**6.1 Glucose oxidase (Amaral et al., 2007)**

The enzyme glucose oxidase is used for quantitative and enzymatic determination of the glucose in food and other materials. The enzyme glucose oxidase test is widely used because it is cheap, stable, and by its specificity well established for glucose.

In this reaction, the glucose is oxidized to gluconic acid and hydrogen peroxide by enzyme glucose oxidase. The hydrogen peroxide reacts with ortho-dianisidine in the presence of peroxidase enzyme to form a colored product (Figure 3). The compound of the orthodianisidine oxidation reacts with sulphuric acid to form a coloured product more stable. The intensity of pink colour measured in 540nm is proportional to the glucose concentration in the sample.

Fig. 3. Reactions of the enzymatic method of glucose oxidase.

#### **6.2 Hexokinase method for simulteneous determination of glucose and fructose (Moerman et al., 2004)**

This method is adequate for determination of monosaccharides glucose and fructose.

The principal enzyme of the method is hexokinase. This enzyme catalyzes the fosforilation of the glucose in glucose-6-phosphate, here upon the second enzyme the glucose-6 phosphate dehydrogenase together with cofactor nicotinamide adenine dinucleotide (NAD) oxidized glucose-6-phosphate to gluconate-6-phosphate and the NAD is reduced to NADH,

Getting an aliquot 80µg as maximum of the sample, add 0.5mL resorcinol reagent, shake in vortex, add 3.5mL hydrochloride acid, shake again, let in water bath at 80°C for 10 minutes,

There are several enzymatic methods for determination of the three principal sugars individually present in nectar - fructose, glucose, and sucrose, and in biological samples like plasma, blood, and urine. A lot of these methods are commercialized in kits and can be used successfully for rapid determination of the sugar from natural products samples. These kits

The enzyme glucose oxidase is used for quantitative and enzymatic determination of the glucose in food and other materials. The enzyme glucose oxidase test is widely used because

In this reaction, the glucose is oxidized to gluconic acid and hydrogen peroxide by enzyme glucose oxidase. The hydrogen peroxide reacts with ortho-dianisidine in the presence of peroxidase enzyme to form a colored product (Figure 3). The compound of the orthodianisidine oxidation reacts with sulphuric acid to form a coloured product more stable. The intensity of pink colour measured in 540nm is proportional to the glucose concentration in

after that, read the absorbance in spectrophotometer at 520nm.

are precise and sensitive, which enable rapid analysis and reliable results.

it is cheap, stable, and by its specificity well established for glucose.

Fig. 3. Reactions of the enzymatic method of glucose oxidase.

**6.2 Hexokinase method for simulteneous determination of glucose and fructose** 

This method is adequate for determination of monosaccharides glucose and fructose.

The principal enzyme of the method is hexokinase. This enzyme catalyzes the fosforilation of the glucose in glucose-6-phosphate, here upon the second enzyme the glucose-6 phosphate dehydrogenase together with cofactor nicotinamide adenine dinucleotide (NAD) oxidized glucose-6-phosphate to gluconate-6-phosphate and the NAD is reduced to NADH,

**6. Enzymatic methods for sugar determination** 

**6.1 Glucose oxidase (Amaral et al., 2007)** 

the sample.

**(Moerman et al., 2004)** 

according to stoichiometry of the second reaction (Figure 4), the spectrophotometric quantity of NADH is corresponding to glucose quantity.

Analysis of glucose is according to the following principle: hexokinase, a first enzyme, catalyzes the phosphorylation of glucose to glucose-6-phosphate, with the participation of the enzyme glucose-6-phosphate dehydrogenase and nicotinamide adenine dinucleotide (NAD) is further specifically oxidized to gluconate-6-phosphate. According to the stoichiometry of the last reaction (Figure 4), the photospectrometrically quantified amount of reduced nicotinamide adenine dinucleotide (NADH) is representative for the amount of glucose. Fructose is always determined subsequently to the glucose determination. Fructose undergoes phosphorylation to fructose-6-phosphate, with the same enzyme hexokinase, which is further converted to glucose-6-phosphate with phosphoglucose isomerase. Further oxidation to gluconate-6-phosphate as described above generates a supplementary amount of NADH that is stoichiometric with the amount of fructose.

