Enzyme oxidation with periodate

Enzymes that contain carbohydrate, such as HRP or GO, may be oxidized with periodate to create reactive derivatives that subsequently can be used to label antibodies or other targeting molecules at their amine groups. The aldehyde-HRP intermediate may be stored for extended periods in a frozen or lyophilized state without loss of activity (either enzymatic or coupling potential). Avoid, however, storage in a liquid state, since polymerization may occur—resulting in precipitation and loss of activity. 

Periodic acid oxidation has proved to be a very useful tool in enzymology since a wide variety of biochemicals contain hydroxyl groups on adjacent carbon atoms. For example, periodate-oxidized ATP (also called adenosine 5 -triphosphate 2, 3 -dialdehyde) has often been used as an alternative substrate or an irreversible inhibitor for a wide variety of ATP-utilizing enzymes. This compound, and many others, are now commercially available, even though they are readily synthesized e.g., periodic acid oxidized ADP, AMP, adenosine, P, P -di(adenosine-5 )pentaphosphate, P, P -di(adenosine-5 )tetraphos-phate, GTP, GDP, GMP, guanosine, CTP, CDP, CMP, etc. In the case of the nucleosides, commercial sources also can supply the dialcohol form of the nucleoside i.e., the nucleoside has first been oxidized with periodic acid and then reduced to the dialcohol with borohydride. 

Poly(ribitol phosphate) synthetase has been found in particulate fractions from Staphylococcus aureus H, and Lactobacillus plantatrum.lt ll-m The bulk of the activity in Lactobacillus plantarum was in crude, cell-wall preparations, and the enzyme is apparently located in the membrane, although intimate association with the wall itself has been suggested. Unlike the natural teichoic acid, the enzymically synthesized ribitol phosphate polymer was readily extracted with phenol hydrolysis by acid and by alkali gave the expected products, and oxidation with periodate indicated a chain length of 5-9 units, a value which compares well with that of 8 units for the natural polymer in the walls of this organism. 

Horseradish peroxidase is coupled to IgG antibody in a two-step procedure. In the first step monosaccharide residues in the enzyme are oxidized with periodate to produce aldehyde groups. Then, in the second step, the aldehyde groups are allowed to react with amino groups in the IgG antibody. The Schiff bases formed are reduced and the conjugate is purified by gel filtration. 

A role for the carbohydrate moieties in the catalytic process of an enzyme was suggested many years ago.24 However, studies with several different enzymes19,149,150 have shown that enzymic activity is affected only slightly if the carbohydrate residues are oxidized with periodate. Furthermore, removal of carbohydrate residues with gly-cosidases,1,15 followed by measurements of activity, has shown that the resulting, modified enzymes had specific activities that were essentially unchanged. With ribonuclease B, it has also been con-... 

D-Glucose Oxidase.— The carbohydrate moieties of D-glucose oxidase from Aspergillus niger have been modified by oxidation with periodate ion. Although ca. 60% of the carbohydrate residues were oxidized at pH 5.6, none of the amino-acid residues was oxidized. The oxidized enzyme retained its full activity and was converted into an immobilized form. 

A -Pyrroline has been prepared in low yield by oxidation of proline with sodium hypochlorite (71), persulfate (102), and periodate (103). A -Pyrroline and A -piperideine are products of enzymic oxidation via deamination of putrescine and cadaverine or ornithine and lysine, respectively (104,105). This process plays an important part in metabolism and in the biosynthesis of various heterocyclic compounds, especially of alkaloids. 

Figure 20.8 Enzymes that are glycoproteins like HRP may be oxidized with sodium periodate to produce reactive aldehyde residues. Conjugation with an antibody then may be done by reductive animation using sodium cyanoborohydride.

Figure 20.14 Periodate oxidation of HRP creates aldehyde groups on the carbohydrate chains of the enzyme. Reaction with a Fab fragment then may be done using reductive amination to produce a lower-molecular-weight complex than would be obtained using intact IgG antibodies.

