L-Prolyl-L-phenylalanine

The stereochemical features of 2,8,14,20-tetrakis(D-leucyl-D-valinamido)resorc[4]arenecarboxylic acid and the N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine-4-nitroanilide polypeptide substrate were investigated by nuclear magnetic resonance spectroscopy. Proton selective relaxation parameters gave the basis for the inhibitory activity of resorcin[4]arene in the hydrolysis of the polypeptide substrate by α-chymotrypsin. Results showed that an interaction between the resorcin[4]arene and α-chymotrypsin does occur, and involves the hydrophobic moiety of the macrocycle. This interaction is further reinforced by polar groups located on the side chains of the resorcin[4]arene, whereas the macrocycle-polypeptide substrate interaction is negligible. Conformational analysis and interaction studies carried out by molecular modeling are in good agreement with the NMR data, thus providing an additional support to the rationalization of the inhibitory potential of resorcin[4]arenes on the α-chymotrypsin activity.

Detergent enzymes account for about 30% of the total worldwide production of enzymes and are one of the largest and most successful applications of modern industrial biotechnology. Proteases can improve the wash performance of household, industrial, and institutional laundry detergents used to remove protein-based stains such as blood, grass, body fluids, and food soils. This article describes two easy and cheap laboratory exercises to study the presence, profile, and basic enzymology of detergent proteases. These laboratory practicals are based on the determination of the detergent protease activity of various commercial detergents using the N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide method and the bovine serum albumin degradation capacity. Students are also required to elucidate the enzymatic subtype of detergent proteases by studying the inhibitory potential of several types of protease inhibitors revealed by the same experimental methodology. Additionally, the results of the exercises can be used to provide additional insights on elementary enzymology by studying the influence of several important parameters on protease activity such as temperature (in this article) and the influence of pH and effects of surfactants and oxidizers (proposed). Students also develop laboratory skills, problem-solving capacities, and the ability to write a laboratory report. The exercises are mainly designed for an advanced undergraduate project in the biochemistry and biotechnology sciences. Globally, these laboratory practicals show students the biotechnological applications of proteases in the detergent industry and also reinforce important enzymology concepts.

Perinatal asphyxia (PA) still occurs in about three to five per 1,000 deliveries in developed countries; 20% of these infants show hypoxic-ischemic encephalopathy (HIE) on brain magnetic resonance imaging (MRI). The aim of our study was to apply metabolomic analysis to newborns undergoing therapeutic hypothermia (TH) after PA to identify a distinct metabotype associated with the development of HIE on brain MRI. We enrolled 53 infants born at >35 weeks of gestation with PA: 21 of them showed HIE on brain MRI (the "HIE" group), and 32 did not (the "no HIE" group). Urine samples were collected at 24, 48 and 72 hours of TH. Metabolomic data were acquired using high-resolution mass spectrometry and analyzed with univariate and multivariate methods. Considering the first urines collected during TH, untargeted analysis found 111 relevant predictors capable of discriminating between the two groups. Of 35 metabolites showing independent discriminatory power, four have been well characterized: L-alanine, Creatine, L-3-methylhistidine, and L-lysine. The first three relate to cellular energy metabolism; their involvement suggests a multimodal derangement of cellular energy metabolism during PA/HIE. In addition, seven other metabolites with a lower annotation level (proline betaine, L-prolyl-L-phenylalanine, 2-methyl-dodecanedioic acid, S-(2-methylpropionyl)-dihydrolipoamide-E, 2,6 dimethylheptanoyl carnitine, Octanoylglucuronide, 19-hydroxyandrost-4-ene-3,17-dione) showed biological consistency with the clinical picture of PA. Moreover, 4 annotated metabolites (L-lysine, L-3-methylhistidine, 2-methyl-dodecanedioic acid, S-(2-methylpropionyl)-dihydrolipoamide-E) retained a significant difference between the "HIE" and "no HIE" groups during all the TH treatment. Our analysis identified a distinct urinary metabotype associated with pathological findings on MRI, and discovered 2 putative markers (L-lysine, L-3-methylhistidine) which may be useful for identifying neonates at risk of developing HIE after PA.