Polypeptides hydrolysis

Further investigations with boar seminal fluid performed by Kalic et al. [54] using a 600-MHz NMR instrument revealed the identification of the amino acids hypotaurine and carnitine. Semiselective HSQC 2D experiments on an 800-MHz speedometer [55] enable one to differentiate between the structurally similar polyamines, spermidine, spermine and their metabolic precursor putrescine, all attracting interest for tumor therapy, in seminal fluid. Recently, Tomlins et al. [56] studied dynamic biochemical processes in incubated human seminal fluid samples The enzymatic hydrolysis of phosphoiylcholine to choline and the conversion of uridine-5 -monophosphate to uridine were found to be very fast, whereas the slow polypeptide hydrolysis to amino acids can be inhibited by the addition of EDTA. It remains to be seen which role the biochemical changes play in the reproductive function. 

The efficiency of catalysts of this type is due to the cyclic transition state in wdiich the concerted transfer of two protons occurs. Enzymes accelerating polypeptide hydrolysis function according to this principle. 

Polyethylene (Section 6 21) A polymer of ethylene Polymer (Section 6 21) Large molecule formed by the repeti tive combination of many smaller molecules (monomers) Polymerase chain reaction (Section 28 16) A laboratory method for making multiple copies of DNA Polymerization (Section 6 21) Process by which a polymer is prepared The principal processes include free radical cationic coordination and condensation polymerization Polypeptide (Section 27 1) A polymer made up of many (more than eight to ten) amino acid residues Polypropylene (Section 6 21) A polymer of propene Polysaccharide (Sections 25 1 and 25 15) A carbohydrate that yields many monosacchande units on hydrolysis Potential energy (Section 2 18) The energy a system has ex elusive of Its kinetic energy... 

The differences in the amino acid chemistry of the hide coUagen and the hair keratin are the basis of the lime-sulfide unhairing system. Hair contains the amino acid cystine. This sulfur-containing amino acid cross-links the polypeptide chains of mature hair proteins. In modem production of bovine leathers the quantity of sulfide, as Na2S or NaSH, is normally 2—4% based on the weight of the hides. The lime is essentially an unhmited supply of alkah buffered to pH 12—12.5. The sulfide breaks the polypeptide S—S cross-links by reduction. Unhairing without sulfide may take several days or weeks. The keratin can be easily hydrolyzed once there is a breakdown in the hair fiber stmcture and the hair can be removed mechanically. The coUagen hydrolysis is not affected by the presence of the sulfides (1—4,7). 

Protein Components. The simplest picture of the proteinaceous components is one of polypeptides, which are composed of a-amino acid residues. It is estimated that wool contains about 170 different types of polypeptides varying in molecular mass from below 10,000 to greater than 50,000 (34). Complete acid hydrolysis of wool yields 18 amino acids, the relative amounts of which vary considerably from one wool to another. Typical figures for two different samples of wool are given in Table 7. 

Carboxypeptidases are zinc-containing enzymes that catalyze the hydrolysis of polypeptides at the C-terminal peptide bond. The bovine enzyme form A is a monomeric protein comprising 307 amino acid residues. The structure was determined in the laboratory of William Lipscomb, Harvard University, in 1970 and later refined to 1.5 A resolution. Biochemical and x-ray studies have shown that the zinc atom is essential for catalysis by binding to the carbonyl oxygen of the substrate. This binding weakens the C =0 bond by... 

Figure 11.4 Serine proteinases catalyze the hydrolysis of peptide bonds within a polypeptide chain. The bond that is cleaved is called the scissile bond. (Ra) and (Rb)j/ represent polypeptide chains of varying lengths.

The most recent advance in treating HIV infections has been to simultaneously attack the virus on a second front using a protease inhibitor. Recall from Section 27.10 that proteases are enzymes that catalyze the hydrolysis of proteins at specific points. When HIV uses a cell s DNA to synthesize its own proteins, the initial product is a long polypeptide that contains several different proteins joined together. To be useful, the individual proteins must be separated from the aggregate by protease-catalyzed hydrolysis of peptide bonds. Protease inhibitors prevent this hydrolysis and, in combination with reverse transcriptase inhibitors, slow the reproduction of HIV. Dramatic reductions in the viral load in HIV-infected patients have been achieved with this approach. 

Remember that, by convention, the NH2 group of each amino acid is written at the left the COOH group is at the right Other reagents split the chain at different points. By correlating the results of different hydrolysis experiments, it is possible to deduce the primary structure of the protein (or polypeptide). Example 23.10 shows how this is done in a particularly simple case. 

A certain polypeptide is shown by arid hydrolysis to contain only three amino acids serine(Ser), alanine(Ala), and methionine(Met) with mole fractions of j, and respectively. Enzymatic hydrolysis yields the following fragments Ser-Ala Ser-Met Ala-Ser. Deduce the primary structure of the polypeptide. 

Deduce the structure of a polypeptide from hydrolysis data. 

On complete hydrolysis, a polypeptide gives two alanine, one leucine, one methionine, one phenylalanine, and one valine residue. Partial hydrolysis gives the following fragments Ala-Phe, Leu-Met, Val-Ala, Phe-Leu. It is known that the first amino acid in the sequence is valine and the last one is methionine. What is the complete sequence of amino acids ... 

Chymotrypsin-like proteinases are serine proteinases that recognize pqDtide residues with aromatic side chains (phyenylalanyl or tyrosyl residues) and that effect hydrolysis of the polypeptide chain on the carboxy-terminal side of these residues. Examples of chymotrypsin-like proteinases are chymotrypsin and cathepsin-G. 

It is not practical to sequence more than about 50 amino acid residues on a single protein in this way and larger proteins need to be broken down into polypeptides with more appropriate lengths to allow complete sequencing to be carried out. This shortening of the polypeptide chain may be carried out using chemical or enzymatic methods, cf. hydrolysis. 

Assuming the sequence of the parent protein is known, it is not necessary to redetermine the whole sequence merely to locate, and sequence, that/those polypeptide(s) that have undergone modification. This can be done by examination of the total-ion-current (TIC) trace before and after protein hydrolysis for the appearance of new polypeptides or to use mass spectrometry methodology to locate those polypeptides that contain certain structural features. Examples are provided here of both methodologies. 

The versatility of the reagent system in the assay of small peptides is nicely illustrated in Figures 3 through 5. Neurotensin, a polypeptide composed of 13 amino acids, and three fragments from partial hydrolysis were derivatized with NDA/CN and separated by... 

---