Purification and Characterization of Tryptophan Hydroxylase

Lærke Tvedebrink Haahr

Research output: Book/ReportPh.D. thesis

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This thesis deals with the purification and characterization of the iron-containing enzyme tryptophan hydroxylase (TPH). TPH exists in two isoforms, called TPH1 and TPH2. Each isoform consists of threestructural distinct domains: the regulatory, the catalytic and the tetramerization domain. TPH catalyzes the hydroxylation of tryptophan to 5-hydroxytryptophan, which is the rate-limiting step in the biosynthesis ofserotonin. Serotonin is an important neurotransmitter, which is involved in a range of psychiatric disorders including depression and obsessive-compulsive disorder. The goal of this project was to developpurification methods for full-length TPH1 and TPH2 as well as to characterize purified TPH variants.
A successful purification method for full-length human TPH1 (hTPH1) was developed, which resulted in pure, active and stable protein. The method includes affinity-purification using maltose binding protein (MBP) tag and high salt concentration in all buffers. It is the first time a successful purification method for hTPH1 has been presented, which also yields stable protein after cleavage from the affinity tag. Furthermore, successful purification procedures were developed for a number of other TPH variants. A purification method for the regulatory and catalytic domains of human TPH1 (rchTPH1) using MBP affinity tag was developed, and revealed that the regulatory domain causes dimerization. Also, a new purification method for the catalytic domain of human TPH1 (chTPH1) using gluthathione S-tranferase (GST) tag was
developed. Two human TPH2 variants containing the regulatory domain were attempted purified using the same general method as for hTPH1, but the yields were very low and the products were unstable and could not be concentrated. The high salt concentration does thus not seem to stabilize the regulatory domain of TPH2. The thermal stability of 4 TPH variants was studied by differential scanning fluorimetry (DSF), and the results gave information on the effect of pH, salt type and ionic strength.
The results from crystallization were unfortunately rather limited. The crystallization procedure for the catalytic domain of gallus gallus TPH1 (cgTPH1) was optimized to faster crystal growth by addition of tryptophan and incubation at room temperature. Crystals without imidazole in the crystallization conditions could be obtained. The solved structures were however of poor quality and did not contribute with any new information on TPH. A strategic approach to crystallization of TPH variants was developed, and the pipeline screening produced leads for rchTPH1 and several crystals for chTPH1.
Four mutants of the catalytic domain of human TPH2 (chTPH2) were made, and it can be concluded that Glu363, which is an iron ligand, is essential for activity, while Tyr358 affects the activity, but is not absolutely required. Circular dichroism and magnetic circular dichroism results on chTPH2 clearly demonstrated that the active site iron changes conformation from 6 to 5 coordinated only when both substrate and cofactor binds. A difference between samples made in glycerol and sucrose was observed. Addition of glycerol to chTPH2 in the Fe(III) form gave a yellow color indicative of interaction. DSF
measurements demonstrated that chTPH2 is significantly more stable in the Fe(II) form compared to the
Fe(III) form. Furthermore, the addition of substrates and cofactor only stabilizes chTPH2 when Fe(II) is present. Isothermal titration calorimetry measurements on chTPH2 determined the dissociation constant of BH2 to 5 μM, while the dissociation constant for tryptophan is somewhat higher. Tryptophan and BH2 bind independently of each other.
Overall, the work performed in this project has made significant contributions to the research on TPH, both
by providing new purification procedures as well as advancements in the knowledge on the mechanism of

Original languageEnglish
Place of PublicationKgs.Lyngby
PublisherDTU Chemistry
Number of pages167
Publication statusPublished - 2012


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