Trypsin composition

Yellow to grayish-yellow powder or crystals. Stable indefinitely in dry form at room temp. Sol in water practically insol in alcohol or glycerol. Readily sol in Sorensen s sodium phosphate bnffer soln. Acts optimally at pH values between 7 and 9. Solns lose 75% of their potency within 3 hrs at room temp. Prepn of stabilized trypsin compositions contg partially hydrolyzed gelatin Sullivan, Martin, U.S. pat. 2,930,736 (I960 to National Drug). 

Several enzymes have been immobilized in sol-gel matrices effectively and employed in diverse applications. Urease, catalase, and adenylic acid deaminase were first encapsulated in sol-gel matrices [72], The encapsulated urease and catalase retained partial activity but adenylic acid deaminase completely lost its activity. After three decades considerable attention has been paid again towards the bioencapsulation using sol-gel glasses. Braun et al. [73] successfully encapsulated alkaline phosphatase in silica gel, which retained its activity up to 2 months (30% of initial) with improved thermal stability. Further Shtelzer et al. [58] sequestered trypsin within a binary sol-gel-derived composite using TEOS and PEG. Ellerby et al. [74] entrapped other proteins such as cytochrome c and Mb in TEOS sol-gel. Later several proteins such as Mb [8], hemoglobin (Hb) [56], cyt c [55, 75], bacteriorhodopsin (bR) [76], lactate oxidase [77], alkaline phosphatase (AP) [78], GOD [51], HRP [79], urease [80], superoxide dismutase [8], tyrosinase [81], acetylcholinesterase [82], etc. have been immobilized into different sol-gel matrices. Hitherto some reports have described the various aspects of sol-gel entrapped biomolecules such as conformation [50, 60], dynamics [12, 83], accessibility [46], reaction kinetics [50, 54], activity [7, 84], and stability [1, 80],... 

FIGURE 14.17 Ultrahigh resolution nano LC-MS separation of base peak chromatogram of 2351 peptides identified in trypsin digest of mouse brain lysate P2 fraction using Micro-Tech XtremeSimple nano-LC and Thermo Electron LTQ. Column 100 cm x 75 fim C18 column, 3 /mi, 8000 psi column head pressure. Solvent composition time 350 min gradient, 5 to 35% B. Solvent A 2% acetonitrile, 0.1% formic acid. Solvent B 95% acetonitrile, 0.1% formic acid. Data analysis Sequest, PeptideProphet, and Protein Prophet. 

Large peptides are partially hydrolyzed with enzymes (trypsin and chymotropsin) or acid to mixtures of di- and tripeptides. From the composition of these small units we establish the sequence of amino acids in the large polypeptide. 

There is currently little understanding of the influence of interfacial composition and (nano)structure on the kinetics of enzymatic hydrolysis of biopolymers and lipids. However, a few preliminary studies are beginning to emerge (McClements et al., 2008 Dickinson, 2008). Thus, for example, Jourdain et al. (2009) have shown recently that, in a mixed5 sodium caseinate + dextran sulfate system, the measured interfacial viscosity increased from qs = 220 mN s m 1 without enzyme to qs = 950 mN s m 1 with trypsin present. At the same time, the interfacial elasticity was initially slightly reduced from (7S = 1.6 mN m 1 to (h = 0.7 mN m, although it later returned to close to its original value. Conversely, in the... 

A model that is consistent with these observations of the action of trypsin and phospholipase A and with the discontinuities in the All-composition curves (Figures 2 and 3) is one in which the lipid monolayer is not a continuous palisade of uniformly oriented lipid molecules but rather an assembly of surface micelles. In this model, proposed by Colacicco (4, 5), the protein first comes into contact with the lipid molecules at the periphery of the surface micelles and then inserts itself as a unit between them. This is the basis for the generalized nonspecific interaction between lipids and proteins which results in increase of surface pressure. One may thus explain the identical All values obtained with films of lecithin and 80 mole % lactoside by picturing the lecithin molecules outside and the lactoside molecules inside the surface micelles. In this model lecithin prevents the bound lactoside from interacting nonspecifically with globulin and produces the same increase in pressure as with a film of pure lecithin. In the mixed micelle the lactose moiety of the lactoside protrudes into the aqueous subphase. Contact of the protein with these or other nonperipheral regions of the surface micelle would not increase the surface pressure. 

