SciresolSciresolJournal of Pharmaceutical Research2454-840510.18579/jopcr/v20i3.ms21041RESEARCH ARTICLESimultaneous Estimation of Protein and Nucleic Acid Using Derivative Spectrophotometric MethodSimultaneous Estimation of Protein and Nucleic AcidAgrawalAtulartirthakkar@gmail.com arthakkar@amity.edu1KaurKiranjot1SharmaRakesh1GoyalAmit1ThakkarArtiartirthakkar@gmail.com arthakkar@amity.edu1ISF College of PharmacyMoga , Punjab, 142 001IndiaAmity Institute of Pharmacy, Amity University Noida, Uttar Pradesh, 201 313India20312021Abstract
Purpose: Simple and precise first derivative zero crossing spectrophotometric method has been developed for the simultaneous estimation of protein and nucleic acid without prior separation. Approach: The concentration ranges for protein and nucleic acid were taken in the range of 60-140 µg mL-1 and 20-100 µg mL-1 respectively in 0.1 M NaOH. Absorption spectra of the samples were recorded between 200 nm to 400 nm against a reagent blank. Zero-order spectra of protein and nucleic acid were stored individually within the above concentration ranges and were derivatized in first order using scaling factor 5 for both the substances. First derivative amplitudes were recorded at 261 nm and 289 nm for estimation of protein and RNA respectively for pure substances and binary mixture. Finding: Limit of detection was found to be 2.90 µg mL-1 and 0.36 µg mL-1 and limit of quantitation was 8.80 µg mL-1 and 1.02 µg mL-1 respectively for protein and nucleic acid respectively. Precision was found to be 1.38 % for protein and 1.25 % for RNA. Reproducibility was found to be 1.54 % for protein and 1.69 % for RNA. Conclusions: Thus, proposed method can be adapted for simultaneous determination of protein and nucleic acid and better alternate technique for immunoassays and electrophoretic methods.
KeywordsFirst derivative zero crossing spectrophotometryProteinNucleic acidRibonucleic acidAnalytical method validationINTRODUCTION
Nucleic acid and protein analysis holds an important place in the field of life sciences 1 as many downstream changes occurring in each step during genetics, epigenetics, transcriptomics, and proteomics. It is very difficult to perform the qualitative and quantitative measurement of such isolated molecular species, and it is often hindered by the limited availability of sample material. Further, it has been observed that generally nucleic acids and proteins are analysed using Flow Cytometry and Mass Cytometry methods.2, 3, 4 Due to these reasons, there is an increasing interest for simultaneous estimation of nucleic acid and proteins from a single sample. 5 In the present study, we developed a method for the simultaneous estimation of nucleic acid and protein that may be useful for qualitative and quantitative estimation of nucleic acid and protein in a given sample. Through this method the correlation between mRNA expression and its corresponding protein levels can be easily understood. It is a quick and easy procedure for all fields of downstream analysis, especially when using limited sample resources. This method can be very fruitful for estimation of nucleic acid and protein levels in tissues of different ages. It can also be used for over-all screening and functioning of the metabolic machinery of an animal organ by quantitative estimation of nucleic acid and protein. 6, 7
In comparison to chromatographic and electrophoresis methods, spectrophotometric methods are simple, less time consuming and more economic. Under computer-controlled instrumentation, derivative spectrophotometry plays a very important role in the multi-component analysis of mixtures. 8, 9 Binary mixtures can be easily resolved by means of a spectrophotometric method, which is based on the simultaneous use of “zero crossing” and “ratio spectra derivative” methods. 10, 11 The main aim of the present research work was to investigate the utility of derivative spectrophotometry and to develop reliable spectrophotometric procedure for the simultaneous estimation of protein and nucleic acid either in laboratory samples, or in dosage forms without prior separation of individual components.
In recent time some of the methods have been published for simultaneous estimation of protein and RNA or DNA. These methods are based on utilizing immunoreactions and DNA hybridizations 12 or using functional nucleic acid molecules 13 or using single molecule arrays. 14 These kinds of methods need specific processing or reagents. The method proposed in the present study is very simple first derivative method for simultaneous estimation of protein and RNA.
MATERIAL AND METHODSInstruments
Spectrophotometric measurements were made on a Perkin Elmer Lambda 35 UV/VIS Spectrophotometer with UV data processing viewer software version 6.0.3 and UV data processing viewer software version 1.0.0.
Reagents
Bovine Serum Protein and RNA (used as model nucleic acid) were obtained from Jenni Biotech. All these chemicals and solvents were of analytical grade and were used without any further purification. All the required solutions were prepared in double distilled water.
Method
Stock solutions, 1 mg mL−1 in 0.1 M NaOH of Standard samples of protein and RNA were freshly prepared individually and further dilutions were made in the same solvent. Binary mixture of protein and RNA (ratio 1:1) was prepared in 0.1 M NaOH. The concentration ranges for protein and nucleic acid were found to be 60-140 µg mL-1 and 20-100 µg mL-1 respectively. Both analytes were tested for stability in solution and during the actual analysis and the behaviour of the analytes remained unchanged up to about 24 h from their preparation at the room temperature (the spectra was unchanged). Absorbance of the derivative spectra was measured at 261 nm, 289 nm for protein and RNA determination, respectively. Both substances were found to be stable during each kind of experimental measurements. Each measurement was done at room temperature.
