Specific and instantaneous one-step chemodetection of histidine-rich proteins by Pauly's stain

Abstract

Histidine-containing proteins are widely found in nature. In most cases about one in 20 residues is a histidine and those containing a higher level are considered as histidine-rich proteins (HRPs) [1,2]. With over 30% of their residues as histidines, HRP II and III of the malaria parasite Plasmodium falciparum show a markedly high density of histidines among proteins [3]. P. falciparum HRP II (PfHRP II) binds as many as 50 monomers of heme [4] and catalyses the detoxification of heme via its polymerization into the nontoxic malaria pigment called hemozoin [5,9]. Such HRPs represent targets of drug action since proven and widely used antimalarial drugs like chloroquine [5] and artemisinin [6,7] antagonize this heme polymerization pathway. Chromatographic detection of HRPs of the malaria parasite is currently being done by tedious and timeconsuming procedures of SDS–PAGE and Coomassie staining or immunologically via Western or dot blots [8]. Due to the significance of HRP II in the diagnosis of malaria, a fast immunochromatographic dipstick test [10–12] is used in the clinics. However this test is far too expensive for routine detection of such proteins in a biochemistry laboratory. Here we report that the diazonium salt-based histidine-reactive Pauly’s stain [13] detects these proteins instantly, with sensitivity comparable to Coomassie ( 200 ng on PAGE) or immunodetection ( 37 ng on nitrocellulose dot blot), with specificity resembling immunodetection and on diverse matrices like Whatman paper, polyacrylamaide gels, nitrocellulose membranes, and Ni–NTA–agarose gels. Recombinant PfHRP II and PfHRP III were expressed and purified by Ni–NTA metal-affinity chromatography [5] and estimated by the BCA method [14]. SDS–PAGE was done as per Laemmli [15]. Pauly’s stain was freshly prepared by mixing equal volumes of prechilled sodium nitrite (Aldrich) (5% w/v in water, reagent A) and sulfanilic acid (Aldrich) (9 g in 90ml of concentrated HCl and 900ml water, reagent B) and incubating the mixture at 25 C for 5min. Finally sodium carbonate (Merck, India) (10%, w/v) in a volume equal to a volume of A+B was added and the mixture allowed to react with the proteins. This procedure stained the HRPs within a couple of minutes with little or no background. Dried gels were found to retain the red colored bands of the Pauly stain for several months. Fig. 1A shows the results of post-SDS–PAGE staining of varying amounts of PfHRP II by the nonspecific Coomassie stain and by the histidine-reactive Pauly stain, respectively. HRP II was detectable at 100 ng by Pauly’s stain and at 10 ng by Coomassie stain. However unlike the Coomassie stain, which requires hours to stain and destain, the Pauly’s stain is accomplished in minutes and requires no destaining. Also while a positive stain at 100 ng suggests out that the detected band is an HRP, one cannot be sure that the trace staining seen at 10 ng with Coomassie originates from HRP or a contaminant non-HRP protein. The Coomassie stain of HRP II shows at least three accessory bands as trace contaminants. Their status as non-HRP contaminants or as HRP degradation products cannot be assessed by Coomassie stain. However the Pauly positivity of these bands suggests that these may be HRP-related Analytical Biochemistry 308 (2002) 405–408