CN104165846A - Calculation method for purity of phycoerythrin - Google Patents
Calculation method for purity of phycoerythrin Download PDFInfo
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- CN104165846A CN104165846A CN201310194791.1A CN201310194791A CN104165846A CN 104165846 A CN104165846 A CN 104165846A CN 201310194791 A CN201310194791 A CN 201310194791A CN 104165846 A CN104165846 A CN 104165846A
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Abstract
The invention relates to a calculation method for purity of phycoerythrin, which belongs to the technical field of expression methods and calculation of the purity of fluorescin. The method is characterized by comprising the following steps: 1) separating and extracting phycoerythrin from marine red alga; 2) carrying out scanning in a whole range of 280 to 800 nm by using an ultraviolet-visible light photometer to obtain the absorption spectrogram of a phycoerythrin solution, wherein a scanning step is 1.0 to 5.0 nm; 3) respectively calculating the areas of a region A, a region B and a region C enclosed by the light absorption values of the phycoerythrin solution and the abscissa number axis in the wavelength scanning interval from 280 to 800 nm according to a figure 1 as shown in the specification; and 4) formulating calculation of the purity P (%) of phycoerythrin, wherein since the area of the region B represents the absorption spectrogram typical of and unique to phycoerythrin and the areas of the region A and the region C represent the absorption spectrogram of other impurity proteins, the purity P (%) of phycoerythrin can be conveniently and accurately calculated by using the formula that P (%) is equal to 100 * the area of the region B/(the area of the region A + the area of the region B + the area of the region C). The calculation method for the purity of phycoerythrin in the invention has the advantages of easiness, convenience and high accuracy and is applicable to acquisition of purity values of different purified phycoerythrin samples and comparison and assessment of advantages and disadvantages of different purified phycoerythrin products.
Description
Technical field
The present invention relates to a kind of computing method of phycoerythrin purity.Belong to fluorescin purity expression and computing technique field.
Background technology
In the cell body of some marine algae, except containing the common chlorophyll a of plant, also contain the special water-soluble auxiliary daylighting pigment of a class, be called phycobniliprotein (Phycobiliprotein), mainly comprise phycoerythrin (Phycoerythrin, PE), phycocyanin (Phycocyanin, PC), phycoerythrocyanin (pec) (Phycoerythrocyanin PEC) and allophycocyanin (Allo-phycocyanin, APC).Phycobniliprotein is not only important Photosynthesis Pigment albumen in frustule body, and has many-sided function and using value widely.In phycobniliprotein, phycoerythrin is most important fluorescin in phycobniliprotein, there is nontoxicity, molar extinction coefficient is large, fluorescence quantum yield is high, stoke shift is large, reddish orange characteristic fluorescence, bias light disturb little, be difficult for cancellation, stable in properties can longer-term preservation etc. feature, be the label that a kind of fluorescent specific is strong.Therefore, phycoerythrin is with a wide range of applications in research fields such as clinical diagnose, immunochemistry and bioengineering.Have hyperfluorescenceZeng Yongminggaoyingguang phycoerythrin molecule can with special antibody or other biomolecule generation combination, when this combination state antibody is attached to the special acceptor site of cell or tissue, the specificity fluorescent that is easy to send by them carries out tracing observation, can be used as respond well fluorescent tracing thing, and this combination state thing to biosome itself without any toxic side effects and infringement, there is well applicable security.Nearest research shows, phycoerythrin can be used as effective photosensitizer, is used for the treatment of tumour, cancer etc.Have many reports and point out, phycoerythrin can effectively be removed the various harmful free radical in body, improves immunity of organisms, has radioresistance, anti-ageing, antitumor action, and its sample markets is large, is the health care product that the current utmost point has development potentiality.In addition, phycoerythrin also can be used as adjuvant, can be widely used in varieties of food items and cosmetic industry, has the effect that strengthens the beautiful color of food or beauty care.According to the marked price of the supplier Sigma company of global biochemical samples maximum, the current market price of highly purified phycoerythrin has not reached 200-800 dollar/milligram not etc., and the price of the ready-made article of some bioprotein molecule and phycoerythrin bond is higher.
