From sewage sludge ash to a recycled feed phosphate – digestibility of precipitated calcium phosphate in broiler chickens and growing pigs

Today, EU is largely (∼92%) dependent on the import of phosphates as most mines are located outside Europe. Because of the limited availability, phosphorus (P) is included on the list of Critical Raw Materials. Precipitated calcium phosphate (PCP) recovered from sewage sludge ash is a novel and sustainable option to replace mined P as raw material in feed phosphates, e.g. monocalcium phosphate (MCP) or dicalcium phosphate, but the digestibility has not yet been tested in vivo. The aim was therefore to determine PCP and MCP apparent ileal digestibility (AID) of P in broiler chickens and apparent (ATTD) and true (TTTD) total tract digestibility of P in growing pigs. A chicken study comprised 240 Ross 308 chickens that were housed in groups of eight from day 21 to day 28. Five diets were used, a basal diet and two test diets, which contributed either 0.075% (low) or 0.150% (high) additional P for each of the test sources (MCP and PCP). The basal and test diets were composed to achieve increasing levels of P and AID was calculated with regression analysis. In the pig study, eight individually housed pigs were used in a change-over study with two experimental periods. The pigs were fed a basal P-free diet in a preperiod to be able to estimate endogenous P losses and then two different diets in two periods using a change-over design, where MCP and PCP were the only P source, providing in total 0.33 (basal diet), 4.42 (MCP) and 3.53 (PCP) g kg-1P, respectively. The AID of P in PCP and MCP for chickens was 58.4 and 75.1% (P = 0.166). The ATTD and TTTD of P in PCP for pigs were 58.4 and 67.2%, respectively, which was lower (P < 0.001) than the corresponding values for MCP (82.1 and 89.1%), respectively. The digestibility of calcium (Ca) did not differ in the chicken diets with high inclusion levels of PCP and MCP (54.7 and 55.3%, respectively, P = 0.535), but was lower for PCP than MCP in the pig study (57.8 and 70.8% respectively, P = 0.001). In conclusion, the digestibility of P in PCP for chickens did not differ from conventional MCP, whereas for pigs, it was lower, but could be a viable alternative to other common sources of P

pKa Determination of a Histidine Residue in a Short Peptide Using Raman Spectroscopy

Determining the pKa of key functional groups is critical to understanding the pH-dependent behavior of biological proteins and peptide-based biomaterials. Traditionally, ¹H NMR spectroscopy has been used to determine the pKa of amino acids; however, for larger molecules and aggregating systems, this method can be practically impossible. Previous studies concluded that the C-D stretches in Raman are a useful alternative for determining the pKa of histidine residues. In this study, we report on the Raman application of the C2-D probe on histidine's imidazole side chain to determining the pKa of histidine in a short peptide sequence. The pKa of the tripeptide was found via difference Raman spectroscopy to be 6.82, and this value was independently confirmed via ¹H NMR spectroscopy on the same peptide. The C2-D probe was also compared to other Raman reporters of the protonation state of histidine and was determined to be more sensitive and reliable than other protonation-dependent signals. The C2-D Raman probe expands the tool box available to chemists interested in directly interrogating the pKa's of histidine-containing peptide and protein systems

Deuterium-Enhanced Raman Spectroscopy for Histidine pKa Determination in a pH-Responsive Hydrogel

We report here a method for the determination of the pKa of histidine in complex or heterogeneous systems amenable to neither solid-state nor solution NMR spectroscopy. Careful synthesis of a fluorenylmethyloxycarbonyl- and trityl-protected, C2-deuterated histidine produces a vibrational-probe-equipped amino acid that can readily be incorporated into any peptide accessible by standard solid-phase methods. The frequency of the unique, Raman-active stretching vibration of this C2-D probe is a clear reporter of the protonation state of histidine. We investigate here a pH-sensitive peptide that self-assembles to form a hydrogel at neutral pH. The pKa of the lone histidine residue in the peptide, which is likely responsible for this pH-dependent behavior, cannot be investigated by NMR spectroscopy because of the supramolecular, soft nature of the gel. However, after synthesizing a C2-deuterated-histidine-containing peptide, we were able to follow the protonation state of histidine throughout a pH titration using Raman difference spectroscopy, thereby precisely determining the pKa of interest

Deamidation and disulfide bridge formation in human calbindin D28k with effects on calcium binding

Calbindin D28k (calbindin) is a cytoplasmic protein expressed in the central nervous system, which is implied in Ca2+ homeostasis and enzyme regulation. A combination of biochemical methods and mass spectrometry has been used to identify post-translational modifications of human calbindin. The protein was studied at 37°C or 50°C in the presence or absence of Ca2+. One deamidation site was identified at position 203 (Asn) under all conditions. Kinetic experiments show that deamidation of Asn 203 occurs at a rate of 0.023 h−1 at 50°C for Ca2+-free calbindin. Deamidation is slower for the Ca2+-saturated protein. The deamidation process leads to two Asp iso-forms, regular Asp and iso-Asp. The form with regular Asp 203 binds four Ca2+ ions with high affinity and positive cooperativity, i.e., in a very similar manner to non-deamidated protein. The form with β-aspartic acid (or iso-Asp 203) has reduced affinity for two or three sites leading to sequential Ca2+ binding, i.e., the Ca2+-binding properties are significantly perturbed. The status of the cysteine residues was also assessed. Under nonreducing conditions, cysteines 94 and 100 were found both in reduced and oxidized form, in the latter case in an intramolecular disulfide bond. In contrast, cysteines 187, 219, and 257 were not involved in any disulfide bonds. Both the reduced and oxidized forms of the protein bind four Ca2+ ions with high affinity in a parallel manner and with positive cooperativity