P4HB, prolyl 4 hydroxylase subunit beta, is a multifunctional and highly abundant enzyme that belongs to the protein disulfide isomerase family. When present as a tetramer consisting of two alpha and two beta subunits, this enzyme is involved in hydroxylation of prolyl residues in preprocollagen. This protein is also a disulfide isomerase containing two thioredoxin domains that catalyze the formation, breakage and rearrangement of disulfide bonds. Source: Recombinant protein corresponding to aa18-508 from human P4HB, fused to His-tag at N-terminal expressed in E. coli. Molecular Weight: ~ 57.5kD(521aa) Specific Activity: >100 A650/cm/min/mg. Enzymatic activity was confirmed by measuring the aggregation of insulin in the presence of DTT. Activity Assay 1. Prepare a 180ul assay buffer into a suitable container: The concentrations are 0.67mM DTT, insulin 5.3mg, 1.2mM EDTA 2. Dilute insulin 8mg/ml in 3mM EDTA, pH 7.0, and incubate diluted insulin for 5 minutes at 37C. 3. Dilute P4HB to 50ug/ml in 0.1% BSA. 4. Mix 40ul diluted P4HB and 10mM DTT 10ul. Amino Acid Sequence: MGSSHHHHHH SSGLVPRGSH MDAPEEEDHV LVLRKSNFAE ALAAHKYLLV EFYAPWCGHC KALAPEYAKA AGKLKAEGSE IRLAKVDATE ESDLAQQYGV RGYPTIKFFR NGDTASPKEY TAGREADDIV NWLKKRTGPA ATTLPDGAAA ESLVESSEVA VIGFFKDVES DSAKQFLQAA EAIDDIPFGI TSNSDVFSKY QLDKDGVVLF KKFDEGRNNF EGEVTKENLL DFIKHNQLPL VIEFTEQTAP KIFGGEIKTH ILLFLPKSVS DYDGKLSNFK TAAESFKGKI LFIFIDSDHT DNQRILEFFG LKKEECPAVR LITLEEEMTK YKPESEELTA ERITEFCHRF LEGKIKPHLM SQELPEDWDK QPVKVLVGKN FEDVAFDEKK NVFVEFYAPW CGHCKQLAPI WDKLGETYKD HENIVIAKMD STANEVEAVK VHSFPTLKFF PASADRTVID YNGERTLDGF KKFLESGGQD GAGDDDDLED LEEAEEPDME EDDDQKAVKD EL Storage and Stability: May be stored at 4C for short-term only. Aliquot to avoid repeated freezing and thawing.. Store at -20C. Aliquots are stable for 6 months after receipt at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
