Resumé
Hvorvidt en naturhistorisk museumsgenstand har været formaldehyd behandlet, kan være en
vigtig information for efterfølgende behandling og analyse. Formaldehyds indflydelse på
enzymaktiviteten af proteasen Savinase er undersøgt. Formaldehyd havde en hæmmende effekt
på enzymerne, både ved tilstedeværelse i enzymopløsningen og som fiksativ bundet i væv. Der er
udviklet en enzymmacerations metode, hvor pH er justeret til basisk område med bufferen
ammoniumcarbonat, hvilket var helt afgørende for at enzymmacerationen kunne forløbe. Denne
sammenhæng mellem pH og enzymaktivitet er anvendt til at udvikle en enzym metode til at
påvise hvorvidt et præparat sandsynligvis er påvirket af formaldehyd behandling eller ej.
Behandling af en vævsprøve med protease enzymer ved 55 oC og neutral pH, viste at ethanol
konserveret væv blev nedbrudt indenfor 24 timer, hvorimod formaldehyd konserveret væv ikke
blev nedbrudt. Det er undersøgt om den basiske enzymmaceration er anvendelig som
forbehandling af formaldehyd konserveret væv før oprensning af mtDNA til PCR analyse. Efter
denne enzymbehandling lykkedes det at analysere DNA fra formaldehyd konserveret væv, men
ikke af så høj kvalitet sammenlignet med DNA fra ukonserveret og ethanol konserveret væv. For at
undersøge strukturændringer som følge af formaldehydbehandling, er der foretaget FT-IR og NMR
analyser af henholdsvis ethanol- og formaldehyd konserveret væv. Her blev der for begge metoder
observeret små forskelle i spektrenes udseende, men afvigelserne var umiddelbart var for små til
at skelne visuelt. Metoderne kunne formodentligt blive mere velegnede med statistisk behandling
af større datamateriale end tilfældet i denne undersøgelse.
Emneord: Formalin, formaldehyd konservering, enzymmaceration, enzymhæmning, enzymaktivitet, skelet
materiale, PCR, polymerase, naturhistoriske samlinger.
Abstract
Whether a natural history museums specimen has been treated with formaldehyde, may be
important information for subsequent analyzing. The influence of formaldehyde on the activity of
the protease Savinase has been investigated. Formaldehyde had an inhibitory effect on the
enzymes, both as a reagent present in the enzyme solution and as a fixative bounded in the tissue.
An enzyme maceration method has been developed with pH adjusted to basic field, using the
buffer ammonia carbonate, which was crucial to start up the maceration. This relationship
between pH and the activity of the enzymes has been used to develop an enzymatic method to
detect whether a sample probably is affected of formaldehyde or not. Treatment of a sample with
protease enzymes at 55 oC and neutral pH level showed that ethanol preserved tissue samples
could be degraded within 24 hours, whereas formaldehyde preserved samples were not degraded.
It has been investigated whether the basic enzyme maceration method is applicable as a pretreatment of formaldehyde preserved tissue, before purification of mtDNA for PCR analysis. After
this enzymatic treatment it was possible to analyse DNA from formaldehyde preserved tissue, but
the quality was lower compared to DNA from non-preserved or ethanol preserved tissue. To
examine the structural changings caused by formaldehyde treatment, ethanol and formaldehyde
preserved tissue have been analysed with FT-IT and NMR. For both methods small disparity
between the spectres were observed, but the differences were too small for visual observation.
These methods might be more suitable using statistic treatment of a greater data material, than
were used in this project.
Keywords. Formalin, formaldehyde preservation, enzyme maceration, enzyme inhibition, enzyme activity,
skeletal materials, PCR, polymerase, natural history collections.