Ovalbumin hydrolysis

GME glycine methyl ester GDM glutamic dimethyl ester EDA ethylendiamine GAM glucosamine HDA hexadecylamine BSA bovine serum albumin OVA ovalbumin CA carbonic anhydrase MYO myoglobin H alkaline hydrolysis for converting ester groups from GME or GDM into free carboxylic acid groups that were subsequently activated with EDC for further modifications. 

R. Montgomery and his colleagues108 fractionated, on Dowex 50, the aspartamidoglycan from ovalbumin. The five components obtained were each subjected to exhaustive hydrolysis with a-D-man-nosidase and 2-acetamido-2-deoxy-/3-D-glucosidase, used separately. The results are shown in Table X. One way in which to explain these results would be to change the position of the extra hexosamine residue in 1, to give the alternative structure 2. 

The differences in the rates of hydrolysis of various linkage types by a particular glycosidase can be used to provide information about this aspect of structure. Jack-bean a-D-mannosidase cleaves a-(l- 2) and a-(l- 6) linkages much faster than a-(l -> 3). Oligosaccharides, obtained by endo-N-acetyl-/J-D-glucosaminidase hydrolysis of ovalbumin, were subjected to acetolysis, which selectively cleaved the a-(l - 6) bonds. A tetrasaccharide isolated after this treatment was then incubated with jack-bean o-d-... 

Table XVI gives a partial list of native proteins that have been hydrolyzed with proteolytic enzymes. A discussion of the interpretation of each example listed is beyond the scope of this review, but a few comments concerning certain features of proteolysis are ivarranted. The mechanism of enzymatic hydrolysis of native proteins was studied in detail by Tiselius and Eriksson-Quensel (1939), who examined the action of pepsin on ovalbumin. Two mechanisms of proteolysis were considered by these workers. In the first mechanism the enzyme hydrolyzes all susceptible peptide bonds in one substrate molecule before hydrolysis of a second molecule begins. This type of mechanism has been described by Lmderstrpm-Lang (1952) as the all or none type. In the second mechanism, the enzyme hydrolyzes the single, most susceptible bond in all substrate molecules before hydrolysis of other bonds occurs. This mechanism is called the zipper type. Hydrolysis of a protein can proceed by either of the two mechanisms or by a mechanism which has features of both types. General aspects of the problem have been reviewed and theoretical equations which describe the kinetics of ea( h mechanism have been derived (Linderstr0m-Lang, 1952, 1953).

It is possible that the toxin was not properly activated, and a new sample of toxin should be activated, using freshly prepared DTT. It is possible that the concentration of NAD in the assay was too low and was hydrolyzed. Because NAD in solution is susceptible to hydrolysis, it should be stored in small portions at -20°C and the stock supply replaced regularly. Mutant toxins may, in fact, be inactive and it is suggested that Assays 3 and 4 be used to confirm or negate this possibility. CT activity may also be affected by salt and/or protein concentrations in the assay. Excessive salt can stimulate toxin activity buffer controls should, therefore, be run in each assay. High protein concentrations may result in decreased or variable toxin activity in Assay 2, the presence of ovalbumin minimizes nonspecific protein effects. 

Protein Hydrolysates. Instead of ethyl hippurate, a peptic hydrolysate of ovalbumin was used as substrate for the resynthesis reaction (64). This substrate (300 mg) was dissolved in water, adjusted to pH 6.0 with NaOH and to 0.9 ml with additional water. An amino acid ester was added to produce a 22.2mM solution and the mixture preincubated at 37°C for 15 min. Papain (3 mg), dissolved in 0.1M L-cysteine (0.1 ml), was combined with the above-mentioned preincubation mixture and incubation carried out at 37°C. After 2 hr, 0.1N NaOH (10 ml) was added to stop the enzymatic reaction and the resulting solution allowed to stand for 3 hr to hydrolyze completely the remaining amino acid ester as well as the ester group from the peptide product. The free amino acid produced from the base-catalyzed hydrolysis of the amino acid ester was determined with an amino acid analyzer. The amount of the amino acid incorporated was obtained by subtracting the determined value from the initial concentration of amino acid ester. The data obtained with the same L-amino acid esters as used in the model experiment (above) are plotted along the ordinate of Figure 3. An excellent correlation is found between the data from the model experiment and those from this experiment using a protein hydrolysate. In Table III data are shown for the extent of covalent incorporation after 2 hr of various amino acid ethyl esters into the protein hydrolysate. There is a close relationship between... 

