Papain specificity

I. SCHECHTEE, A. Bergee On the active site of proteases. 3. Mapping the active site of papain specific peptide inhibitors... 

There has been no attempt to explain all the features of papain specificity nor to indicate the number of steps from I to IV in the scheme of Figure 28. Indeed, it is likely that other groups on the substrate also interact with the enzjrme. Postulation of a three-point interaction may be necessary to explain the optical specificity of papain. The scheme of Figure 28 may be regarded as a minimal working hypothesis. 

Different enzymes have different specificities. Some, such as amylase, are specific for a single substrate, but others operate on a range of substrates. Papain, for instance, a globular protein of 212 amino acids isolated from papaya fruit, catalyzes the hydrolysis of many kinds of peptide bonds. In fact, it s this ability to hydrolyze peptide bonds that makes papain useful as a meat tenderizer and a cleaner for contact lenses. 

The class of proton transfer (PT) reactions plays a major role in many biological processes, including various enzymatic reactions. This class of reactions will be served here as a general example and an introduction for more complicated reactions. As a specific demonstration let s consider a proton transfer between Cys 25 and His 159 in papain. This reaction can be formally described as... 

Next we evaluate the PDLD + EVB surface for the enzymatic reaction using eq. (5.17). The resulting surface is shown in Fig. 5.6. As seen from the ligure, the protein can reduce Aby stabilizing the ionic state more than water. In fact, in the specific case of papain the protein inverts the stabilization of the covalent and ionic states relative to their order in solution. 

Proteolytic enzyme from the latex of Carica papaya with an approximate molecular weight of 27000. It is differentiated from papain in electrophoresis behavior, in solubility and in substrate specifity. Isolation by acidify of papaya-latex with HCl, salting out with NaCl and following chromatographic purification. The formulation contains L-cysteine as reducing agent. 

Irrespective of the descaling effect, development of a soft lustre depends on application of a softener. These experiments were positive in demonstrating the possibility of descaling the fibres and in perceptibly improving lustre under mild conditions. In particular, it was shown that papain is effective at the remarkably low concentration of 50 mg/1, showing a high degree of specificity after chlorination. 

Soya Proteins. Early attempts to make albumen substitutes from soya protein also ran into problems. A bean flavour tended to appear in the finished product. A solution to these problems has been found. Whipping agents based on enzyme modified soy proteins are now available. The advantage of enzymatic modification is that by appropriate choice of enzymes the protein can be modified in a very controlled way. Chemical treatment would be far less specific. In making these materials the manufacturer has control of the substrate and the enzyme, allowing the final product to be almost made to order. The substrates used are oil-free soy flakes or flour or soy protein concentrate or isolate. The enzymes to use are chosen from a combination of pepsin, papain, ficin, trypsin or bacterial proteases. The substrate will be treated with one or more enzymes under carefully controlled conditions. The finished product is then spray dried. 

During the last ten years, it has become apparent that calcium-dependent papain-like peptidases called calpains (EC 3.4.22.17) represent an important intracellular nonlysosomal enzyme system [35][36], These enzymes show limited proteolytic activity at neutral pH and are present in virtually every eukaryotic cell type. They have been found to function in specific proteolytic events that alter intracellular metabolism and structure, rather than in general turnover of intracellular proteins. Calpains are composed of two nonidentical subunits, each of which contains functional calcium-binding sites. Two types of calpains, i.e., /i-calpain and m-calpain (formerly calpain I and calpain II, respectively), have been identified that differ in their Ca2+ requirement for activation. The activity of calpains is regulated by intracellular Ca2+ levels. At elevated cytoplasmic calcium concentrations, the precursor procal-pain associates with the inner surface of the cell membrane. This interaction seems to trigger autoproteolysis of procalpain, and active calpain is released into the cytoplasm [37]. 

G. Lowe, Y. Yuthavong, Kinetic Specificity in Papain-Catalyzed Hydrolyses , Biochem. J. 1971, 124, 107-115. 

In another approach the specificity of an existing enzyme has been changed. Levine and Kaiser (57) have transformed a protease, papain, into a redox enzyme by alkylation of the active site thiol with (27), a derivative of xanthine. 

Among the specific enzymes whose activity has been reported to be decreased after in vitro ozone exposure are papain, glyceraldehyde-3-phosphate dehydrogenase, lysozyme, ribonuclease, and acetylcholinesterase. The latter enzyme appears to be particulady susceptible to free-radical and oxidative states. A loss in acetylcholinesterase activity has been reported in the red cells of humans and mice that inhaled ozone. However, there are only minimal amounts of this enzyme in lupg tissue, and, although it has been suggested that acetylcholinesterase is important in bronchial tract ciliary activity, there is no direct evidence to support this conjecture. 

This peptidase family Cl enzyme [EC 3.4.22.15] is an lysosomal endopeptidase with specificity akin to papain. Cathepsin L displays a higher activity toward protein substrates than does cathepsin B. 

This enzyme [EC 3.4.22.6], also known as papaya proteinase II, is a member of the peptidase family Cl. It is the major endopeptidase of papaya (Carica papaya) latex. It has a specificity similar to that of papain. In addition, there are a number of chromatographic forms of the enzyme. 

Figure 4.5. Schematic representation of enzyme specific cieavage of immunogiobuiin G (igG) by pepsin and papain. Treatment of igG with pepsin produces two unique fragments. Fab, with two antigen binding sites and Fj without binding sites. Treatment of igG with papain generates two Fab and one F fragments.

All peptidases catalyze the general reaction depicted in Figure C2.2.2, the hydrolysis of a peptide bond. The different peptidases are unique with respect to their specificity that is, their ability to accommodate particular sets of amino acids in the vicinity of a potentially scissile peptide bond. Some peptidases have very broad specificities, such as papain, which has few limi-... 

It should be noted that ascorbic acid is more stable at pH 4-5 than at pH 7, at which the folacin vitamers are more stable. Additional protection from oxidation can be achieved by degassing the extraction solution with an inert gas, such as helium. Homogenization is followed immediately by protein precipitation and release of bound folacin vitamers. This can be accomplished by mild acidification, heating, addition of organic compounds such as trichloroacetic acid, and/or enzymatic (e.g., papain) hydrolysis. The specific conditions used for homogenization and protein precipitation are dictated by the food matrix and the expected profile of folacin vitamers. 

---