Crystallization of insulin derivatives with addition of halides

Abstract

Ovaj diplomski rad sastoji se od istraživačkog i metodičkog dijela. U istraživačkom dijelu rada opisana je kristalizacija halogenidnih i nehalogenidnih derivata goveđeg i ljudskog inzulina primjenom metode difuzije para viseće kapi. Korištene su Hampton Research VDX ploče od 24 spremnika. Ploče su držane u mraku na temperaturi od 20 °C. Varijacijom koncentracije precipitantnih reagensa, koncentracije proteina i veličine kapi dobiveni su monokristali derivata inzulina. Dodatkom natrijevog klorida i soli odgovarajućeg metala kristaliziraju sljedeći derivati inzulina: cinkov(II) klor-derivat goveđeg i ljudskog inzulina; kobaltov(II) klor-, niklov(II) klor-, manganov(II) klor-, bakrov(II) klor- i molibdenov(VI) klor-derivat ljudskog inzulina. Uz dodatak natrijevog jodida kristalizira cinkov(II) jod-derivat ljudskog inzulina, a uz dodatak soli odgovarajućeg metala kristaliziraju vanadijev(IV) i molibdenov(VI) derivat ljudskog inzulina. Strukture cinkovog(II) klor-, cinkovog(II) jod- i kobaltovog(II) klor-derivata ljudskog inzulina određene su rentgenskom strukturnom analizom, čime se dobio uvid u koordinaciju metalnih iona u inzulinu kao i u konformaciju molekule inzulina. Navedeni derivati kristaliziraju u trigonskom sustavu, u prostornoj grupi R3. Cinkov(II) klor-derivat pripada 4Zn tipu (T3R3 f tipu). Cinkov(II) jod-derivat pripada 2Zn tipu, ali i T3R3 tipu koji je inače atipičan za 2Zn tip. Kobaltov(II) klor-derivat ljudskog inzulina pripada 2Zn tipu (T6 tipu). U metodičkom dijelu rada pod naslovom Integrirani pristup poučavanju probavnih enzima, predložen je nastavni materijal za integrirani način izvođenja nastavne jedinice „Probavni enzimi“. Načinjen je pregled nastavnih programa iz kemije i biologije za osnovnu i srednju školu s osvrtom na probavne enzime i obrazovne ishode koji se očekuju za pojedino obrazovno razdoblje. Priložena je uputa za nastavnike, prijedlozi pokusa i testovi za samoocjenjivanje (grupe A i B) te zadaci za provjeru znanja. Nastavni materijal je prilagođen obrazovnom razdoblju četvrtog razreda gimnazije u završnom programskom dijelu.This thesis consists of two parts: the research and methodological one. In the research part of the thesis crystallization of halogen and non-halogen derivatives of bovine and human insulin by the hanging drop vapour diffusion method is described. Hampton Research VDX plates consisting of 24 wells were used. The plates were kept in the dark at 20 oC. Single crystals of insulin derivatives were obtained by variations in the concentration of the precipitant solution, protein concentration and drop size. The following insulin chloro derivatives were crystallized with addition of NaCl and a metal salt in the precipitant solution: zinc(II) chloro-derivative of bovine and human insulin; cobalt(II) chloro-, nickel(II) chloro-, manganese(II) chloro-, copper(II) chloro- and molybdenum(VI) chloro-derivatives of human insulin. By addition of NaI the zinc(II) iodo derivative of human insulin was crystallized. By addition of a metal salt vanadium(IV) and molybdenum(VI) derivatives of human insulin were crystallized. The X-ray diffraction experiment was conducted on the zinc(II) chloro, zinc(II) iodo and cobalt(II) chloro derivatives of human insulin to determine their molecular and crystal structure. The three mentioned derivatives were found to crystallize in the trigonal system, R3 space group. The zinc(II) chloroderivative exhibits the 4Zn form (T3R3 f form). The zinc(II) iodo-derivative is of the 2Zn type, but also in the T3R3 form, usually atypical for the 2Zn type; and the cobalt(II) chloro-derivative exhibits the 2Zn type (T6 form). In the methodological part of the Thesis entitled Integrated approach to teaching digestive enzymes, teaching material for the integrated lesson unit “Digestive enzymes” is suggested. An overview of the teaching curriculum in chemistry and biology for elementary and high school, with the reference to digestive enzymes, was made. Instructions for teachers, tests for self-evaluation (groups A and B) with experiments and a knowledge assessment test for fourth grade high school students are enclosed