Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Illumination of goat alpha-lactalbumin (GLA) with 280 or 295 nm light results in tryptophan-mediated photolysis of disulfide bonds within the protein. The photolysis is not dependent on the absence or presence of Ca(2+) and is observed as well on illumination of native and of partially unfolded GLA. However, photolysis of native GLA results in a partial unfolding of the protein. The latter phenomenon is most clearly observed on fluorescence measurements at low temperatures (near 3 degrees C). The photolysis induces some dimerization and oligomerization, but most GLA molecules remain monomeric. To obtain more information about the reaction products, the illuminated protein is treated with iodoacetamide to label the free thiol groups, it is fragmented with trypsin, and the fragments are analyzed by mass spectrometry. Via this approach, we observe that the cleavage of disulfide bonds is restricted to Cys6-Cys120 and Cys73-Cys91 bonds. The photolytic cleavage of either of these disulfide bonds results in the formation of a single free thiol, a phenomenon restricted to Cys120 and Cys91, respectively. We also found indications that a thioether linkage is formed between Cys73 and Trp60. The alkylsulfenylation of Trp60 presumably results from a combination of primary thiyl and tryptyl radicals.
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PMID:Photoexcitation of tryptophan groups induces reduction of two disulfide bonds in goat alpha-lactalbumin. 1220 76

As a potential tool for proteomics and protein characterization, in-gel cysteine- and arginine-specific cleavage is demonstrated by means of trypsin or endoproteinase Lys-C for six model proteins (lysozyme, alpha-lactalbumin, beta-lactoglobulin, ribonuclease A, albumin, and transferrin), ranging in size from 14 kDa to 79 kDa. Chemical modifications of cysteine (aminoethylation with bromoethylamine or N-(iodoethyl)-trifluoroacetamide, and subsequent guanidination) and lysine (acetylation) prior to tryptic digestion releases peptides delineated by cysteine or arginine residues. Peptide products are analyzed by MALDI-TOF-MS, ESI-MS, and ESI- and MALDI-MS/MS (with a quadrupole time-of-flight instrument). Complications induced by acrylamide alkylations of cysteines were avoided by substituting lower pH bis-tris polyacrylamide gels for tris-glycine. Sequence coverages from 35 to 86% were obtained and amino acid compositions of generated peptides could be confirmed by comprehensive y- and b-ion series. Detailed information about, in particular, cysteine rich proteins after gel electrophoresis were obtained. The chemistries for modification and cleavage specificities at cysteine residues provide an alternative means to characterize and identify proteins separated by gel electrophoresis.
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PMID:In-gel derivatization of proteins for cysteine-specific cleavages and their analysis by mass spectrometry. 1271 30

Prolonged exposure of Ca(2+)-loaded or Ca(2+)-depleted human alpha-lactalbumin to ultraviolet light (270-290 nm, 1 mW/cm(2), for 2 to 4 h) results in a 10-nm red shift of its tryptophan fluorescence spectrum. Gel chromatography of the UV-illuminated samples reveals two non-native protein forms: (1) a component with a red-shifted tryptophan fluorescence spectrum; and (2) a component with kynurenine-like fluorescent properties. The first component has from 0.6 to 0.9 free DTNB-reactive SH groups per protein molecule, which are absent in the native protein and is characterized by slightly lowered Ca(2+)-affinity (2 x 10(8) M(-1) versus 8 x 10(8) M(-1) for the native protein) and absence of observable thermal transition. The second component corresponds to the protein with photochemically modified tryptophan residues. It is assumed that the UV excitation of tryptophan residue(s) in alpha-lactalbumin is followed by a transfer of electrons to the Sbond;S bonds, resulting in their reduction. Mass spectrometry data obtained for trypsin-fragmented UV-illuminated alpha-lactalbumin with acrylodan-modified free thiol groups reveal the reduction of the 61-77 and 73-91 disulfide bridges. The effect observed has to be taken into account in any UV-region spectral studies of alpha-lactalbumin.
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PMID:Ultraviolet illumination-induced reduction of alpha-lactalbumin disulfide bridges. 1278 9

