Intrinsic protein absorbance

Texturization is not measured directly but is inferred from the degree of denaturation or decrease of solubility of proteins. The quantities are determined by the difference in rates of moisture uptake between the native protein and the texturized protein (Kilara, 1984), or by a dyebinding assay (Bradford, 1976). Protein denaturation may be measured by determining changes in heat capacity, but it is more practical to measure the amount of insoluble fractions and differences in solubility after physical treatment (Kilara, 1984). The different rates of water absorption are presumed to relate to the degree of texturization as texturized proteins absorb water at different rates. The insolubility test for denaturation is therefore sometimes used as substitute for direct measurement of texturization. Protein solubility is affected by surface hydrophobicity, which is directly related to the extent of protein-protein interactions, an intrinsic property of the denatured state of the proteins (Damodaran, 1989 Vojdani, 1996). 

We have observed three types of effects on the structures of enzymes as the temperature is lowered in cryosolvents (1) no apparent change in the protein conformation, with the possible exception of decreased mobility of the surface side chains (2) conformational transitions, usually marked by little effect on the catalytic properties and (3) increased association of subunits. In most cases no detectable effects of decreasing temperature on the enzyme s structure have been detected by such procedures as monitoring the intrinsic fluorescence (16), or intrinsic visible absorbance in the case of flavin enzymes (Fink and... 

The most widely used method to study retinoid-protein interactions is fluorescence titration. The strong absorbance of retinol and retinol analogs in the region of emission of protein tryptophans is associated with an efficient energy transfer from excited tryptophans to the bound retinoid, and leads to a substantial quenching of intrinsic protein fluorescence when retinoids interact with... 

The vitamin deficiency is often due to failure to absorb B12 from the stomach and can be alleviated by giving mg doses with extracts of hog s stomach which contains the intrinsic anti-pernicious anaemia factor (a muco-protein), which promotes the absorption. 

Fluorescent probes are divided in two categories, i.e., intrinsic and extrinsic probes. Tryptophan is the most widely used intrinsic probe. The absorption spectrum, centered at 280 nm, displays two overlapping absorbance transitions. In contrast, the fluorescence emission spectrum is broad and is characterized by a large Stokes shift, which varies with the polarity of the environment. The fluorescence emission peak is at about 350 nm in water but the peak shifts to about 315 nm in nonpolar media, such as within the hydrophobic core of folded proteins. Vitamin A, located in milk fat globules, may be used as an intrinsic probe to follow, for example, the changes of triglyceride physical state as a function of temperature [20]. Extrinsic probes are used to characterize molecular events when intrinsic fluorophores are absent or are so numerous that the interpretation of the data becomes ambiguous. Extrinsic probes may also be used to obtain additional or complementary information from a specific macromolecular domain or from an oil water interface. 

Very small amounts of cobalamin are required each day (<5 i.g) and the diet normally provides plenty more than the minimum, so dietary B12 deficiency is uncommon, except in very strict vegetarians. Pernicious anaemia arises when a defect in the stomach results in too little secretion of a protein called intrinsic factor, without which, cobalamin cannot be absorbed in the ileum of the small intestine. 

Compared to absorbance detection, direct detection of proteins rich in aromatic amino acids by the intrinsic fluorescence of tryptophan and tyrosine residues provides enhanced sensitivity without the complexity of pre- or postcolumn derivatization. The optimal excitation wavelengths for these amino acids are in the 270- to 280-nm range. 

Nutritional Deficiency-Related Dementias. We have already mentioned that chronic alcoholics are subject to thiamine deficiency that can cause dementia. It usually occurs only after heavy, prolonged abuse of alcohol. In developed countries, the other key nutritional concern is vitamin deficiency. Vitamin deficiency can surprisingly strike even those with a healthy diet. Such people are missing a vital protein, intrinsic factor, which would enable them to absorb it from their digestive tract. 

INTRINSIC AND EXTRINSIC FLUORESCENCE. Intrinsic fluorescence refers to the fluorescence of the macromolecule itself, and in the case of proteins this typically involves emission from tyrosinyl and tryptopha-nyl residues, with the latter dominating if excitation is carried out at 280 nm. The distance for tyrosine-to-tryp-tophan resonance energy transfer is approximately 14 A, suggesting that this mode of tyrosine fluorescence quenching should occur efficiently in most proteins. Moreover, tyrosine fluorescence is quenched whenever nearby bases (such as carboxylate anions) accept the phenolic proton of tyrosine during the excited state lifetime. To examine tryptophan fluorescence only, one typically excites at 295 nm, where tyrosine weakly absorbs. [Note While the phenolate ion of tyrosine absorbs around 293 nm, its high pXa of 10-11 in proteins typically renders its concentration too low to be of practical concern.] The tryptophan emission is maximal at 340-350 nm, depending on the local environment around this intrinsic fluorophore. 

