Pronase digestion

Nasr SH, Galgano SJ, Markowitz GS, et al. Immunofluorescence on pronase-digested paraffin sections a valuable salvage technique for renal biopsies. Kidney Int. 2006 70 2148-2151. 

If pronase digestion of the biotinylated protein is to be done, heat 100 pi of the sample at 56°C for 10 minutes, then add 10 pi of the pronase solution. Allow the sample to digest enzymatically at room temperature overnight. If no pronase digestion is desired, simply use the biotinylated protein solution prepared in step 3 without further treatment. 

Recently, sulfur mustard has been shown to alkylate a cysteine residue in human serum albumin (10). The site of alkylation was identified in a tryptic digest of albumin from blood exposed to [14C]sulfur mustard. A sensitive method for its analysis was developed based on Pronase digestion of alkylated albumin to the tripeptide S-[2-[(hydroxyethyl)thio]ethyl-Cys-Pro-Phe, and detection using micro-LC-MS-MS. In vitro exposure of human blood to > 10 nM sulfur mustard could be detected employing this method. The analytical procedure was successfully applied to albumin samples from Iranian casualties of the Iraq-Iran war. 

Incubation of l,l,2,2,-tetrachloro[l,2- 4C]ethane with a reconstituted monooxygenase system or with intact rat liver microsomes led to the formation of a metabolite capable of binding covalently to proteins and other nucleophiles. The only soluble metabolite detected upon incubation of 1,1,2,2-tetrachloroethane with a reconstituted system was dichloroacetic acid. Pronase digestion of the C-labcllcd microsomal proteins indicated the presence of several derivatized amino acids, which were hydrolysed by alkali to yield dichloroacetic acid. The results are consistent with biotransformation of 1,1,2,2-tetrachloroethane by cytochrome P450 to dichloroacetyl chloride, which can bind covalently to various nucleophiles or hydrolyse to dichloroacetic acid (Halpert, 1982). 

Binding of sarin and soman to a tyrosine residue present in blood has been observed by Black et al. (51) When sarin or soman was incubated with human plasma, phosphonylated tyrosine was observed by LC/MS after Pronase digestion, in addition to phosphonylated serine. The precise site of this residue has not yet been confirmed but it is associated with the albumin fraction. A phosphonylated tryptic peptide [/-PrO(CH3)P(0)]-Tyr-Thr-Lys, consistent with albumin, has been identified but this sequence is also present in other proteins. Before the advent of modem mass spectrometry, diisopropyl fluorophosphate was reported to bind... 

After Pronase digestion of plasma, the analytes were concentrated on a C18 or C8 cartridge and analyzed by LC/MS/MS. The adducts have been detected in the blood of guinea pigs 24 h after being exposed to 0.5 LD50 doses of sarin and soman. It is not known if they are formed in cases of human exposure. 

Noort et al. (53) recently demonstrated that phosgene binds effectively to albumin and hemoglobin upon in vitro exposure of human blood to [14C]phos-gene (53). Upon Pronase digestion of globin, one of the adducts identified was the pentapeptide 0=C-(Val-Leu)-Ser-Phe-Ala, representing amino acids 1-5 of a-globin, with a hydantoin function between the N-terminal valine and leucine. This adduct... 

Figure 6. Trace level LC/electrospray tandem MS analysis of (S-HETE)Cys-Pro-Phe in pronase digest of albumin (20mg) after purification on Sep-Pak Cl8, measuring the transition m/z 470 (MH+) -a 105. Albumin was isolated from nonexposed blood (A) or from human blood that was exposed to 1 nM (B). Panel C represents the 1 nM digest after spiking with synthetic (S-ffETE)Cys-Pro-Phe. The arrow indicates the peak for (S-ElETE)Cys-Pro-Phe... 

Kim and coworkers examined glycopeptides from normal, and cancerous, colonic mucosa.869 Pronase digestion of disrupted cell-membranes gave a soluble glycopeptide fraction. The fractions obtained from normal tissues inhibited Dolichos biflorus, but not Ricinus communis, hemagglutination, whereas the reverse was true of fractions obtained from malignant, colonic mucosa. No further analysis was carried out.869... 

Initial efforts by Noort et al. (1996,1997) to detect protein adducts of sulfur mustard focused on the 4-(2-hydrox-yethylthioethyl)-L-aspartate, 5-(2-hydroxyethylthioethyl)L-glutamate, the cysteine and the N-terminal valine adduct and two histidine adducts, Nl- and N3-(2-hydroxyethylth-ioethyl)-L-histidine, respectively. Acidic hydrolysis and pronase digestion were used to release these adducts from... 

Grafts isolated by pepsin and trypsin digestion had stronger amide absorption bands than after pronase digestion. Pronase, because of its broad specificity, is capable of hydrolyzing the collagen trunks more extensively than the other two enzymes. Thus, the grafts isolated by... 

The method including heat-denaturation, pronase digestion, ODS silica gel treatment, and immunoaffinity... 

Figure 2. Glycopeptides from pronase digestion of ribonuclease B. Two series are observed corresponding to the dipeptide RN and single amino acid N.

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