Figure 23.10 Glycoproteins may be oxidized with sodium periodate to generate aldehyde residues. These may be specifically labeled using a hydrazide-streptavidin derivative through hydrazone bond formation. Subsequent detection may be done using biotinylated enzymes.

The precise technical name of HCN is Hydrocyanic Acid. The cyanides are true protoplasmic poisons, combining in the tissues with the enzymes associated with cellular oxidation. They thereby render the oxygen unavailable to the tissues, and cause death through asphyxia. Inhaling concentrations of more than 180 ppm of HCN will lead to unconsciousness in a matter of minutes, but the fatal effects would normally be caused by carbon monoxide poisoning after HCN has made the victim unconscious. Exposure to HCN concentrations of 100 to 200 ppm for periods of 30 to 60 minutes can also cause death. 

Two aldehydic nucleotide derivatives have found use as affinity labels. The magnesium salt of (64), formed by oxidation of ATP with periodate, is a competitive inhibitor of pyruvate carboxylase with respect to [Mg. ATP2-],100 and (65), obtained from the / -anomer of 5-formyluridine-5 -triphosphate on treatment with alkali, is a non-competitive and reversible inhibitor of DNA-dependent RNA polymerase from E. coli.101 In each case, addition of borohydride gives stoicheiometric covalent linkage of the nucleotide to the enzyme, with irreversible inactivation. It is thought that condensation with lysine occurs to give a Schiff s base intermediate, which undergoes subsequent reduction. 

Carbohydrates are easily oxidized by periodate to aldehydes which react with primary amines or hydrazines. If the formed hydrazide carries a specific ligand, e.g., biotin or digoxigenin, these ligands immobilized via the blotted macromolecule are very sensitively detected by the respective enzyme conjugates. 

Covalent immobilization of lipase on nylon fibers has been done, using the enzymes carbohydrate groups as chemical link. Oxidation of the lipases carbohydrates with periodate provides aldehyde groups for the binding to hydrazide activated nylon (Lopez, Braun Klein, 1996). 

More definite evidence for the transient existence of the un-cyclized l-(jS-aminoethyl)-3,4-benzoquinones has been obtained recently by Kodja and Bouchilloux,77 78 who noted that a transient yellow color (Amax ca. 385 mp) was occasionally observed during the enzymic oxidations of catecholamines (particularly in unbuffered systems at low temperatures). This phenomenon was probably due to the formation of the transient o-quinones. (The absorption maximum of o-benzoquinone, the effective chromophore of the open-chain quinones, is known to occur at ca. 390 mp.79) An absorption maximum at 390 mp is characteristic of the formation of the dopa-quinone chromophore during oxidation of small C -terminal tyrosine peptides in the presence of tyrosinase.37 48 Similar spectroscopic features were observed when the oxidations were carried out with lead dioxide in sulfuric acid solutions (pH> 1). If the initial oxidation was carried out for a short period of time, it was possible to regenerate the original catecholamines by reduction (e.g. with sodium bisulfite, potassium iodide, and zinc powder) and to show that the 385 mp peak disappeared.77,78 Kodja and Bouchilloux were also able to identify 2,4-dinitrophenylhydrazones of several of the intermediate non-cyclized quinones by paper chromatography and spectroscopy (Amax n weakly acid solution ca. 350 mp with a shoulder at ca. 410 mp).77,78... 

A small proportion of O-D-glucosylribitol was produced directly by hydrolysis of the teichoic acid with alkali ( see Fig. 16) this product is identical with that obtained by dephosphorylation of the hydrolysis mixture. The major products of such a hydrolysis with alkali were the isomeric monophosphates (58) and (59), in which R = 0-D-glucopyranosyl, both of which gave the O-D-glucosylribitol on enzymic dephosphorylation. The isomer (58) reduced 3 molar proportions of periodate, and the ribitol residue was oxidized, whereas the isomer (59) reduced 2 molar proportions of periodate, the ribitol residue being resistant to oxidation. Small proportions of the diphosphates (56) and (57) were also produced. Oxidation of the diphosphate (57) with periodate, followed by treatment with alkali to remove the aldehydic residues, gave a ribitol diphosphate. 

---