‭12‬‭(5).‬

‭https://doi.org/10.1098/rsbl.2015.0976‬

‭Heda,‬‭R.,‬‭Tombazzi,‬‭C.‬‭R.,‬‭&‬‭Toro,‬‭F.‬‭(2022,‬‭May‬‭8).‬‭Physiology,‬‭Pepsin‬‭.‬‭National‬‭Library‬‭of‬

‭Medicine.‬‭https://www.ncbi.nlm.nih.gov/books/NBK537005/‬

‭Huey,‬‭R.‬‭B.,‬‭Kearney,‬‭M.‬‭R.,‬‭Krockenberger,‬‭A.,‬‭Holtum,‬‭J.‬‭A.‬‭M.,‬‭Jess,‬‭M.,‬‭&‬‭Williams,‬‭S.‬‭E.‬

‭(2012).‬‭Predicting‬‭organismal‬‭vulnerability‬‭to‬‭climate‬‭warming:‬‭roles‬‭of‬‭behaviour,‬

‭physiology‬‭and‬‭adaptation.‬‭Philosophical‬‭Transactions‬‭of‬‭the‬‭Royal‬‭Society‬‭B:‬‭Biological‬

‭Sciences‬‭,‬‭367‬‭(1596),‬‭1665–1679.‬‭https://doi.org/10.1098/rstb.2012.0005‬

‭International‬‭Panel‬‭on‬‭Climate‬‭Change.‬‭(2023).‬‭Climate‬‭Change‬‭2023‬‭Synthesis‬‭Report.‬

‭Kiew,‬‭P.‬‭L.,‬‭&‬‭Don,‬‭M.‬‭M.‬‭(2012).‬‭Screening‬‭and‬‭Empirical‬‭Kinetic‬‭Models‬‭of‬‭Collagen‬

‭Extraction‬‭from‬‭Selected‬‭Malaysian‬‭Freshwater‬‭Fish.‬‭Journal‬‭of‬‭Food‬‭Processing‬

‭Engineering‬‭,‬‭36‬‭(4),‬‭428-438.‬‭Retrieved‬‭January‬‭7,‬‭2023,‬‭from‬

‭https://onlinelibrary.wiley.com/doi/10.1111/j.1745-4530.2012.00683.x‬
 ‭20‬

‭Kines,‬‭K.,‬‭&‬‭Krupczak,‬‭T.‬‭(2016).‬‭Nutritional‬‭Interventions‬‭for‬‭Gastroesophageal‬‭Reflux,‬

‭Irritable‬‭Bowel‬‭Syndrome,‬‭and‬‭Hypochlorhydria:‬‭A‬‭Case‬‭Report.‬‭Integrative‬‭Medicine‬

‭(Encinitas,‬‭Calif.)‬‭,‬‭15‬‭(4),‬‭49–53.‬

‭https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4991651/‬

‭Lindsey,‬‭R.,‬‭&‬‭Dahlman,‬‭L.‬‭(2023,‬‭January‬‭18).‬‭Climate‬‭Change:‬‭Global‬‭Temperature‬‭|‬‭NOAA‬

‭Climate.gov.‬

‭https://www.climate.gov/news-features/understanding-climate/climate-change-global-tem‬

‭perature‬

‭Lyon,‬‭P.,‬‭Strippoli,‬‭V.,‬‭Fang,‬‭B.,‬‭&‬‭Cimmino,‬‭L.‬‭(2020).‬‭B‬‭Vitamins‬‭and‬‭One-Carbon‬

‭Metabolism:‬‭Implications‬‭in‬‭Human‬‭Health‬‭and‬‭Disease.‬‭Nutrients‬‭,‬‭12‬‭(9),‬‭2867.‬

‭https://doi.org/10.3390/nu12092867‬

‭Mazumder,‬‭S.‬‭K.,‬‭Das,‬‭S.‬‭K.,‬‭Rahim,‬‭S.‬‭M.,‬‭&‬‭Ghaffar,‬‭M.‬‭A.‬‭(2018).‬‭Temperature‬‭and‬‭diet‬

‭effect‬‭on‬‭the‬‭pepsin‬‭enzyme‬‭activities,‬‭digestive‬‭somatic‬‭index‬‭and‬‭relative‬‭gut‬‭length‬‭of‬

‭Malabar‬‭blood‬‭snapper.‬‭Aquaculture‬‭Reports‬‭,‬‭9‬‭(9),‬‭1–9.‬

‭https://doi.org/10.1016/j.aqrep.2017.11.003‬

‭Moraes,‬‭G.,‬‭&‬‭de‬‭Almeida,‬‭L.‬‭C.‬‭(2020).‬‭Chapter‬‭11‬‭-‬‭Nutrition‬‭and‬‭functional‬‭aspects‬‭of‬

‭digestion‬‭in‬‭fish.‬‭Biology‬‭and‬‭Physiology‬‭of‬‭Freshwater‬‭Neotropical‬‭Fish,‬‭3(4),‬‭251-271.‬

‭https://www.sciencedirect.com/science/article/abs/pii/B9780128158722000117‬

‭Raoufinia,‬‭R.,‬‭Mota,‬‭A.,‬‭Keyhanvar,‬‭N.,‬‭Safari,‬‭F.,‬‭Shamekhi,‬‭S.,‬‭&‬‭Abdolalizadeh,‬‭J.‬‭(2016).‬

‭Overview‬‭of‬‭Albumin‬‭and‬‭Its‬‭Purification‬‭Methods.‬‭Advanced‬‭Pharmaceutical‬‭Bulletin‬‭,‬

‭6‬‭(4),‬‭495–507.‬‭https://doi.org/10.15171/apb.2016.063‬
 ‭21‬

‭Sholeh,‬‭M.‬‭M.,‬‭Ambarsari,‬‭L.,‬‭Nurcholis,‬‭W.,‬‭&‬‭Nurhayati,‬‭T.‬‭(2020).‬‭Characterization‬‭of‬

‭ammonium‬‭sulfate‬‭fraction‬‭of‬‭pepsin‬‭from‬‭fish‬‭stomach.‬‭IOP‬‭Conference‬‭Series:‬‭Earth‬

‭and‬‭Environmental‬‭Science‬‭.‬‭https://doi.org/10.1088/1755-1315/404/1/012039‬