Table III. Molar Composition of Core Protein (Peptide) of BSM, Trypsin and /3-Elim. Glycopeptides (Pro = 3)...

The two major peptides obtained from BSM by the two different processes ( -elimination and trypsin digestion, respectively) show amino acid compositions that closely resemble that of the original BSM, except for some deviation in the serine content. 

At this point you should be able to deduce the sequence of five amino acids at the C-terminus of eledoisin.) (4) Trypsin hydrolysis gives two peptides, P and Q, with the indicated compositions ... 

At this point, you can deduce two possible sequences for Q.) (5) Trypsin hydrolysis of L gives a peptide of composition Ala, Asp, Phe which, with 2,4-dinitrofluorobenzene, gives the 2,4-dinitrophenyl derivative of aspartic acid. (6) Partial acid hydrolysis of eledoisin gives several dipeptides, among them Ser-Lys and Pro-Ser. 

From a rare fungus you have isolated an octapeptide that prevents baldness, and you wish to determine the peptide sequence. The amino acid composition is Lys(2), Asp, Tyr, Phe, Gly, Ser, Ala. Reaction of the intact peptide with FDNB yields DNP-alanine plus 2 moles of e-DNP-lysine on acid hydrolysis. Cleavage with trypsin yields peptides the compositions of which are (Lys, Ala, Ser) and (Gly, Phe, Lys), plus a dipeptide. Reaction with chymotrypsin releases free aspartic acid, a tetrapeptide with the composition (Lys, Ser, Phe, Ala), and a tripeptide the composition of which, following acid hydrolysis, is (Gly, Lys, Tyr). What is the sequence ... 

Bhatty, R.S. 1993. Further compositional analyses of flax Mucilage, trypsin inhibitors and hydrocyanic acid. J. Am. Oil Chem. Soc. 70, 899-904. 

The mobile phase used for the CZE separations of the trypsin digests was 0.1 M tricine and 0.02 M morpholine adjusted to pH 8.15. The mobile phase composition for the linearity and precision data was 0.01 M tricine, 0.0058 M morpholine, and 0.02 M NaCl adjusted to pH 8.0. The mobile phase buffers used to determine peak shape and electrophoretic mobility vs. pH all contained 0.02 M NaCl and 0.01 M of the buffer (pH 3.0, citrate pH 4.0, formic acid pH 5.0, acetate pH 6.0, MES pH 7.0, MOPS pH 8.0, tricine pH 9.0, borate and pH 10.0, glycine) and were adjusted to the indicated pH with acid or base. The column was rinsed with mobile phase between injections or successively with 0.1 M sodium hydrooxide and mobile phase when the mobile phase composition was changed. 

Tryptic Maps of Relaxin and Relaxin B-chain. Digestion of the A-chain of human relaxin with trypsin can theoretically result in the release of five fragments that of the B-chain in the release of six fragments as illustrated in Table II. A typical tryptic map of relaxin B-chain is shown in Figure 2. The peptide was reduced and carboxymethylated with iodoacetic acid before enzymatic digestion. The peptide assignments were made after analysis of the peaks by amino acid hydrolysis for amino acid composition and confirmed by fast atom bombardment mass spectrometry (FAB-MS) as shown in Table IH... 

Fig. 1 Summary of the data used to establish the complete amino acid sequence of Er-1 mating pheromone. The peptides have been designated and numbered according to the type of digest ana the theoretical order in which they appear in the sequence. Designations are CNBr, cyanogen bromide T, trypsin V8, . aureus V8 protease CT, chymotrypsin. Peptiaes indicated by two numbers connected with a hyphen result from partial cleavage. Residues directly identified by automated Edman degradation and carboxypeptidase Y digestion (CP-Y) are marked by right and left arrows, respectively, residues identified by amino acid composition are indicated by dashed lines. Taken from ref. 13 and reproduced by permission of the American Society of ...

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