Similar conclusions may be drawn from the rates of hydrolysis of gramicidin (Synge, 1945 Christensen and Hegsted, 1945) the effect being more marked at 37° than at the boiling temperature. On the contrary, however, it was found that the yield of free amino acids during the hydrolysis of ovalbumin with 1 N HCl was almost exactly theoretical (Warner, 1942b). 

Thus Synge and Tiselius (1947) were able to fractionate the components of tyrocidin, both by elution and displacement methods. In a study of the partial hydrolysis of ovalbumin Moring-Claesson (1948) was able to separate by adsorption the unchanged protein from the breakdown products. The former was adsorbed much more strongly on alumina and less strongly on carbon than the smaller peptides and amino acids. 

Antibodies have been produced to both retronecine and monocrotaline and detected using an avidin-biotin antibody. Retronecine was obtained from the hydrolysis of monocrotaline, succinylated and coupled to bovine serum albumin or ovalbumin. Recently an ELISA lateral flow device (dipstick test) based on gold colloidal polyclonal antibodies has been developed for jacobine and lycopsamine in honey and animal feed [59]. 

The rather infrequent occurrence of the cysteinylglycine sequence implied by these studies makes unlikely any major contribution of GSH as a directly utilized periodicity in protein synthesis. Nevertheless, experiments are in progress, using C and S tagged GSH, to determine whether or not the isolatable cysteinylglycine groupings which have been shown to occur in ovalbumin and ribonuclease are derived from GSH without prior cellular hydrolysis to the free component amino acids. 

While many examples of limited proteolysis are known, e.g., tiypsin on the B-chain of insulin (Sanger and Tuppy, 1951), zymogen activation (Neurath, 1957), ovalbumin-plakalbumin conversion (Ottesen, 1958), etc., a reversible equilibrium, as in step 1, has not yet been demonstrated except possibly in the pepsinogen-pepsin conversion (Herriott, 1938,1941). It probably does not exist because the peptide is too short the argument for a critical size of the peptide, in order to observe reversibility, has been presented in Chapter III. However, recent experiments of Richards (1958) on the subtilisin digestion of ribonuclease, wherein the 20 residue N-terminal tail is removed by hydrolysis, indicate that side-chain hydrogen bonding may possibly be involved in the association between the tail and the core. 

Natural melanins usually occur in the form of melanoproteins and thioether linkages, such as those mentioned above, may be important in the overall molecular structure. However, in this regard several attempts to demonstrate the formation of addition products between the oxidation products of either 5,6-dihydroxyindole or DOPA with certain proteins and peptides including ovalbumin [218, 224] or bovine serum albumin [224] have been unsuccessful. More recently, however, it has been shown that when tyrosine was oxidised in the presence of bovine lens protein, brown or black melanoproteins were formed [225]. On hydrolysis these pigments gave rise to a compound with similar properties to those of a (110)-type compound, which could have been formed from the oxidation of DOPA in the presence of cysteine. The thiol groups of the protein may react with some of the intermediates produced by the oxidation of tyrosine [225]. Reactions such as this may be involved in the formation of cataracts in the eye [225]. 

Miscellaneous.—An e db-j8-D-acetamidodeoxyglucanase obtained from culture filtrates of Streptomyces griseus rapidly released oligosaccharides from Saccharo-myces cerevisiae )8-fructofuranosidase, bovine pancreatic ribonuclease I and deoxyribonuclease 1, and sulphitolysed ovalbumin these glycoproteins contain a D-mannopyranosyl residue attached to di-iV-acetylchitobiose, which, in turn, is linked to an L-asparagyl residue. Analysis of the hydrolysis products showed that the enzyme cleaved between the 2-acetamido-2-deoxy-D-glucopyranosyl residues. 

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