First derivative zero crossing spectrophotometry
The absorption spectra of the samples were recorded between 200 nm to 400 nm against a reagent blank (the same of the samples without the substance to be determined) using a 1.0 cm quartz cell. The zero-order spectra of analytes were stored individually within the above concentration ranges and were derivatized in first order using scaling factor 5 for both the substances. The first derivative amplitudes were recorded at 261 nm and 289 nm for estimation of protein and RNA respectively for pure analyte and binary mixture.
Method Validation
The proposed method was validated in the limelight of ICH Guidelines for linearity, range, accuracy, precision, limit of detection and limit of quantification. Consequently, the following were performed.
Linearity and Range
A calibration curve was plotted over a concentration range of 60 to 140 µg mL-1 for protein and 20-100 µg mL-1 for RNA. The first derivative amplitudes were recorded at 261 nm and 289 nm for estimation of protein and RNA respectively for pure analyte and binary mixture. The Linearity was constructed by plotting concentration against absorbance with each reading.
Accuracy
For studying the accuracy recovery experiments were carried out by the standard addition method. This study was performed by addition of known amounts of protein and RNA to a known concentration of the binary solution. The amounts of standard recovered were calculated in terms of mean recovery with the upper and lower limits of % R.S.D.
Precision
Intra-day precision and inter-day precision for the developed method were measured in terms of % R.S.D. The experiments were repeated three times a day for intra-day precision and on three different days for inter-day precision. Except lowest and highest, five middle concentrations were taken for determination of the precision (concentrations for protein: 70, 80, 90, 100, 120 µg mL-1 and RNA: 30, 40, 60, 70, 80 µg mL-1). The concentration values for both intra-day precision and inter-day precision were calculated five times separately and % R.S.D were calculated. Finally, the mean of % R.S.D. (% R.S.D. = [S/X] 100, where S is standard deviation and X is mean of the sample analysed) was reported.
Limit of detection (LOD) and limit of quantitation<bold id="strong-905b8477159940a0be5adcb8afb0d66e"> </bold>(LOQ)
LOD and LOQ were calculated according to the 3s/m and 10s/m criterions, respectively, where s is the standard deviation of the absorbance (n = 10) of the sample and m is the slope of the corresponding calibration curve.
Reproducibility
The reproducibility of the method was determined by the use of different instruments (UV-VIS Spectrophotometer, UV-1700 Pharmaspec, Shimadzu) and different analyst.
RESULTS AND DISCUSSION
The absorption spectra of the two analytes, protein and RNA standards overlapped closely are shown in Figure 1. For this reason, the determination of these analytes was not possible by direct measurements of absorbance in zero-order spectra. On the other hand, derivative spectroscopy shows more resolution and makes it possible to analyse each analyte without interference of another analyte. Therefore, for simultaneous estimation of both analyte, first derivative zero crossing spectrophotometry method have been developed and validated.
<bold id="strong-94e65ac4ed3849c8b7223feea459c0c6"/>Zero order spectra ofprotein and RNA Standards.
First derivative zero crossing spectrophotometry
Derivative spectrophotometry has the ability to detect and measure the minor spectral features are enhanced and are measurable. This enhancement of spectra can distinguish very subtle changes in the spectrum and also there is increase in the number of peaks than the zero order spectra that would help in analysing the analyte with more selectively. Thus, it would be helpful to measure analyte easily in the binary mixtures. Thus, in the present method, in contrast to zero-order spectra, first derivative spectra have shown more resolution in terms of zero crossing points shown in Figure 2.
<bold id="strong-f9b35f5949f545a4ba89d47ab41fab6b"/>First derivative spectra of Standardprotein and RNA showing more resolution.
The first derivative wavelengths were 261 nm for protein estimation and 289 nm for RNA estimation. At 261 nm there is no interference of RNA, and protein was determined at 261 nm (Figure 3).
<bold id="strong-bd4d51ed6c4e4b5aac215fd2f3180a13"/>Protein estimation at 261 nm as there is no contribution of RNA at this wavelength.
At 289 nm, Protein shows zero absorbance; therefore, RNA was determined without any interference (Figure 4).
<bold id="strong-4aefe3fce4704cf3aa899c905cdb0c53"/>RNA estimation at 289 nm as there is no contribution of Protein at this wavelength.
The developed method was validated, and all the validation parameters and results of accuracy are shown in Table 1 and Table 2 respectively. The method was found to be linear in the range of 60 – 140 µg mL-1 and 20 – 100 µg mL-1. Also, method was precise and reproducible.
The developed and validated method provides a simple, fast and reliable means of simultaneous estimation of protein and nucleic acids in formulations containing protein and nucleic acid without any pre-treatment. This method does not require sophisticated instruments or methods. It is a quick and easy procedure for all fields of downstream analysis, especially when using limited sample resources and ensures reproducible outcome.
ACKNOWLEDGEMENTS
The authors express their sincere thanks to ISF College of Pharmacy, Moga, Punjab (India) for providing the basic research facilities.
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