Quantitation of heme oxygenase 1: Heme titration increases yield of purified protein

Abstract

Free heme binds to heme oxygenase as a prosthetic group and substrate in the conversion of heme to biliverdin, carbon monoxide, and free iron. Current methods for quantifying heme oxygenase-1 (HO-1) involve reconstitution of the enzyme with heme, followed by a hydroxyapatite column to remove the excess heme. As a result of the hydroxyapatite chromatography, there are significant losses of purified protein. We have developed a method which allows accurate quantitation of HO-1 using a heme titration and elimination of the final hydroxyapatite column, increasing the amount of purified protein.Heme oxygenase-1 (HO-1) is an inducible enzyme responsible for the oxidative cleavage of heme to biliverdin, carbon monoxide and free iron (1). Heme is a heterocyclic porphyrin with an iron center which serves as cofactor or prosthetic group for a number of proteins classified as hemoproteins. The binding of heme to HO-1 is unique because heme serves as both cofactor and substrate, and is degraded upon the addition of reducing equivalents. Recombinant HO-1 proteins from a variety of species have been expressed and purified, with human and rat most commonly studied (2,3). The deletion of 23 C-terminal amino acids, which serve as the membrane spanning domain (4), allowed for a swift purification of truncated human HO-1 (sHO-1) in large quantities. Standard purification procedures of sHO-1 produce a purified protein that must be reconstituted with heme to calculate the specific HO-1 concentration (3). Excess heme is removed from the HO-1-heme complex via passage through a final hydroxyapatite (HA) column. The HO-1-heme spectrum includes a Soret band at 405 nm, which is commonly utilized to quantify the complex with an extinction coefficient of 140 mM −1 cm −1 (5). The HO-1-heme complex produces a distinct absorption spectrum in comparison to the apoprotein. This report describes an improved method to quantify HO-1 using a heme titration, eliminating the final HA column, and effectively increasing the total purified protein.For these studies, truncated, human HO-1 was purified by a previously described method, with minor modifications (3). Following the expression of sHO1 in E. coli strain DH5α, the media appeared green in color, due to the accumulation of biliverdin (6). The bacterial cell pellet remained green throughout the purification procedure prior to the ammonium sulfate precipitation. The increase in ionic strength forced the disassociation of the biliverdin from the sHO-1, forming a colorless protein product. Following standard column chromatography, the Correspondence should be addressed to: Wayne L. Backes, Ph.D., Department of Pharmacology& The Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar St., New Orleans, LA 70112, Tel. 504-568-6557; FAX 504-568-6888; E-mail: wbacke@lsuhsc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the man...

C-Terminal Membrane Spanning Region of Human Heme Oxygenase-1 Mediates a Time-Dependent Complex Formation with Cytochrome P450 Reductase

“…The full-length HO-1 expression system was kindly provided by Dr. Mahin Maines (University of Rochester), and mutated at position 254 as described previously (46). Both HO-1 and sHO-1 were purified in the apo- form and quantified using the heme titration method (47). In this manuscript, HO-1 refers to the R254K mutant containing the C-terminal membrane binding domain as described previously (46), whereas sHO-1 refers to the form lacking this C-terminal region.…”

Human Cyclooxygenase-2 Is a Sequence Homodimer That Functions as a Conformational Heterodimer

“…, where I 0 is the concentration of added heme; ␣, the fraction of enzyme sites occupied by heme, is given by ␣ ϭ ⌬/⌬ max , where ⌬ is the optical change measured (27); in this plot, the slope is equal to the K d for heme binding, and the intercept (e 0 ) represents the concentration of heme-binding sites. Titrations were also performed in the presence of palmitic acid and several COX inhibitors.…”

“…Aliquots of a 200 M hematin solution in 20 mM Tris-HCl, pH 8.3, containing 40 mM KCl, 0.1% C 10 E 6 , and 2% DMSO were added to a quartz cuvette containing 200 l of 5 M apohuPGHS-2 in the same buffer solution without DMSO at room temperature (26,27). The increase in absorbance at 408 nm was plotted in a linear form using the equation…”

“…, where I 0 is the concentration of added heme; ␣, the fraction of enzyme sites occupied by heme, is given by ␣ ϭ ⌬/⌬ max , where ⌬ is the optical change measured (27). The slope is equal to the mixture did not change the profile of oxygenated products.…”

Human cyclooxygenase-1 activity and its responses to COX inhibitors are allosterically regulated by nonsubstrate fatty acids

“…Aliquots of heme solutions in 20 mM Tris HCl, pH 8.3, containing 40 mM KCl, 0.02% C 10 E 6 , and 2% DMSO, were added to a quartz cuvette containing 200 µl of 5 M apo-huPGHS-1 in the same buffer solution without DMSO at room temperature ( 40,41 ). The increase in absorbance at 412 nm was transformed to a binding fraction and then plotted using Grapher TM 7 software in a linear form using the Scatchard equation in which the slope is equal to the Ϫ 1/ K d for heme binding.…”