JPH0137118B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to dihydrofluorate reductase that has been treated so that it can be stored stably for a long period of time as a lyophilized product. dihydrofluorate reductase. Dihydrofluorate reductase is a substance useful as a reagent used to measure the blood concentration of the antifolate methotrexate administered to cancer patients. This substance is commercially available as a freeze-dried powder in a sealed bottle, and is an unstable enzyme substance that must be kept at low temperatures during storage and transportation. Furthermore, even if the present enzyme is simply freeze-dried, the enzyme activity decreases during the operation. As a result of intensive research, the present inventors discovered that the stability of the present substance can be greatly improved by adding albumin to the substance and freeze-drying the substance, thereby completing the invention. The amount of albumin added is not particularly limited, but
0.002 to 0.5% is appropriate. Example 1 Ice-cold 0.05M Tris buffered solution containing 0.1M KCl (pH 7.5, adjusted with 6NHCl) to achieve the desired enzyme concentration for the enzyme dihydrofluorate reductase
Dissolve, prepare, and examine enzyme activity. (under ice-cooled stirring) of the buffer volume used in the above solution.
Add and dissolve albumin at a ratio of 0.1% (W/V). Dispense the desired dose into each vial in a cold room and pre-freeze at -70° to -80°C for about 6 hours. Lyophilize for 24 hours and seal the vial with a rubber stopper under vacuum to obtain a lyophilized product of dihydrofluorate reductase. Example 2 The effect of adding albumin to prevent the decrease in activity due to freeze-drying of an enzyme solution was investigated. The results are shown in the table. The albumin-free enzyme decreases by approximately 20% (average activity is 80.3% of pre-freezing activity) just by freezing to -10°C, and by approximately 40% (average activity is 59.5% of pre-lyophilizing activity) when further freeze-dried. This caused a decrease in activity. In contrast, albumin, 0.02%, 0.1%,
The average activity of the enzyme lyophilized with the addition of 0.5% was 96.9%, 106.4%, and 102.9%, respectively, and no decrease due to lyophilization was observed by adding albumin. Enzyme activity was determined by absorbance measurement, and the decrease in absorbance over 4 minutes and the activity rate of the treated sample relative to the pre-frozen enzyme sample were determined. The enzyme activity values in the table were measured for enzyme preparations without albumin addition and for those that were freeze-dried after adding albumin at various concentrations.

[Table] Bovine serum albumin was used as albumin.
Three enzyme standard samples were examined.
Example 3 The stability of an enzyme containing 0.1% albumin was compared with a commercially available product containing no additive. The results are shown in the table.
Enzyme activity was 87 after 1 month with no albumin added.
% decreased to 79% after 3 months, whereas the albumin-added product according to the present invention showed an activity of 98% or more even after 3 months. All samples used were freeze-dried products packed in sealed bottles. Enzyme activity was expressed by absorbance measurement as a decrease in absorbance over 2 minutes. The enzyme activity values in the table are the average values obtained by repeatedly measuring three samples of each sample three times.

【table】

[Table] Sample number 1: Commercial product without albumin addition
Sample numbers 2 and 3: Albumin addition
As albumin, 0.1% bovine serum albumin was used.

Claims (1)

[Claims] 1. A stabilized dihydrofluorate reductase, which is obtained by freeze-drying an aqueous solution of dihydrofluorate reductase to which albumin has been added.