Separation and Extraction phycoerythrin from marine red alga, because the algae source material that relates to is different, or due to the separation and Extraction, purification process and the technology property of there are differences that adopt, thereby certainly exists obtained phycoerythrin purity and has very large difference.Because the purity of phycoerythrin relates to the fluorescence property of phycoerythrin, be a very important physical and chemical index value, be directly connected to institute's separation and Extraction phycoerythrin at qualitative superiority-inferiority.To so far, express traditionally and what calculate that the phycoerythrin purity of separation and Extraction adopts is to determine phycoerythrin at the light absorption value at 280nm, 565nm and 625nm wavelength, use A
565/ A
280ratio and A
625/ A
565than the phycoerythrin purity of value representation separation and Extraction.But the method for this expression and calculating phycoerythrin purity is a kind of very rough phycoerythrin purity calculated value, because be only, with the light absorption value on 3 single wavelengths, expresses phycoerythrin purity.In order to reflect more comprehensively, rationally, exactly the phycoerythrin purity of separation and Extraction, need to set up a kind of method of better expression and calculating phycoerythrin purity.
Summary of the invention
Technical matters to be solved by this invention is exactly for background technology recited above, adopt accurate, suitable computing method, obtain the Reinheitszahl P (%) of phycoerythrin sample, according to the Reinheitszahl size of calculating, the otherness of the different phycoerythrin samples that can compare easily and accurately separation and Extraction aspect purity.
The technical solution used in the present invention and technical characterictic:
Get 4 milliliters of the phycoerythrin sample solutions of separation and purification from marine red alga, with ultraviolet-visible spectrophotometer, from the omnidistance scanning of 280-800nm, scanning step 1.0-5.0nm, obtains the abosrption spectrogram of phycoerythrin solution, sees shown in accompanying drawing 1.As shown in Figure 1, calculate respectively by the light absorption value of phycoerythrin solution and horizontal ordinate number axis at interval a-quadrant area, B region area and the C region area that encloses bag of 280-800nm length scanning.A branch be phycoerythrin solution in the absorption value of 280nm wavelength light, b branch is the absorption value of phycoerythrin solution when just starting to occur its typical absorption spectra, the absorption value that c branch is phycoerythrin solution when finishing to occur its typical absorption spectra.The absorption value that phycoerythrin solution obtains through ultraviolet-visible spectrophotometer scanning is transferred to behind Excel interface, implements the calculating of each region area.The implication of the respective area that it is concrete and calculating operation process are as follows:
The implication of a-quadrant area and calculating: a-quadrant area refers to from a branch to b branch, the area that the light absorption value of phycoerythrin solution and horizontal ordinate surround.In fact a-quadrant area has represented that in the phycoerythrin solution of separation and Extraction, the optical absorption spectra of small molecular weight impurity albumen and some other impurity molecule forms.The computing method of its area are: when scanning phycoerythrin solution, the light absorption value that under every adjacent two step-lengths, (step-length is 1.0-5.0nm) obtains is as calculating the upper base of each small trapezoidal area of this a-quadrant and going to the bottom, and two adjacent step-lengths are exactly the height while calculating each small trapezoidal area.Calculate small trapezoidal areas all in a-quadrant, calculate the total value that small trapezoidal areas all in a-quadrant is added.
Implication and the calculating of B territory area: B region area refers to from b branch to c branch, the area that the light absorption value of phycoerythrin solution and horizontal ordinate surround.In fact B territory area is exactly to have represented that typical case, distinctive phycoerythrin absorption spectrum form.The computing method of its area are: when scanning phycoerythrin solution, the light absorption value that under every adjacent two step-lengths, (step-length is 1.0-5.0nm) obtains is as calculating the upper base of this each small trapezoidal area of B region and going to the bottom, and two adjacent step-lengths are exactly the height while calculating each small trapezoidal area.Calculate small trapezoidal areas all in B region, calculate the total value that small trapezoidal areas all in a-quadrant is added.
The implication of C region area and calculating: C region area refers to from c branch to 800nm wavelength, the area that the light absorption value of phycoerythrin solution and horizontal ordinate surround.In fact C region area represented in the phycoerythrin solution of separation and Extraction, and other phycobniliprotein that may exist (for example phycocyanin) absorption spectrum forms.The computing method of its area are: when scanning phycoerythrin solution, the light absorption value that under every adjacent two step-lengths, (step-length is 1.0-5.0nm) obtains is as calculating the upper base of this each small trapezoidal area of C region and going to the bottom, and two adjacent step-lengths are exactly the height while calculating each small trapezoidal area.Calculate small trapezoidal areas all in C region, calculate the total value that small trapezoidal areas all in C region is added.
Obtain after the numerical value of above-mentioned 3 region areas, then with formula, calculate purity P (%)=100*B region area/(a-quadrant area+B region area+C region area) of the phycoerythrin solution of separation and Extraction.