All methodology of this analysis is performed following instructions of each enzymatic kit. For these enzymatic analysis must carried out the assays criteriously, because the order of addiction of reagents, the time of analysis and reading on spectrophotometer are determinants for an adequate analysis.

Fig. 4. Enzymatic reactions in enzymatic methods using hexokinase.

Spectrophotometry as a Tool for Dosage Sugars in Nectar of Crops Pollinated by Honeybees 281

opened flowers had generally only glucose and fructose. These reports confirmed early researches (Wykes, 1953; Bailey et al., 1954) that suggested a relation between three monosaccharides and different species with flower. In another research, the nectar was divided in four different classes in function to sucrose/hexose rate - S/H: sucrose dominant - S/H >0.999, rich in sucrose - 0.5<0.999, rich in hexose - 0.1<0.499 and hexose dominant -

In the research of Alves (2004) and Alves et al. (2010), the means of sucrose.hexose-1 (S/H) per flower for all treatments were: 0.91μg.μL-1, for covered area with Africanized honeybee colony – rich in sucrose; 0.74μg.μL-1, semicovered area with free insects visitation – rich in sucrose; 0.86μg.μL-1, uncovered area with free insect visitation – rich in sucrose; and 3.05μg .μL-1, for covered area without Africanized honeybee colony – sucrose dominant. However, Severson e Erickson (1984) reported in several cultivars of soybean values from 1.2:1.0:1.4 to 1.2:1.0:6.7, with sucrose predominance, which sucrose concentration in nectar ranged from 97 to 986μg.μL-1, these means are higher than those reported by Alves et al. (2010) who found 12.06μg.μL-1. This range suggests that sugar concentration in soybean nectar is influenced by other environment factors independently of pollinator action. Robacker et al. (1983) reported that edaphic and climatic factors affect the number of flowers and another floral characteristics during soybean growing. So, the environmental conditions that generate an increase in number and size of flowers, higher anthesis period, colourness more intense, and also greater nectar production are the factors responsible by became flowers

Cruden et al. (1983) suggested that the maximum nectar accumulation occurs before or at the beginning of pollination activity. Such fact can be verified in siratro, since the highest sugar concentration was found at 8:30 a.m. (Figure 5) time in which the bee visitation started (Toledo et al., 2005). Variations in the siratro nectar sugar content measured along the day were observed (Figure 6 – Toledo et al., 2005) and probably associated with the intensity of foraging by honeybees, which is directly related to the nectar quantity and quality (Heinrich,

In flowers exposed to pollinators, it is possible that nectar secretion ceases if there is not pollinator in the area or can be reabsorbed in old or pollinated flowers (Cruden et al., 1983). A nectar production without reabsorption may be have an impact on reproductive biology (Galleto & Bernardello, 1995). Therefore, plants reabsorb nectar from aging flowers and utilize its carbon in developing seeds and this is a reproductive advantage (Zimmerman,

Chiari et al. (2005) studying the pollination of Africanized honeybees on soybean flowers (*Glycine max* L. Merrill) var. BRS 133, measured through the manual refractometer the sugar concentration as total solids and concluded that data found presented a big uniformity, different of the results obtained by Sheppard et al. (1978) that observed big variations in these concentrations and attributed these differences to the variation in the soil composition and other environmental conditions, like precipitation. Despite this, the mean values found by Chiari et al. (2005) were 21.33 ± 0.22% in uncovered area and 22.33 ± 0.38% in covered area with honeybees and differed to each other (P=0.0001). Besides, the medium amounts of total sugar and glucose measured in the nectar of the flowers were 14.33 ± 0.96mg/flower

1979; Hagler, 1990) or to its sugar composition (Waller, 1972; Abrol & Kapil, 1991).

and 3.61 ± 0.36mg/flower, respectively, in the same research.

S/H<0.1 (Baker & Baker, 1983).

more attractive to honeybees (Alves et al., 2010).

1988).