Chemical modification of the proteins bovine serum albumin, alpha-lactalbumin, beta-lactoglobulin and chicken lysozyme by 3-hydroxyphthalic anhydride (3-HP) yielded compounds which exerted antiviral activity in vitro as compared with the native unmodified proteins. Of the three enveloped viruses tested, human herpes simplex virus type 1 (HSV-1), bovine parainfluenza virus type 3 and porcine respiratory corona virus, only HSV-1 proved sensitive to the 3-HP-proteins. All of the chemically modified proteins presented antiviral activity against HSV-1 when assayed before, during or after infection. However, to achieve HSV-1 inhibition, significantly higher concentrations of the modified proteins were required if present before infection as compared to during or after infection. Our results suggest that multiple mechanisms are involved in the inhibition of HSV-1 infection. Proteolytical digestion of albumin, alpha-lactalbumin, beta-lactoglobulin and lysozyme by trypsin, chymotrypsin and pepsin yielded several peptide fragments with antiherpetic activity. Chemical modification of these peptide fragments by 3-HP generated peptides with antiviral activity, however, this was almost always combined with a cytotoxic effect on the Vero cells. Overall, our results suggest that targeted chemical modification of some natural products might provide compounds effective against HSV-1 infection.
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PMID:The antiviral activity of naturally occurring proteins and their peptide fragments after chemical modification. 1283 57

A nanoliter enzyme microreactor was developed for on-line capillary electrophoresis (CE) peptide mapping of proteins, allowing picomole quantities of proteins to be digested. The enzyme microreactor was formed by immobilizing trypsin onto a monolithic capillary column, which was prepared by in situ polymerization of glycidyl methacrylate and ethylene dimethacrylate in a capillary. Highly efficient digestion of three protein standards was demonstrated. The detection of peptide fragments in CE was enhanced by post-column derivatization and laser-induced fluorescence detection. The microreactor has a volume of about 30 nL and is coupled with a separation capillary via a fluid joint for on-line digestion. The overall analysis, including digestion and separation, lasted only about 16 min. Column efficiencies > 300 000 plates/m were obtained for most peaks in the electropherogram of an on-line peptide mapping experiment of denatured alpha-lactalbumin under optimal conditions.
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PMID:On-line protein digestion and peptide mapping by capillary electrophoresis with post-column labeling for laser-induced fluorescence detection. 1517 54

We have investigated the antioxidant activity of hydrolysates from whey proteins bovine alpha-lactalbumin (alpha-La) and beta-lactoglobulin A (beta-Lg A) by commercial proteases (pepsin, trypsin, chymotrypsin, thermolysin, and Corolase PP). Corolase PP was the most appropriate enzyme to obtain antioxidant hydrolysates from alpha-La and beta-Lg A (ORAC-FL values of 2.315 and 2.151 micromol of Trolox equivalent/mg of protein, respectively). A total of 42 peptide fragments were identified by HPLC-MS/MS in the beta-Lg A hydrolysate by Corolase PP. One of the sequences (Trp-Tyr-Ser-Leu-Ala-Met-Ala-Ala-Ser-Asp-Ile) possessed radical scavenging (ORAC-FL value of 2.621 micromol of Trolox equivalent/micromol of peptide) higher than that of butylated hydroxyanisole (BHA). Our results suggest that whey protein hydrolysates could be suitable as natural ingredients in enhancing antioxidant properties of functional foods and in preventing oxidation reaction in food processing.
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PMID:Preparation of antioxidant enzymatic hydrolysates from alpha-lactalbumin and beta-lactoglobulin. Identification of active peptides by HPLC-MS/MS. 1568 6