Accompanying drawing explanation
Fig. 1 is the scan light spectrogram of phycoerythrin of the present invention, and while calculating phycoerythrin purity, the determining of the area territory that the different material that the scanning optical spectrum of phycoerythrin presents occupies.Be determining of a-quadrant area: from a branch to b branch, the area that the light absorption value of phycoerythrin solution and horizontal ordinate surround, has represented that, in the phycoerythrin of separation and Extraction, the optical absorption spectra of small molecular weight impurity albumen and some other impurity molecule forms.Determining of B region area: from b branch to c branch, the area that the light absorption value of phycoerythrin solution and horizontal ordinate surround, has represented typical case, distinctive phycoerythrin absorption spectrum.Determining of C region area: from c branch to 800nm wavelength, the area that the light absorption value of phycoerythrin solution and horizontal ordinate surround, has represented in the phycoerythrin of separation and Extraction, and other phycobniliprotein that may exist (for example phycocyanin) absorption spectrum forms.
Embodiment and enforcement illustration
Below in conjunction with embodiment, the invention will be further described.Following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.The computing method that the present invention proposes, equally applicable to other phycobniliprotein, phycocyanin for example, the purity of allophycocyanin is calculated.
Specific embodiment card 1:
Get 4 milliliters of phycoerythrin sample 1 solution of separation and purification from marine red alga, use ultraviolet-visible photometer, from the omnidistance scanning of 280-800nm, scanning step 1.0nm, obtain the abosrption spectrogram of sample 1 phycoerythrin solution, record this Sample Purification on Single liquid and be respectively 0.6915,1.5884,0.5366 at the light absorption value of 280nm, 565nm and 625nm wavelength; Purity ratio A
565/ A
280be 2.2970, purity ratio A
625/ A
565be 0.3378.According to computing method of the present invention, the a-quadrant area that abosrption spectrogram is calculated is that 51.81, B region area is that 146.22, C region area is 12.43, and this sample phycoerythrin purity calculating is 69.47%.
Specific embodiment card 2:
Get 4 milliliters of phycoerythrin sample 2 solution of separation and purification from marine red alga, use ultraviolet-visible photometer, from the omnidistance scanning of 280-800nm, scanning step 5.0nm, obtain the abosrption spectrogram of sample 1 phycoerythrin solution, record this Sample Purification on Single liquid and be respectively 0.2333,0.3508,0.0555 at the light absorption value of 280nm, 565nm and 625nm wavelength; Purity ratio A
565/ A
280be 1.5036, purity ratio A
625/ A
565be 0.1582.According to computing method of the present invention, the a-quadrant area that abosrption spectrogram is calculated is that 20.29, B region area is that 36.58, C region area is 4.23, and this sample phycoerythrin purity calculating is 59.86%.
Specific embodiment card 3:
Get 4 milliliters of phycoerythrin sample 3 solution of separation and purification from marine red alga, use ultraviolet-visible photometer, from the omnidistance scanning of 280-800nm, scanning step 1.0nm, obtain the abosrption spectrogram of sample 1 phycoerythrin solution, record this Sample Purification on Single liquid and be respectively 0.1265,0.2781,0.0147 at the light absorption value of 280nm, 565nm and 625nm wavelength; Purity ratio A
565/ A
280be 2.1984, purity ratio A
625/ A
565be 0.0529.According to computing method of the present invention, the a-quadrant area that abosrption spectrogram is calculated is that 9.626, B region area is that 27.19, C region area is 0.97, and this sample phycoerythrin purity calculating is 71.96%.
Specific embodiment card 4:
Get 4 milliliters of phycoerythrin sample 4 solution of separation and purification from marine red alga, use ultraviolet-visible photometer, from the omnidistance scanning of 280-800nm, scanning step 1.0nm, obtain the abosrption spectrogram of sample 1 phycoerythrin solution, record this Sample Purification on Single liquid and be respectively 0.0796,0.2337,0.0005 at the light absorption value of 280nm, 565nm and 625nm wavelength; Purity ratio A
565/ A
280be 2.9359, purity ratio A
625/ A
565be 0.0021.According to computing method of the present invention, the a-quadrant area that abosrption spectrogram is calculated is that 5.495, B region area is that 19.21, C region area is 0.01, and this sample phycoerythrin purity calculating is 77.82%.
Specific embodiment card 5:
Get 4 milliliters of phycoerythrin sample 5 solution of separation and purification from marine red alga, use ultraviolet-visible photometer, from the omnidistance scanning of 280-800nm, scanning step 1.0nm, obtain the abosrption spectrogram of sample 1 phycoerythrin solution, record this Sample Purification on Single liquid and be respectively 0.3107,1.4598,0.0195 at the light absorption value of 280nm, 565nm and 625nm wavelength; Purity ratio A
565/ A
280be 4.6984, purity ratio A
625/ A
565be 0.0134.According to computing method of the present invention, the a-quadrant area that abosrption spectrogram is calculated is that 23.59, B region area is that 119.75, C region area is 0.6965, and this sample phycoerythrin purity calculating is 83.14%.
