Spectrophotometric determination of neutrophil cytochrome b₅₅₈ of chronic granulomatous disease

Abstract

Background: Chronic granulomatous disease (CGD) is an inherited disease characterized clinically by severe recurrent bacterial infections from infancy. This disease is a disorder of the formation of superoxide (02-) b j the neutrophil NADPH oxidase system, mostly due to defects in cytochrome b,,, (cyt b,,,), which is one oj the oxidase components. Diagnosis of CGD has been performed by the assay of the 0,forming activity. immunological determination of defects in the oxidase components, and or spectrophotometry of cyt bS5, However, spectrophotometric analysis of the b-type heme is difficult with small amounts of blood from infant CGD patients, as the limited amounts of neutrophils are contaminated with a relatively high ratio of hemoglobin (Hb) that interferes with the heme spectrum of cyt b,,,. This report presents an accurate method fol the spectrophotometric analysis of cyt b,,, in a small amount of CGD neutrophils that were treated with CO gas in ; I safe procedure instead of the previously reported CO-bubbling method.Methalr and Results: The difference of the reduced minus oxidized cyt bS58 spectrum was measured undet no interference from oxy Hb at the a and bands and differentiated as d[AA]ldh ( h = wavelength) to obtain further evidence for the defects of the cyt b,,, heme spectrum. The interference from CO-insensitive met Ht was eliminated by subtracting the absorption peak at the Soret (y) band of the contaminating met Hb, which was estimated from the CO-treated and untreated spectra of the same, hemolyzed sample. ConclusWns: This spectrophotometric method is feasible for the determination of abnormality and heme content of cyt b,,, with a small amount of CGD neutrophils in 10-20 mL of blood even in the presence of contaminating Hb.Key words chronic granulomatous disease, cytochrome b,,,, oxyhemoglobin, methemoglobin, myeloperoxidase.Phagocyte specific cyt bSSX is an essential component of the membrane-bound NADPH oxidase that generates 0; in response to invading microorganisms.' The important role of the NADPH oxidase has been further emphasized by the demonstration of a major defect in the oxidase of neutrophils from patients with chronic granulomatous disease (CGD) who suffer from recurrent severe infectious diseases.IJ The absence of the heme spectrum of cyt bgSk in neutrophils of patients with X-linked recessive CGD was discovered in 1978., Over the past two decades, further detailed information on cyt b,,, has been obtained by extensive studies on this heme protein of patients with X-linked CGD. Cyt b,,, is a membrane-bound heterodimer of a 91 kDa glycosylated subunit (gp91-phox) and a 22kDa subunit (~22-phox);~,' both of the subunits are lacking in neutrophils of X-linked CGD. In addition to the classic X-linked CGD, patients with autosomal recessive CGD have normal levels of cyt h,,,, but lack cytosolic factors; that is. a 47 kDa protein (+7-phox) and a 67 kDa protein (p67-phox), which are both required for the activation of the NADPH oxidases8 The b type cytochrome is also absent in autosomal CG...

A novel mutation at a probable heme-binding ligand in neutrophil cytochrome b 558 in atypical X-linked chronic granulomatous disease

“…The mother (M) has 52% of p22-phox and about 30% of gp91-phox, which varied because the electrophoretic migration pattern differed from those of the controls. We also carried out an identical immunoblot anal-ysis with the same antibodies on other male patients with typical X-linked recessive CGD and found the complete absence of both subunits of cyt b 558 in their neutrophils (Kiryu et al 1998;M. Kaneda et al in preparation).…”

“…It is known that both subunits of cyt b 558 are remarkably unstable when the synthesis of either one is disrupted (Parkos et al 1989). Indeed, we have observed the complete absence of both subunits in the neutrophils of several patients (males) with typical X-linked CGD by immunoblot analysis (Kiryu et al 1998;M. Kaneda et al in preparation).…”

“…The spectrum of the patient revealed the absence of the α peak at 558 nm of cyt b 558 . A small shoulder at the γ band (425 nm) was derived from slightly contaminating methemoglobin (Kiryu et al 1998). The difference spectra of the normal and the CGD neutrophils were differentiated as d[∆ A]/dλ, where ∆ A is the absorption difference and λ is the wavelength, as shown in Fig.…”

“…The spectra obtained were further data processed on a computer (FMV-5150T4) with a specialized software (Origin, microsoftware, Northampton, Mass.) to obtain differential spectra (for details, see Kiryu et al 1998). …”

“…In contrast, the present male patient with atypical X-linked CGD shows small amounts of both subunits of cyt b 558 . Recently, we have developed a sensitive and accurate spectrophotometric technique for the analysis of the heme spectrum of cyt b 558 of CGD neutrophils (Kiryu et al 1998). This technique has been applied to the neutrophils of our patient to examine whether the heme is absent or present in the cyt b 558 .…”