A Naphthyl phosphate

An enzyme-amplified detection scheme, based on tire coupling of a streptavidin-alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalysed the hydrolysis of the elecn oiiractive a-naphthyl phosphate to a-naphtlrol this product is elecU oactive and has been detected by means of differential... 

Babson proposed a-naphthyl phosphate as an essentially specific substrate for the activity of prostatic acid phosphatase in serum (104). However Marshall, Price, and Amador found that this substrate is not specific for the prostatic enzyme because urine of human females contain 50 times more acid a-naphthyl phosphatase than male serum and 50% as much activity as male urine. Platelets have significant activity and the serum activity can increase to abnormal values following clotting. These workers also observed elevated activities in females with skeletal metastases of the breast. In 50 hospitalized male patients who had no evidence of prostatic cancer and 25 hospitalized female patients, the incidence of false positive results was 12%, a magnitude sufficient to preclude meaningful clinical interpretation (105). 

A disposable electrochemical enzyme-amplified genosensor was described for specific detection of Salmonella (Del Giallo et al., 2005). A DNA probe specific for Salmonella was immobilized onto screen-printed carbon electrodes and allowed to hybridize with a biotinylated PCR-amplified product of Salmonella. The hybridization reaction was detected using streptavidin conjugated-AP where the enzyme catalyzed the conversion of electroinactive a-naphthyl phosphate to electroactive a-naphthol, which was detected by differential pulse voltammetry. 

The iron(II)-iron(III) form of purple acid phosphatase (from porcine uteri) was kinetically studied by Aquino et al. (28). From the hydrolysis of a-naphthyl phosphate (with the maximum rate at pH 4.9) and phosphate binding studies, a mechanism was proposed as shown in Scheme 6. At lower pH (ca. 3), iron(III)-bound water is displaced for bridging phosphate dianion, but little or no hydrolysis occurs. At higher pH, the iron(III)-bound OH substitutes into the phosphorus coordination sphere with displacement of naphthoxide anion (i.e., phosphate hydrolysis). The competing affinity of a phosphomonoester anion and hydroxide to iron(III) in purple acid phosphatase reminds us of a similar competing anion affinity to zinc(II) ion in carbonic anhydrase (12a, 12b). 

Most investigators utilize p-nitrophenyl or a-naphthyl phosphate as substrate. The determination of serum prostatic acid phosphatase was developed by Fishman and Lemer (34) based on the d-(+)-tartrate inhibition of prostatic enzyme discussed below. Babson et al. (35, 36) demonstrated that a-naphthyl phosphate was much more easily split by prostatic than red cell phosphatase. Table V (35) shows the results obtained when prostatic or red cell phosphatase was added to human serum which had been incubated at pH 8.6 for 1 hr at 37° to destroy all endogeneous phosphatase activity. The table shows the superiority of a-naphthyl phosphate as substrate. 

A spectrofluorometric method for the estimation of acid phosphatase has been devised. It uses a-naphthyl phosphate as substrate thus, it is somewhat more specific for prostatic acid phosphatase than most (37). 

The purple acid phosphatases (PAPs) are a class of phosphoprotein phosphatases which possess a p-oxo(hydroxo)-bridged dinuclear iron centre. An enzyme has been isolated from beef spleen which is purple in colour, while a violet phosphatase has been characterised from red kidney beans (KBPase). This latter enzyme consists of two subunits with M = 58200 and contains two equivalents of Zn(II) and Fe(III) per dimer which are essential for catalytic activity. KBPase hydrolyses nucleosidetriphos-phates as well as activated phosphomonoesters such as 4-nitrophenylphosphate or a-naphthyl phosphate (Beck et al., 1986). As with the beef spleen enzyme, KBPase is inhibited by tetrahedral oxoanions such PO and AsO . 

The pattern of hydrolysis of various substrates by these three isoenzymes also showed marked differences. When the velocity of hydrolysis of p-nitrophenyl phosphate was arbitrarily set at 100, the rates for isoenzyme I were a-naphthyl phosphate, 59 pyridoxine 5-phosphate, 40. 

Procedure for Estimation of Alkaline Phosphatase Activity with a-Naphthyl Phosphate as Substrate... 

Fia. 12. Lineweaver-Burk plot relating the reciprocals of velocity and substrate concentration. For human intestine the substrate was a-naphthyl phosphate, and the L-phenylalanine concentration was 0.005 M. For human placenta the substrate was phenyl phosphate, and the L-phenylalanine concentration was 0.0025 M. 

Most of the substrates that have been used in measuring alkaline phosphatase activity have also been used to measure acid phosphatase, e.g. p-nitrophenylphosphate, phenylphosphate and a-naphthyl phosphate. In most cases of acid phosphatase estimation, it is the level of the prostatic phosphatase which needs to be known, and so specific inhibitors are included in the reaction mixtures. The prostatic isoenzyme is strongly inhibited by tartrate and so in many methods it is tartrate-labile acid phosphatase which is measured. On the pther hand, the red cell enzyme which contributes significantly to the total serum activity is inhibited by formaldehyde and cupric ions. Many laboratories therefore measure formaldehyde-stable acid phosphatase as an indication of prostatic acid phosphatase. 

Bettazzi et al. used an electrochemical low-density DNA array in combination with polymerase chain reaction (PCR) in order to investigate the presence of hazelnut major allergens. Cor a 1.04 and Cor a 1.03, in foodstuff. Unmodified PCR products were captured at the electrode interface via sandwich hybridization with surface-tethered probes and biotinylated signaling probes. The resulting biotinylated hybrids were coupled to a streptavidin-alkaline phosphatase conjugate and then exposed to an a-naphthyl phosphate solution. Differential pulse voltammetry (DPV) was used to detect the a-naphthol signal with detection limits as 0.3 and 0.1 nmol for Cor a 1.03 and Cor 1.04, respectively. The results are comparable with the ones obtained with classical ELISA tests. 

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