Tool the present embodiment card 6:
Get 4 milliliters of phycoerythrin sample 6 solution of separation and purification from marine red alga, use ultraviolet-visible photometer, from the omnidistance scanning of 280-800nm, scanning step 5.0nm, obtain the abosrption spectrogram of sample 1 phycoerythrin solution, record this Sample Purification on Single liquid and be respectively 0.1483,0.8348,0.0046 at the light absorption value of 280nm, 565nm and 625nm wavelength; Purity ratio A
565/ A
280be 5.6291, purity ratio A
625/ A
565be 0.0055.According to computing method of the present invention, the a-quadrant area that abosrption spectrogram is calculated is that 10.06, B region area is that 67.41, C region area is 0.055, and this sample phycoerythrin purity calculating is 86.95%.
Specific embodiment card 7:
Get 4 milliliters of phycoerythrin sample 7 solution of separation and purification from marine red alga, use ultraviolet-visible photometer, from the omnidistance scanning of 280-800nm, scanning step 1.0nm, obtain the abosrption spectrogram of sample 1 phycoerythrin solution, record this Sample Purification on Single liquid and be respectively 0.2928,1.4943,0.0098 at the light absorption value of 280nm, 565nm and 625nm wavelength; Purity ratio A
565/ A
280be 5.1035, purity ratio A
625/ A
565be 0.0066.According to computing method of the present invention, the a-quadrant area that abosrption spectrogram is calculated is that 20.13, B region area is that 117.76, C region area is 0.5902, and this sample phycoerythrin purity calculating is 85.03%.
From the result of calculation of above enforcement illustration, clearly illustrate that, the computing method that propose according to patent of the present invention, can obtain the phycoerythrin Reinheitszahl information state of different purification of samples, B region area is larger, a-quadrant area and C region area are less, the purity of the phycoerythrin sample obtaining is higher, and such product quality is just better.Above-mentioned purity result of calculation shows that the purity of sample 6 phycoerythrin is the highest, reaches 86.95%; And the purity of sample 2 phycoerythrin is minimum, be only 59.86%; The Reinheitszahl of other sample phycoerythrin is between the highest and minimum.Thereby the method that adopts patent of the present invention to propose, can calculate easily and accurately the Reinheitszahl of sample phycoerythrin, and can, according to the Reinheitszahl situation of different samples, evaluate the superiority-inferiority of phycoerythrin product quality.
Claims (1)
1. the computing method of a phycoerythrin purity, it is characterized in that: from marine red alga, separation and Extraction goes out phycoerythrin, with ultraviolet-visible spectrophotometer, from the omnidistance scanning of 280-800nm, scanning step 1.0-5.0nm, obtain the abosrption spectrogram of phycoerythrin solution. according to the abosrption spectrogram obtaining, from a branch (280nm), start to 800nm, the region area of absorption spectrum and bag that horizontal ordinate encloses is divided into 3 region areas, be divided into a-quadrant area, B region area and C region area. wherein a-quadrant area refers to from a and divides the b branch of lighting to just starting to occur typical phycoerythrin absorption spectra, the region area of absorption spectra and bag that horizontal ordinate encloses, this region area has represented in the phycoerythrin of separation and Extraction, the optical absorption spectra of small molecular weight impurity albumen and some other impurity molecule forms.B region area refers to from just starting to occur that the b branch of typical phycoerythrin absorption spectra is to the end point c branch of typical phycoerythrin absorption spectra, the region area of absorption spectra and bag that horizontal ordinate encloses, this region area has represented that typical case, distinctive phycoerythrin absorption spectrum form.C region area refers to from c and divides and light to 800nm, the region area of absorption spectra and bag that horizontal ordinate encloses, and this region area represented in the phycoerythrin of separation and Extraction, other phycobniliprotein that may exist (for example phycocyanin) absorption spectra forms.Using the light absorption value that obtains under every adjacent two step-lengths as calculating the upper base of each small trapezoidal area of regional and going to the bottom, the height of step-length when calculating each small trapezoidal area.All small trapezoidal area in summation regional, obtains respectively a-quadrant area, B region area and C region area.The calculation expression of the purity P of phycoerythrin (%) is P (%)=100*B region area/(a-quadrant area+B region area+C region area).
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