Covalent nucleophile-electrophile interactions have been established to be important for recognition of substrates by several enzymes. Here, we employed an electrophilic amidino phosphonate ester (EP1) to study the nucleophilic reactivity of the following proteins: albumin, soluble epidermal growth factor receptor (sEGFR), soluble CD4 (sCD4), calmodulin, casein, alpha-lactalbumin, ovalbumin, soybean trypsin inhibitor and HIV-1 gp120. Except for soybean trypsin inhibitor and alpha-lactalbumin, these proteins formed adducts with EP1 that were not dissociated by denaturing treatments. Despite their negligible proteolytic activity, gp120, sEGFR and albumin reacted irreversibly with EP1 at rates comparable to the serine protease trypsin. The neutral counterpart of EP1 reacted marginally with the proteins, indicating the requirement for a positive charge close to the electrophilic group. Prior heating resulted in altered rates of formation of the EP1-protein adducts accompanied by discrete changes in the fluorescence emission spectra of the proteins, suggesting that the three-dimensional protein structure governs the nucleophilic reactivity. sCD4 and vasoactive intestinal peptide (VIP) containing phosphonate groups (EP3 and EP4, respectively) reacted with their cognate high-affinity binding proteins gp120 and calmodulin, respectively, at rates exceeding the corresponding reactions with EP1. Reduced formation of EP3-gp120 adducts and EP4-calmodulin adducts in the presence of sCD4 and VIP devoid of the phosphonate groups was evident, suggesting that the nucleophilic reactivity is expressed in coordination with non-covalent recognition of peptide determinants. These observations suggest the potential of EPs for specific and covalent targeting of proteins, and raise the possibility of nucleophile-electrophile pairing as a novel mechanism stabilizing protein-protein complexes.
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PMID:Broadly distributed nucleophilic reactivity of proteins coordinated with specific ligand binding activity. 1570 8

Bovine milk alpha-lactalbumin (alpha-La) was mixed with soybean saponin, and the resulting mixture was hydrolyzed by trypsin. Saponin increased the tryptic-hydrolysis level of alpha-La only at relatively high phosphate buffer concentrations (> or = 0.05 M). T(1) experiments with acetylated soybean saponin demonstrated that there were some interactions between alpha-La and saponin not only at high concentrations of phosphate buffers but even at low concentrations as well. Circular dichroism spectra of alpha-La showed that the tertiary structure of alpha-La was changed through interactions with saponin only at high buffer concentrations. Furthermore, by analyzing the tryptic peptides from an alpha-La/saponin mixture, hydrolyzing rates at all or some of K5, R10, and K16 of alpha-La were accelerated by saponin interactions. The increase in the tryptic hydrolysis of alpha-La by saponin addition was considered due to modification of the tertiary structure of alpha-La by saponin.
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PMID:Characterization of tryptic hydrolysis of alpha-lactalbumin/saponin mixture and structural change of alpha-lactalbumin interacting with soybean saponin. 1570 70

Variability in the protein composition of breast milk has been observed in many women and is believed to be due to natural variation of the human population. Single nucleotide polymorphisms (SNPs) are present throughout the entire human genome, but the impact of this variation on human milk composition and biological activity and infant nutrition and health is unclear. The goals of this study were to characterize a variant of human alpha-lactalbumin observed in milk from a Filipino population by determining the location of the polymorphism in the amino acid and genomic sequences of alpha-lactalbumin. Milk and blood samples were collected from 20 Filipino women, and milk samples were collected from an additional 450 women from nine different countries. alpha-Lactalbumin concentration was measured by high-performance liquid chromatography (HPLC), and milk samples containing the variant form of the protein were identified with both HPLC and mass spectrometry (MS). The molecular weight of the variant form was measured by MS, and the location of the polymorphism was narrowed down by protein reduction, alkylation and trypsin digestion. Genomic DNA was isolated from whole blood, and the polymorphism location and subject genotype were determined by amplifying the entire coding sequence of human alpha-lactalbumin by PCR, followed by DNA sequencing. A variant form of alpha-lactalbumin was observed in HPLC chromatograms, and the difference in molecular weight was determined by MS (wild type=14,070 Da, variant=14,056 Da). Protein reduction and digestion narrowed the polymorphism between the 33rd and 77th amino acid of the protein. The genetic polymorphism was identified as adenine to guanine, which translates to a substitution from isoleucine to valine at amino acid 46. The frequency of variation was higher in milk from China, Japan and Philippines, which suggests that this polymorphism is most prevalent in Asia. There are SNPs in the genome for human milk proteins and their implications for protein bioactivity and infant nutrition need to be considered.
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PMID:Detection of a single nucleotide polymorphism in the human alpha-lactalbumin gene: implications for human milk proteins. 1586 26

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.
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PMID:Selectivity of tryptophan residues in mediating photolysis of disulfide bridges in goat alpha-lactalbumin. 1647 97


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