Pharmacokinetics The parietal cells of the stomach secrete intrinsic factor, which regulates the amount of vitamin B-12 absorbed in the terminal ileum. Bioavailability of oral preparations is approximately 25%. Vitamin B12 is primarily stored in the liver. Enterohepatic circulation plays a key role in recycling vitamin B-12 from mainly bile. If plasma-binding proteins are saturated, excess free vitamin B- 2 will be excreted in the kidney. 

ATR spectroscopy in the infrared has been used extensively in protein adsorption studies. Transmission IR spectra of a protein contain a wealth of conformational information. ATR-IR spectroscopy has been used to study protein adsorption from whole, flowing blood ex vivo 164). Fourier transform (FT) infrared spectra (ATR-FTIR) can be collected each 5-10 seconds165), thus making kinetic study of protein adsorption by IR possible 166). Interaction of protein with soft contact lens materials has been studied by ATR-FTIR 167). The ATR-IR method suffers from problems similar to TIRF there is no direct quantitation of the amount of protein adsorbed, although a scheme similar to the one used for intrinsic TIRF has been proposed 168) the depth of penetration is usually much larger than in any other evanescent method, i.e. up to 1000 nm water absorbs strongly in the infrared and can overwhelm the protein signal, even with spectral subtraction applied. 

In this unit, Basic Protocol 1 presents a procedure using casein as substrate. The Alternate Protocol describes the modification of this procedure for use with a denatured hemoglobin substrate. Basic Protocol 2 presents a procedure using a chromaphore-conjugated casein derivative, azocasein. For quantitation, the authors have chosen to use either the BCA-based colorimetric assay unitbli) for soluble protein/peptides (in Basic Protocol 1) or the intrinsic absorbance of the chromaphore-conjugated peptide products (in Basic Protocol 2). 

Vitamin B12. Vitamin B12 is a larger molecule than the other vitamins, and it can be absorbed via the intestine, which involves binding to specialized transport proteins.44 After oral administration, vitamin B12 binds to intrinsic factor (IF) produced from the parietal cells in the stomach and proximal cells in the duodenum. The vitamin B12—IF complex passes down the small intestine until it reaches the ileum, where the complex binds to a specific IF receptor located on the apical membrane of the villous enterocyte. The complex is then internalized via RME, vitamin B12 is released from IF by the action of cathepsin L on IF, and free vitamin B12 consequently forms the complex with transcobal-amin II to be delivered into the basolateral side of the membrane via the transcytotic pathway. 

Vitamin B12 can be absorbed when present in physiological amounts only if it is first bound to a specific protein—the so-called intrinsic factor—that tightly binds to the vitamin. The complex then passes through the jejunum to the ileum, which contains receptor sites for the vitamin B12/intrinsic factor complex. Calcium ions are required for the reaction between ileal receptors and the intrinsic factor/vitamin B12 complex. The reaction is inhibited by EDTA and reduced by a pH below 5.4. The vitamin appears to be separated from intrinsic factor at the ileal receptor sites and is then bound to another protein carrier, transcobalamin II, which transports the vitamin and permits its uptake by a number of tissues. The subject has been well reviewed by Jacob and her colleagues (Jl). Removal of 60 cm of ileum may impair vitamin B12 absorption and with the loss of 180 cm absorption is almost always affected. 

Patients with exocrine pancreatic dysfunction may malabsorb vitamin B12 because a considerable part of the dietary vitamin may bind to R-proteins in the stomach. The R-proteins are normally broken down by pancreatic enzymes. The vitamin is released and binding to intrinsic factor then takes place. If the R-proteins are not degraded, then the vitamin B12 will remain bound to these proteins and will not be absorbed (M10). Harms and his colleagues (H27) measured vitamin B12 absorption in 19 children with exocrine pancreatic insufficiency and found the average absorption to be 8.0% compared to 59.2% in a control group. Adding pancreatin to the test dose of radiolabeled vitamin B12 increased absorption of the vitamin to an average of 61%. 

Brugge et al. (B21) described a simple dual-isotope test for pancreatic exocrine function. They gave their patients radiolabeled [57Co]Bl2 bound to R-protein and [58Co]B12 bound to intrinsic factor if significantly less of the R-protein-bound vitamin was absorbed compared to the intrinsic-factor-bound vitamin this provided good evidence for pancreatic exocrine dysfunction. 

This observation led to the discovery that a dietary compound (extrinsic factor) was absorbed only after combination with a protein secreted by the normal stomach (intrinsic factor [IF]), and that the IF was missing from the secretions of the atrophic stomach found in patients with PA. The extrinsic factor, later named vitamin B12, was obtained in crystalline form in 1948, and its structure was defined by x-ray crystallography by Dorothy Hodgkins, an accomplishment for which she received the Nobel Prize for Chemistry in 1964. 

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